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ENHANCING ORAL BIOAVAILABILITY OF

FAMOTIDINE AND ROXITHROMYCIN (BCS-IV DRUGS) BY

NANO-EMULSIFYING DRUG DELIVERY SYSTEM

By

MUHAMMAD SHAFIQUE

DEPARTMENT OF PHARMACY

UNIVERSITY OF MALAKAND

2012-2016

ENHANCING ORAL BIOAVAILABILITY OF FAMOTIDINE

AND ROXITHROMYCIN (BCS-IV DRUGS) BY NANO-

EMULSIFYING DRUG DELIVERY SYSTEM

This dissertation is submitted

As partial fulfillment of the requirement for the

Degree of

Doctor of Philosophy in Pharmacy

By

MUHAMMAD SHAFIQUE

DEPARTMENT OF PHARMACY

UNIVERSITY OF MALAKAND

2012-2016

In the name of AllahThe Most Gracious,

Merciful and Compassionate

i

APPROVALThe Department of Pharmacy, University of Malakand, accepts this thesis entitled

"Enhancing Oral Bioavailability of Famotidine and Roxithromycin (BCS-IV

Drugs) by Nano-emulsifying Drug Delivery System" submitted by Mr.

Muhammad Shafique in its present form and it is satisfying the dissertation

requirements for the degree of PhD in Pharmacy.

Approved By:

External Examiner-I: ________________________________________________

External Examiner-II: ________________________________________________

Supervisor: ______________________________________________________

Prof. Dr. Mir Azam KhanDepartment of PharmacyUniversity of Malakand

Co-supervisor: ______________________________________________________

Dr. Waheed S. KhanPrincipal Scientist National Institue for Biotechnology and Genetic EngineeringFaisalabad

Head of Department: ______________________________________________

Prof. Dr. Munasib KhanChairmainDepartment of PharmacyUniversity of Malakand

Dated: _____________________

ii

CERTIFICATE

It is certified that Mr. Muhammad Shafique having enrollment number

20120030104 has carried out all the work related to the thesis entitled "Enhancing

Oral Bioavailability of Famotidine and Roxithromycin (BCS-IV Drugs) by Nano-

emulsifying Drug Delivery System" under my supervision at the Department of

Pharmacy, University of Malakand in partial fulfillment for the award of PhD degree.

Date: _________________ Supervisor:_______________________

Dr. Mir Azam KhanProfessorDepartment of PharmacyUniversity of Malakand

_____________________________

Prof. Dr. Munasib KhanChairmanDepartment of PharmacyUniversity of Malakand

iii

DECLARATION

I hereby declare that I have produced the work presented in this thesis, during the

scheduled period of study. I also declare that this thesis does not incorporate, without

acknowledgement, any material previously submitted for a degree in any university

and that to the best of my knowledge and belief it does not contain any material

previously published or written by another person where due reference is not made in

the text. If a violation of HEC rules on research has occurred in this thesis, I shall be

liable to punishable action under the plagiarism rules of the HEC.

Date: _________________

____________________________

Muhammad Shafique

iv

RIGHT TO THESIS

All parts of this thesis are reserved to the researcher. No part of this research may be

produced or transmitted to any form or by any means, electronic or mechanical,

including photocopy, recording or any information storage and retrieval system,

without permission in writing from researcher.

Muhammad Shafique

v

Dedicated To my Father Jehan zeb

khan (late)

vi

ACKNOWLEDGEMENT

First of all many thanks to Almighty Allah for His un-countable blessings upon me

and helped-out me to successfully accomplish such a tedious job. After this, I want to

convey deepest thanks to countless people as without their support and help this work

would not have been possible.

First and foremost, I am cordially thankful to my research supervisor Prof.

Dr. Miz Azam Khan and co-supervisor Dr. Waheed Samreez Khan (Principal

Scientist, NIBGE) for their extreme dedication to my research work in many ways.

You taught me what it means to be a mentor, I really appreciate it. Your guidance,

confidence, motivation and encouragement to think independently helped me to

grow as a nano-scientist throughout my professional career. Research in the

field of Nanotechnology was a very big challenge and without your valuable

help, it was impossible to materialize the ideas and fruition this work.

I am grateful to the representatives of Graduate Science Committee (GSC)

(Prof. Dr. Waqar Ahmad, Prof. Dr. Munasib Khan, Dr. Mohammad Shoaib, Dr.

Muhammad Junaid, Dr. Abdul Sadiq) for playing integral role in making

corrections and in-time valuable advices. I am also vey thankful to the faculty of

Department of Pharmacy (Dr. Farhat Ullah, Dr. Syed Wadood Ali Shah, Dr.

Muhammad Ayyaz, Dr. Nasiara Karim, Dr. Rukhsana Ghaffar, Mr. Jahangir

Khan, Mr. Aziz Ur Rehman, Mr. Jamil Anwar & Mr. Mubashir Ahmad )

To Dr. Shahzeb Khan and Dr. Munasib Khan, thank you for your

willingness to help me in non-traditional manner, it has not gone unnoticed.

I am thankful to my lab-mates and great research group, especially Mr.

Maqsood ur Rehman, Dr. Abdullah, Mr. Muhammad Ibrar and Mrs. Mahwish

Kamran for their co-operation, friendship and sharing knowledge.

I am cordially thankful to Higher Education Commission (HEC), Islamabad

and also to the Director of National Institute of Biotechnology and Genetic

Engineering (NIBGE), Faisalabad. To Dr. Ayesha Ihsan (Senior Scientist,

NIBGE), thank you for accepting me into your lab and also all scientists of Nano-

Biotechnology Group for letting me to be a part of their research group at NIBGE.

I would like to express my heartfelt thanks to the management of Alliance

Pharmaceuticals (Pvt) Ltd, Peshawar, Pakistan and Polyfine Chempharma (Pvt) Ltd, viii

Peshawar, Pakistan for providing the drug samples (raw). I thank to Mr. Abdullah

and Mr. Khan Malok (Centralized Resource Laboratory, University of Peshawar,

Pakistan) and Mr. Zahir-ur-Rehman Ferozsons (Laboratories Limited, Nowshera,

Pakistan) for their help in characterization and industry relevant research work.

On a personal note, I would like to thank my sister and brothers (Dr.

Muhammad Fawad and Muhammad Jawad) and all family members and friends for

their support and love. To my Mother, you taught me that I could achieve any target

in my life and supported me in every step I have taken. Thanks all for helping me to

come true my fetching dream of big dreams. I love you all.

MUHAMMAD SHAFIQUE

ix

ABSTRACT

Pre-dosage forms of Famotidine and Roxithromycin available in the market suggest

that their treatment may not facilitate patients due to poor water solubility and

permeability which ultimately leads to their low oral bioavailability. To reduce the

draw backs associated with their systemic administration, Solid Lipid Nanoparticles

(SLNs) loaded with Famotidine and Roxithromycin were fabricated as a mean of

achieving boosted oral bioavailability.

During fabrication of Solid Lipid Particles (SLNs), emulsion was employed as

the most important precursor. Stearic acid was employd as the solid lipid phase and

Tween® 80 as surfactant. Polyethylene glycol and polyvinyl alcohol were used as co-

surfactants. Different results in term of particles size and polydispersity index

(PDI) were obtained by varying experimental conditions, i.e. concentration of

surfactant, concentration of co-surfactant and stirring time. SLNs were fabricated

via three different techniques (Solvent Injection, Solvent Emulsification Evaporation

and Hot Melt Encapsulation) using nano-template engineering technology.

Solvent Injection technique was employed for Fabrication of SLNs loaded

with Roxithromycin and Famotidine. SLNs loaded with Roxithromycin and

Famotidine demonstrated particle size 169.6±2.3 nm & 162.7±2.3 nm, PDI

0.462±0.02 & 0.352±0.03, zeta potential -32.6±1.9 mV & -34.35±2 mV, percent

entrapment efficiency 84.36±1.3% & 85±2.7%, percent drug loading capacity

2.709±0.43% & 2.74±0.33% respectively.

Solvent Emulsification Evaporation method being used for preparation of

SLNs loaded with Roxithromycin and Famotidine. SLNs loaded with Roxithromycin

and Famotidine showed particle size 126.27±2.1 nm & 111.9±1.3 nm, PDI

0.435±0.01 & 0.464±0.03, zeta potential -36.72±2 mV & -33.46±2 mV, percent x

entrapment efficiency 83.61±2.3% & 84±2.7%, percent drug loading capacity

2.677±0.13% & 2.709±0.13% respectively.

Hot Melt Encapsulation technique, which avoids the use of organic solvent

was also being employed for Fabrication of SLNs loaded with Roxithromycin and

Famotidine. SLNs loaded with Roxithromycin and Famotidine demonstrated particle

size 179.7±2.3 nm & 174.8±2.1 nm, PDI 0.424±0.03 & 0.419, zeta potential -

38.16±1.6 mV & -36.35 mV, percent entrapment efficiency 86% & 87±2.1%, percent

drug loading capacity 2.77% & 2.81±0.13% respectively.

During further characterization of loaded SLNs formulations, the white

patches in the micrographs of Scanning Electron Spectroscopy (SEM) verified

identical, spherical shaped and nano-metric size particles. SEM also showed that the

particles size was in concordance to the data attained from Dynamic Light Scattering

analysis. Fourier Transform Infrared Spectroscopy revealed no drugs-excipients

interaction. Moreover, characterization via using Powdered X-Ray Diffractometer and

Differential Scanning Calorimetry confirmed successful reduction in the crystalline

nature of the loaded SLNs formulations. In-vitro drug release study was conducted

and enhanced sustained release was found with maximum drug pay-load. Different

mathematical kinetic models were employed to the drug release data to confirm the

drug release kinetics and mechanism. During stability study, SLN dispersions stored

at different conditions confirmed maximum stability at refrigerated condition,

showing a consistent particles size and polydispersity.

Moreover, tray drying technique as alternative to lyophilization was

investigated and found that this technique can also be employed for SLNs drying

purpose, especially for bulk production. Scanning Electron Microscopy (SEM) was

conducted for the samples being prepared by tray drying technique in order to

xi

compare with the lyophilized samples, the white patches in the micrographs of both

samples were almost similar in size and shape. To acquire proper solid dosage form,

loaded SLNs nano-suspensions were processed to obtain dried powder followed by

conversion to granules and consequently filled in capsule shells.

Comparative in-vitro study of the prepared capsules was conducted for

dissimilarity (f1) and similarity (f2) factors determination. Dissimilarity factor greater

than 65 (f1>65) showing a remarked difference compared to the marketed products.

Comparative in-vivo study of the SLNs nano-suspension as well as prepared

capsules with the marketed product has also been conducted. This study showed

massive difference, in terms of increased Cmax as well as AUC0→24 compared to the

marketed products.

Overall, these results indicate that the developed Nanoparticulate Drug

Delivery System of SLNs is smart enough showing significantly improved oral

bilavailability with sustained drug release profile for Famotidine and Roxithromycin.

xii

LIST OF ACRONYMS

AUC Area under curve

BBB Blood brain barrier

Cmax Peak plasma concentration

DDS Drug delivery system

DSC Differential scanning calorimetry

DLS Dynamic light scattering

DLC Drug loading capacity

EE Encapsulation Efficiency

e.g. exempli gratia

FT-IR Fourier transform infrared

g Grams

GIT Gastrointestinal tract

HPH High pressure homogenization

HPLC High performance liquid chromatography

hr Hour

kg Kilogram

LOD Loss on drying

min Minutes

ml Milliliter

NP Nanoparticle

nm Nanometer

o/w Oil-in Water

PBS Phosphate buffer saline

xiii

PIT Phase inversion temperature

PCS Photon correlation spectroscopy

PDI Polydispersity index

PEG Polyethylene glycol

ppm part per million

PVP Polyvinylpyrrolidone

P-XRD Powder X-Ray Diffraction

RES Reticulo-endothelial system

rpm Revolutions per minute

SEM Scanning electron microscopy

SD Standard Deviation

SLNs Solid Lipid Nanoparticles

TEM Transmission Electron Microscopy

Tmax Maximum plasma concentration time

UV Ultraviolet

w/o Water-in Oil

w/o/w Water in-Oil-in Water

ZP Zeta potential

xiv

TABLE OF CONTENTS

CHAPTER 1 INTRODUCTION.............................................................................1

1.1 DRUG DEVELOPMENT AND BIOAVAILABILITY ISSUES..............................1

1.2 REASONS FOR POOR ORAL BIOAVAILABILITY.............................................2

1.3 APPROACHES FOR IMPROVING ORAL BIOAVAILABILITY........................4

1.4 NANOTECHNOLOGY................................................................................................5

1.5 NANOPARTICULATE DRUG DELIVERY SYSTEM............................................7

1.5.1 TYPES OF NANOPARTICULATE DRUG DELIVERY SYSTEM......................7

1.5.2 APPROACHES FOR NANOPARTICLES PRODUCTION.................................14

1.5.3 MERITS OF NANOPARTICULATE DRUG DELIVERY SYSTEM..................15

1.5.4 DE-MERITS OF NANOPARTICULATE DRUG DELIVERY SYSTEM...........16

1.6 DESCRIPTION OF SOLID LIPID NANOPARTICLES (SLNS)..........................17

1.6.1 COMPONENTS OF SLNS.....................................................................................18

1.6.2 DRUG INCORPORATION MODELS OF SLNS.................................................20

1.6.3 INFLUENCE OF DIVERSE PARAMETERS ON SLNS......................................21

1.6.4 FATE OF SLNS......................................................................................................23

1.6.5 FABRICATION TECHNIQUES OF SLNS...........................................................24

1.6.6 SECONDARY STEPS IN SLNS FABRICATION................................................30

1.6.7 ANALYTICAL CHARACTERIZATION OF SLNS.............................................31

1.6.8 CONVERSION OF SLNS DISPERSION TO SOLID DOSAGE FORM.............36

1.6.9 IN-VIVO STUDY OF SLNS...................................................................................37

1.6.10 ROUTES OF ADMINISTRATION FOR SLNS....................................................38

1.6.11 ADVANTAGES OF SLNS.....................................................................................40

1.6.12 DISADVANTAGES OF SLNS..............................................................................40

1.7 ROXITHROMYCIN...................................................................................................41

1.8 FAMOTIDINE............................................................................................................42

1.9 MOTIVATION TO DO THIS WORK.....................................................................45

1.9.1 STATEMENT OF THE PROBLEM......................................................................45

1.9.2 AIMS AND OBJECTIVES OF THE STUDY.......................................................46

CHAPTER 2 LITERATURE REVIEW...............................................................47

2.1 SLNS FABRICATION TECHNIQUES....................................................................48

2.1.1 SOLVENT INJECTION (SI) TECHNIQUE..........................................................48

2.1.2 SOLVENT EVAPORATION TECHNIQUE.........................................................52

xv

2.1.3 HOT MELT ENCAPSULATION TECHNIQUE..................................................55

2.2 FORMULATIONS OF ROXITHROMYCIN..........................................................56

2.2.1 CONVENTIONAL FORMULATIONS................................................................56

2.2.2 MODIFIED FORMULATIONS............................................................................57

2.3 FORMULATIONS OF FAMOTIDINE....................................................................60

2.3.1 CONVENTIONAL FORMULATIONS................................................................60

2.3.2 MODIFIED FORMULATIONS............................................................................61

2.4 DEFICIENCES IN ROXITHROMYCIN AND FAMOTIDINE FORMULATIONS 63

2.5 PROPOSED APPROACH FOR ROXITHROMYCIN AND FAMOTIDINE......64

CHAPTER 3 MATERIALS AND METHODS....................................................66

3.1 MATERIALS...............................................................................................................66

3.1.1 CHEMICALS.........................................................................................................66

3.1.2 INSTRUMENTS USED.........................................................................................67

3.2 METHODS..................................................................................................................70

3.2.1 METHODS FOR SLNS FABRICATION..............................................................70

3.2.1.1 Method-I (Solvent Injection Method).............................................................70

3.2.1.2 Method-II (Solvent Evaporation Method)......................................................73

3.2.1.3 Method-III (Hot Melt Encapsulation Method)...............................................75

3.3 LYOPHILIZATION...................................................................................................78

3.3.1 TRAY DRYING AS ALTERNATIVE TO LYOPHILIZATION.........................78

3.4 CHARACTERIZATION............................................................................................79

3.5 CONVERSION OF NANO-SUSPENSION TO CAPSULES.................................83

3.5.1 TRAY DRYING.....................................................................................................83

3.5.2 MOISTURE LEVEL DETERMINATION............................................................83

3.5.3 GRANULATION...................................................................................................84

3.5.4 CAPSULE SHELLS FILLING..............................................................................86

3.6 COMPARATIVE IN-VIVO STUDY OF PREPARED NANOFORMULATIONS

WITH MARKETED PRODUCTS............................................................................87

3.6.1 ORAL DRUG ADMINISTRATION.....................................................................87

3.6.2 COLLECTION OF BLOOD SAMPLES...............................................................87

3.6.3 ANALYSIS OF PLASMA DRUG CONCENTRATION VIA HPLC...................87

3.6.4 ANALYSIS OF DRUG DATA..............................................................................88

3.7 DISSIMILARITY (F1) AND SIMILARITY (F2) FACTORS...................................89

xvi

CHAPTER 4 RESULTS AND DISCUSSION......................................................90

4.1 SOLVENT INJECTION METHOD.........................................................................90

4.1.1 PARTICLE SIZE ANALYSIS...............................................................................90

4.1.2 SCANNING ELECTRON MICROSCOPY (SEM)...............................................93

4.1.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY (FT-IR)....................95

4.1.4 POWDERED X-RAY DIFFRACTOMETRY (P-XRD)........................................96

4.1.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC).....................................98

4.1.6 ENTRAPMENT EFFICIENCY AND DRUG LOADING CAPACITY...............99

4.1.7 IN-VITRO STUDY...............................................................................................101

4.1.8 KINETIC MODELING........................................................................................102

4.1.9 STABILITY STUDY...........................................................................................103

4.1.10 COMPARATIVE IN-VIVO STUDY OF PREPARED NANO-SUSPENSION

WITH MARKETED PRODUCT.........................................................................105

4.2 SOLVENT EMULSIFICATION EVAPORATION METHOD...........................108

4.2.1 PARTICLE SIZE ANALYSIS.............................................................................108

4.2.2 SCANNING ELECTRON MICROSCOPY (SEM).............................................111

4.2.3 FOURIER TRANSFORM INFRARED MICROSCOPY (FT-IR)......................112

4.2.4 POWDERED X-RAY DIFFRACTOMETRY (P-XRD)......................................114

4.2.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC)...................................115

4.2.6 ENTRAPMENT EFFICIENCY AND DRUG LOADING CAPACITY.............116

4.2.7 IN-VITRO STUDY...............................................................................................118


4.2.9 STABILITY STUDY...........................................................................................121

4.2.10 COMPARATIVE IN-VIVO STUDY OF PREPARED NANO-SUSPENSION

WITH MARKETED PRODUCT.........................................................................122

4.3 HOT MELT ENCAPSULATION METHOD........................................................125

4.3.1 PARTICLE SIZE ANALYSIS.............................................................................125

4.3.2 SCANNING ELECTRON MICROSCOPY (SEM).............................................128

4.3.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY (FT-IR)..................129

4.3.4 POWDERED X-RAY DIFFRACTOMETRY (P-XRD)......................................131

4.3.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC)...................................132

4.3.1 ENTRAPMENT EFFICIENCY AND DRUG LOADING CAPACITY.............134

4.3.2 IN-VITRO STUDY...............................................................................................135


4.3.4 STABILITY STUDY...........................................................................................138

xvii

4.3.5 COMPARATIVE IN-VIVO STUDY OF SLNS NANO-SUSPENSION WITH

MARKETED PRODUCT........................................................................................139

4.3.6 COMPARATIVE IN-VIVO STUDY OF PREPARED CAPSULES WITH MARKETED

PRODUCT.............................................................................................................141

4.3.7 DISSIMILARITY FACTOR (F1) AND SIMILARITY FACTOR (F2)................144

CONCLUSION.....................................................................................................................148

REFERENCES......................................................................................................................150

xviii

LIST OF TABLES

Table 1.1: Definitions for solubility terms.................................................................................1

Table 1.2: Particle sizes with surface area and percentage of surface molecules......................5

Table 1.3: Roles of various drug delivery systems..................................................................14

Table 1.4: Lipid and excipient employed for the fabrication of SLNs....................................19

Table 2.1: List of drugs loaded into SLNs via different techniques........................................55

Table 3.1: Formulations of blank SLNs (Solvent Injection method)......................................71

Table 3.2: Formulations of RTM-SLNs (Solvent Injection method)......................................72

Table 3.3: Formulations of FTD-SLNs (Solvent Injection method).......................................72

Table 3.4: Formulations of blank SLNs (Solvent evaporation method)..................................74

Table 3.5: Formulations of RTM-SLNs (Solvent evaporation method)..................................74

Table 3.6: Formulations of FTD-SLNs (Solvent evaporation method)...................................75

Table 3.7: Formulations of blank SLNs (Hot Melt Encapsulation method)............................76

Table 3.8: Formulations of RTM-SLNs (Hot Melt Encapsulation method)............................77

Table 3.9: Formulations of FTD-SLNs (Hot Melt Encapsulation method).............................77

Table 3.10: Excipients employed during wet granulation.......................................................84

Table 3.11: Excipients employed for granules coating............................................................85

Table 4.1: Melting point of RTM, Stearic Acid and RFSi-4 nanoformulation.......................98

Table 4.2: Melting point of FTD, Stearic Acid and FFSi-4 nanoformulation.........................99

Table 4.3: R2 value of different kinetic models for RTM-SLNs formulations......................103

Table 4.4: R2 value of different kinetic models for FTD-SLNs formulations........................103

Table 4.5: Stability study of RTM Loaded SLNs (RFSi-4 nanoformulation).......................104

Table 4.6: Stability study of FTD Loaded SLNs (FFSi-4 nanoformulation).........................104

Table 4.7: Comparative in-vivo pharmacokinetic parameters of RTM.................................106

Table 4.8: Comparative in-vivo pharmacokinetic parameters of FTD..................................106

Table 4.9: Melting point of RTM, Stearic Acid and RFSe-4 nanoformulation.....................116

Table 4.10: Melting point of FTD, Stearic Acid and FFSe-4 nanoformulation....................116

Table 4.11: R2 value of different kinetic models for RTM-SLNs formulations....................120

Table 4.12: R2 value of different kinetic models for FTD-SLNs formulations.....................120

Table 4.13: Stability study of RTM Loaded SLNs (RFSe-4 nanoformulation)....................121

Table 4.14: Stability study of FTD Loaded SLNs (FFSe-4 nanoformulation)......................122

Table 4.15: Comparative in-vivo pharmacokinetic parameters of RTM...............................123

Table 4.16: Comparative in-vivo pharmacokinetic parameters of FTD................................123

Table 4.17: Melting point of RTM, Stearic Acid and RFH-4 nanoformulation....................133

Table 4.18: Melting point of FTD, Stearic Acid and FFH-4 nanoformulation.....................133

xix

Table 4.19: R2 value of different kinetic models for RTM-SLNs formulations....................137

Table 4.20: R2 value of different kinetic models for FTD-SLNs formulations.....................138

Table 4.21: Stability study of RTM Loaded SLNs (RFH-4 nanoformulation).....................139

Table 4.22: Stability study of FTD Loaded SLNs (FFH-4 nanoformulation).......................139

Table 4.23: Comparative in-vivo pharmacokinetic parameters for RTM..............................140

Table 4.24: Comparative in-vivo pharmacokinetic parameters for FTD...............................141

xx

LIST OF FIGURES

Figure 1.1: Approaches for shifting solubility and permeability of drugs to BCS-I drugs 4

Figure 1.2: Surface area enlargement with particle size reduction....................................6

Figure 1.3: Types of nanoparticulate drug delivery system..............................................8

Figure 1.4: Bottom-up and top-down approaches...........................................................15

Figure 1.5: Comparision of SLNs and Liquid Lipid Emulsions......................................18

Figure 1.6: Drug incorporation models...........................................................................21

Figure 1.7: Techniques for SLNs fabrication..................................................................24

Figure 1.8: Different characterization parameters of SLNs............................................31

Figure 1.9: DLS of Large particles (A) and small particles (B)......................................32

Figure 1.10: Mechanism of action of Roxithromycin......................................................42

Figure 1.11: Mechanism of action of Famotidine............................................................44

Figure 3.1: Schematic diagram of Solvent Injection technique.......................................73

Figure 3.2: Schematic diagram of Solvent Evaporation technique.................................75

Figure 3.3: Schematic diagram of Hot Melt Encapsulation technique............................78

Figure 4.1: Particle size and PDI of blank SLNs formulations (SI).................................92

Figure 4.2: Particle size (A) & Zeta Potential (B) of RFSi-4 nanoformulation...............93

Figure 4.3: Particle size (A) & Zeta Potential (B) of FFSi-4 nanoformulation................93

Figure 4.4: SEM micrograph of RFSi-4 nanoformulation...............................................94

Figure 4.5: SEM micrograph of FFSi-4 nanoformulation................................................94

Figure 4.6: FT-IR Spectra of (A) unprocessed RTM (B) RFSi-4 Nanoformulation........96

Figure 4.7: FT-IR Spectra of (A) unprocessed FTD (B) FFSi-4 Nanoformulation.........96

Figure 4.8: P-XRD of unprocessed RTM and RFSi-4 nanoformulation..........................97

Figure 4.9: P-XRD of unprocessed FTD and FFSi-4 nanoformulation...........................97

Figure 4.10: EE% and DLC% of RTM-SLNs formulations (SI)...................................100

Figure 4.11: EE (%) and DLC (%) of FTD-SLNs formulations....................................100

Figure 4.12: Drug Release from RTM-SLNs Formulations...........................................101

Figure 4.13: Drug Release from different FTD-SLN formulations................................102

Figure 4.14: Comparative in-vivo drug release study of RTM.......................................106

Figure 4.15: Comparative in-vivo drug release study of FTD........................................107

Figure 4.16: Particle size and PDI of blank SLNs formulations....................................109

Figure 4.17: Particle size (A) and Zeta Potential (B) of RFSe-4 nanoformulation........111

Figure 4.18: Particle size (A) and Zeta Potential (B) of FFSe-4 nanoformulation........111

Figure 4.19: SEM micrographs of RFSe-4 nanoformulation.........................................112

Figure 4.20: SEM micrograph of FFSe-4 nanoformulation...........................................112

xxi

Figure 4.21: FT-IR Spectra of (A) unprocessed RTM (B) RFSe-4 nanoformulation....113

Figure 4.22: FT-IR Spectra of (A) unprocessed FTD (B) FFSe-4 nanoformulation......114

Figure 4.23: Diffractogram of unprocessed RTM and RFSe-4 nanoformulation..........115

Figure 4.24: Diffractogram of unprocessed FTD and FFSe-4 nanoformulation............115

Figure 4.25: EE (%) and DLC (%) of RTM-SLNs formulations...................................118

Figure 4.26: EE (%) and DLC (%) of FTD-SLNs formulations....................................118

Figure 4.27: Drug Release from RTM-SLNs formulations............................................119

Figure 4.28: Drug Release from FTD-SLNs formulations.............................................119

Figure 4.29: Comparative in-vivo drug release study of RTM.......................................124

Figure 4.30: Comparative in-vivo drug release study of FTD........................................124

Figure 4.31: Particle size and PDI of unloaded SLNs (HME).......................................126

Figure 4.32: Particle size (A) and Zeta potential (B) of RFH-4 nanoformulation.........127

Figure 4.33: Particle size (A) and Zeta potential (B) of FFH-4 nanoformulation..........128

Figure 4.34: SEM micrograph of RFH-4 nanoformulation............................................129

Figure 4.35: SEM micrograph of FFH-4 nanoformulation............................................129

Figure 4.36: FT-IR Spectra of (A) unprocessed RTM (B) RFH-4 nanoformulation.....130

Figure 4.37: FT-IR Spectra of (A) Unprocessed FTD (B) FFH-4 nanoformulation......131

Figure 4.38: P-XRD of unprocessed RTM and RFH-4 nanoformulation......................132

Figure 4.39: P-XRD of unprocessed FTD and FFH-4 nanoformulation........................132

Figure 4.40: EE% and DLC% of RTM-SLNs formulations..........................................135

Figure 4.41: EE% and DLC% of FD-SLNs formulations..............................................135

Figure 4.42: Drug release from RTM-SLNs Formulations............................................136

Figure 4.43: Drug release from FTD-SLNs nanoformulations......................................137

Figure 4.44: Comparative in-vivo drug release of RTM................................................142

Figure 4.45: Comparative in-vivo drug release of FTD..................................................143

Figure 4.46: Drug release profile of RFH-4 nanoformulation and marketed drug.........145

Figure 4.47: Drug release profile of FFH-4 nanoformulation and marketed drug.........146

xxii

CHAPTER No. 1 INTRODUCTION

Chapter 1 INTRODUCTION

1.1 DRUG DEVELOPMENT AND BIOAVAILABILITY ISSUESIn the 19th century and earlier eras, extracts of natural products and derivatives of

botanical species, presented the major resource of folk medicine [1]. Efforts were

being made from time to time in order to identify the actives in the natural products

and acoordingly seperated them in pure form. In the end of the 19th century, pure bio-

active organic molecules were seperated from medicinal plants for medicinal purpose

i.e. in 1874, salicyclic acid (precursor of aspirin) was seperated from bark of willow.

As a result, the 1st pharmaceutical drug (aspirin) was synthesized in the last half of the

19th century. From that instance, efforts are being made by scientists and synthesized

different drugs which passed several stages to be shaped into final product which is

efficacious and safe. But approximately 40% of these drug products, available in the

market-place in conventional dosage form are reported to have poor oral

bioavailability [2]. Poor oral bioavailability is due to low solubilization of the

marketed drugs with low permeability across the gastro-intestinal tract (GIT) which

ultimately leads to effect the drug safety and efficacy [3].

That’s why; first of all seven different specified terms were designated for

diverse degree of solubility. Definitions for these terms are given in Table 1.1.

Table 1.1: Definitions for solubility termsS. No Term Parts of solvent for one part of solute (in

ml)1 Insoluble > 10,0002 Very slightly soluble

1000 - 10,000

3 Slightly soluble

100 – 1000

4 Sparingly 30 – 100

1


soluble5 Soluble 10 – 306 Freely soluble 1 – 107 Very soluble < 1

1.2 REASONS FOR POOR ORAL BIOAVAILABILITYDuring drugs development process, significant physico-chemical parameters such as

aqueous solubility, permeability, dissociation constant and stability are mainly

considered. As, oral drug absorption is preliminary affected by aqueous solubility and

gastro-intestinal permeability, which in turn affect oral bioavailability. Thus, these

two factors are principally figured-out during drug development process.

For to determine the aqueous solubility of a drug, the highest unit dose is

dissolved in 250 mL phosphate buffer (pH 1-8). Generally high-solubility is

considered for those drugs which have dose/solubility volume of solution less than or

equal to 250 mL. While during determination of drugs permeability, the drug is said

to be highly permeable when the extent of absorption from the gastro intestinal tract

(GIT) is almost greater than 90% in the absence of any reported in-stability in the

GIT.

Taking into consideration the solubility and permeability issues,

biopharmaceutics classified the drugs by a system termed as Biopharmaceutical

Classification System (BCS). This system classified the drugs into the following four

different groups [4]:

i. BCS-I: High Solubility - High Permeability

ii. BCS-II: Low Solubility - High Permeability

iii. BCS-III: High Solubility - Low Permeability

iv. BCS-IV: Low Solubility - Low Permeability

This system suggests that BCS-I drugs have high solubility and permeability

and thus, should have no oral bioavailability issues. BCS-I drugs and in some 2


instances BCS-III drugs having 85% dissolution in 0.1N HCl in 15 minutes can

guarantee that the rate limiting step for drug absorption is not dissolution but is gastric

emptying. During fasting condition, the mean gastric emptying time is 15-20 minutes.

A drug should not have any oral bioavailability problems if it is 85% dissolved in 15

minutes.

In BCS-II drugs, for drug absorption, the rate limiting step is dissolution.

While for BCS-III drugs, the rate limiting step is permeability. BCS-IV drugs have

significant issues in oral administration as this class has both solubility and

permeability problems.

BCS Classification of orally administered drugs according to WHO model list

is as follow [5].

i. BCS Class-I Drugs

Zidovudine, Theophylline, Stavudine, Salbutamol, Riboflavin Vitamin, Pyrazinamide,

Propranolol, Primaquine, Prednisolone, Phenobarbital, Phenoxy-Methylpenicillin,

Metronidazole, Levodopa, Fluconazole, Diazepam, Cyclophosphamide, Chloroquine,

Amiloride, Digoxine, Doxycycline.

ii. BCS Class-II Drugs

Carbamazepin, Dapsone, Griseofulvin, Ibuprofen, Nifedipine, Phenytoin,

Sulfamethoxazole, Nitrofurantoin, Trimethoprim, Valproic Acid, Rifampicin,

Praziquantel, Nalidixic Acid, Iopanoic Acid.

iii. BCS Class-III Drugs

Abacavir, Acetylsalicylic Acid , Aciclovir, Allopurinol, Atenolol, Captopril,

Chloramphenicol, Cimetidine, Sodium Cloxacillin, Codeine Phosphate, Colchicine,

Ergotamine Tartrate, Hydralazine, Hydrochlorothiazide, Levothyroxine, Metformine,

3


Methyldopa, Paracetamol, Penicillamine, Promethazine, Propylthiouracil,

Pyridostigmine, Thiamine Vitamin.

iv. BCS Class-IV Drugs

Aluminium hydroxide, indinavir, nelfinavir, ritonavir, acetazolamide, azathioprine,

Famotidine, Roxithromycin.

1.3 APPROACHES FOR IMPROVING ORAL BIOAVAILABILITY

Creative formulation efforts are required to produce a finished drug product having

acceptable pharmacokinetic attributes. Physical or chemical alteration of these

drugs is required to solve their oral bioavailability issues followed by targeted

drug delivery and controlled release. Scientists’ through-out the globe have worked

and tried different techniques for solving the solubility and permeability issues of the

marketed drug products. Different techniques were employed to drugs of different

classes for shifting to Class-IV drugs (high solubility, high permeability). The

resultant successful techniques are envisioned in Figure 1.1:

4


Figure 1.1: Approaches for shifting solubility & permeability of drugs to BCS-I drugs

1.4 NANOTECHNOLOGYAccordingly, from the last few decades, efforts are being made to produce modified

drug delivery system to solve the problems associated with poor oral bioavailability.

Fast development opened numerous new vistas in pharmaceutical sciences, especially

in the field of Pharmaceutical Nanotechnology. In Pharmaceutical Nanotechnology,

scientists mainly focused on particle size and employed numerous techniques for their

size reduction. As, modification in particle size influences many key characteristics of

drug material and is a valuable indicator of performance and quality. In

Pharmaceutical Nanotechnology, the drugs particle size is reduced to nano-scale

which dramatically increased the total surface area of given material with increased

percentage of surface molecules. Different particle sizes with different surface area

and percentage of surface molecules are envisioned in Table 1.2 [6].

5


Table 1.2: Particle sizes with surface area and percentage of surface moleculesS. No Particle diameter

(nm)Surface Area

(nm2)Surface molecules (%)

1 10,000 1.26 × 109 0.03

2 1,000 1.26 × 107 0.30

3 100 1.26 × 105 2.974 10 1260 27.10

5 1 12.6 100.00

The above mentioned table, clearly indicated that particles size is inversely

proportional to the percentage of surface molecules. By reducing the particle size upto

1 nm, 100% molecules of the given material will be exposed on its surface.

Additionally, particle size reduction causing increase in surface area is shown

mathematically and diagrammatically in Figure 1.2.

Figure 1.2: Surface area enlargement with particle size reduction

Pharmaceutical Nanotechnology is a classical approach based on Noyes-

Whitney equation. It states that dissolution rate is proportional to the surface area of

the drug particles, which are being in contact with the dissolution medium. That’s

why Nanotechnologial techniques are used to boost the solubility of hydrophobic

drugs by reducing their particle size as the total surface area of the processed drug

6


increased. The relationship between drug dissolution and its surface area is ruled

by Noyes-Whitney equation as,

Dissolution Rate= A . Dh

(C s−Cb)

Whereas,A is surface areaD is diffusivityh is boundary layer thicknessCs is saturation solubilityCb is bulk concentration [7].

Gravitational force is much more smaller on nanoparticles compared to

microparticles, thus small size particles (nanoparticles) cannot precipitate out easily

because of less gravitational force. As, nanoparticles, due to their small size are less

prone to gravitational settling and thus, can easily be suspended in liquid

formulations. According to Stokes’ law, the settling velocity (v) of a particle is given

by,

v=d2 g( ps−pl)

18 μl

Whereas, d is particle diameter g is gravitational acceleration (9.8 m.sec-1 at sea level) ρs is solid density ρl is liquid density (997 kg.m-3 for water at 25ºC)μl is liquid viscosity (0.00089 Pa.sec-1 for water at 25ºC)

Resistance to sedimentation causes random thermal Brownian motion, keeping

the particles to be suspended in solution, thus gives more homogeneous suspension

with extended shelf-life and negates the need for shaking before use.

7


1.5 NANOPARTICULATE DRUG DELIVERY SYSTEMRecently numerous systems have been developed for drug delivery using the same

idea of nanotechnology i.e. particles size reduction (nanoparticles). Using

Nanoparticulate Drug Delivery Systems, new formulations of drugs have been

fabricated having elevated aqueous solubility and permeability with temporal and

targeted site-specific delivery. In recent years, the number of pharmaceutical products

and patents in this field are increasing extensively.

1.5.1 TYPES OF NANOPARTICULATE DRUG DELIVERY SYSTEM

Nanotechnology is an umbrella term, covering a number of drug delivery systems.

Different types of nanoparticulate drug delivery system are discussed below and

envisioned in Figure 1.3:

Figure 1.3: Types of nanoparticulate drug delivery system

i. Polymeric Nanoparticles

Polymeric nanoparticles (PNs) are fabricated by embedding the drug into

biodegradable polymeric matrix or drug adsorption on its surface. Now-a-days

PNs have gained much more importance, due to increased systemic circulation

8


half-life, reduced in-activation of loaded drug and decrease up-take by the phagocytic

system. FDA has also approved limited polymers for human use. As inside the human

body, these approved polymers simply degraded into monomers and cleared via

normal metabolic pathway i.e. poly-glycolic-acid (PGA), poly-lactic-co-glycolic-acid

(PLGA) and poly-lactic-acid (PLA) are hydrolyzed inside the body into glycolic acid

or lactic acid [8]. As compared to liposome, PNs are much more advantageous having

better therapeutic potential as well as long term storage stability. PNs protect the

encapsulated drugs from the external environment compared to liposomes [9].

ii. Metallic and Inorganic Nanoparticles

Numerous metallic and inorganic materials have been employed as carriers for

formulating nano-particulate drug delivery system. Among metallic, gold

nanoparticles are commonly used due to sophisticated photoelectric and optical

properties [10, 11]. Furthermore, gold nanoparticles have some additional specific

characteristics such as inertness, high physical stability, non-toxicity, the ease of

conjugation and modification with numerous functional groups like thiol, amine and

di-sulfide. In addition to drug delivery, gold nanoparticles are also used in

chemotherapy of cancer, as x-ray contrast agent, in the oligo-nucleotide delivery,

thiol conjugated RNA delivery, gene and insulin delivery [12-17]. But, these

nanoparticles cannot be employed for controlled drug release.

iii. Magnetic Nanoparticles

This system is being made by attachment of drug with magnetic nanoparticles. Thus,

we can say that magnetic nanoparticles comprise of two components, a magnetic

9


material (iron, magnetite, cobalt and nickel) and a chemical component (drug) that has

functionality [18]. Iron oxide as carrier has marvelous super paramagnetic effects,

naturally biodegradable and has capability of contrast agent in magnetic resonance

based imaging [19]. This system has achieved great attention in the field of

pharmaceutical drug delivery as it has immune-biocompatibility, controllable and

acceptable particle size and also has the ability of site-specific delivery [20].

iv. Liposomes

Lipid based nanoparticulate drug delivery system, comprises of two layered

membrane having multi-lamellar vesicles (MVL, >500 nm), large uni-lamellar

vesicles (LUV, 100-500 nm) and small uni-lamellar vesicles (SUV, 20-100 nm). this

system can be used for the loading of both hydrophilic and lipophilic drugs [21].

Liposomes fabricated with phosphatidylcholine have improved oral absorption, drug

compatibilities, ease of preparation, increase solubility of drugs and good pk profile

[22]. But the problems associated with this system includes its instability for oral

route of drug administration due to GIT physiological condition, particles hydrolysis

and aggregation, content leakage, poor controlled release, low encapsulation and drug

loading capacity. Transferosomes and ethosomes are also liposomes which are

formulated using surfactant and ethanol in order to make them more flexible.

Liposomal drug delivery system using viral protein as a carrier is termed as virosome

and is used specially for immunization and administered via mucosa, intra-muscular

and intra-dermal routes [23, 24].

v. Dendrimers

Dendrimers are tree-shaped nanoparticles having significant control over size and

polydispersity [25]. They have amazing surface functionality, thus, have numerous

applications like gene delivery, drug delivery, imaging agents in MRI and biological

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https://en.wikipedia.org/wiki/Chemical

https://en.wikipedia.org/wiki/Nickel

https://en.wikipedia.org/wiki/Cobalt

https://en.wikipedia.org/wiki/Iron


adhesives [26-29]. They cross barriers by transcellular and paracellular ways, thus can

be used for oral, ocular, transdermal and intravenous routes [30, 31]. Tumor cells can

be targeted with dendrimer as it has small size and non-immunogenic nature, thus the

vasculature can be escaped but not below the threshold for renal filtration [32].

Dendrimers have controlled drug release behavior with high drug loading capacity.

Drug release is greatly influenced by nature of bond between drug and dedrimeric

material and also by the pH of the medium used for drug release. Here, the chief issue

is toxicity as drug can be released at wrong place at wrong time.

vi. Nanocrystals

Crystalline nanoparticles are termed as nanocrystals. Nowadays, there is possibility

for to fabricate nanocrystals of metals, semi-conductors and other materials by

numerous techniques [33]. Drug nanocrystal can be fabricated in the desired size and

shape, using three different principles: milling, homogenization and precipitation as

well as a combination thereof. Amazing characteristic of drug nanocrystal is its

composition of 100% drug only. Carrier materials employed in fabrication of

polymeric nanoparticles are not being used for nanocrystals [34]. Nanocrystal when

dispersed in liquid medium leads to nano-suspension. For nano-suspensions,

dispersion medium can be non-aqueous, aqueous solutions or water. In dispersion,

particles must be stabilized. Nanocrystals dispersions are either charge-stabilized or

sterically stabilized [35]. To attain maximum solubility level, amorphous state and

nanometric size particles will be ideal. In the strictest sense, nanocrystal should not

be called to amorphous drug nanoparticle. But, often someone termed it as

nanocrystal despite in amorphous state.

vii. Carbon Nanotubes

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Todays, carbon nanotubes as nanoparticulate drug delivery system has great

significance in pharmaceutical sciences due to protein transporters or vaccines drug

delivery [36, 37]. They are fabricated via rolling up a single layer sheet of graphene or

sometimes multiple layers to make concrete cylinder [38]. In the initial stages of

fabrication, they were poorly water soluble and were toxic e.g. Pristine. Later on

carbon nanotubes were modified using different methodologies to enhance water

solubility. Numerous molecules such as nucleic acid, peptides, proteins and different

therapeutic agents are associated to carbon nanotubes [39].

viii. Quantum Dots

Quantum dots comprise of coated cores, consist of groups II-VI or III-V atoms of the

periodic table having reduced leakage of the metals from the core [40, 41]. These

metals have significant fluorescent and optical characteristics, hence have extreme

importance in the field of diagnosis [41]. Commonly employed metals are cadmium

telluride, cadmium selenide and indium arsenide used for both preventive and

diagnostic reasons [42]. Zinc oxide and titanium oxide are employed as sun-screen

agents [43]. Upon connection with photon, they get excited and release the absorbed

energy in ultra violet (UV) visible or near infrared region due to which they are

traced. Quantum dots are used for labeling of biological macro-molecules, due to

small size. Near tumor tissues, they produce hyperthermia due to their non-invasive

radiofrequency [44].

ix. Nanogels

Nanogels are cross-linked polysaccharide-based particles and are termed as hydrogels

if consist of water swell-able polymer chains [45, 46]. Water content is high in

hydrogels. They have desired mechanical properties and bio-compatibility with a

unique interior network for loading of bio-active molecules. They are non-toxic,

12


biodegradable and have stability for prolonged circulation in the blood stream. This

review illustrates the current developments of nanogels as drug delivery carrier for

biomedical and biological applications. Diverse synthetic strategy for the fabrication

of nanogel (based on chemical and physical cross-linking) is detailed, including

micro-molding and photolithographic methods, modification of bio-polymers,

continuous micro-fluidics, heterogeneous free-radical and controlled/living radical

polymerization [47]. Beside drug delivery, their exclusive characteristics propose high

potential for the utilization in the field of biomedical implants, tissue engineering and

bio-nanotechnology [48].

x. Polymeric Micelles

Polymeric micelles are developed as carriers for hydrophobic drugs having low

permeability or/and low long term stability [49, 50]. Polymeric micelles have

amphiphilic composition, in which the lipophilic drugs are loaded in the

lipophilic aggregate's core while the outer lipophobic coronas serves as

stabilizing layer. It is reported that amphiphilic polymer increased the

solubilization of hydrophobic drug. Like, hydroxypropylcelluloseg-

polyoxyethylene alkyl ether micelles enhanced the cyclosporine-A solubility with

no cytotoxicity in Caco-2 cells [51, 52]. Self assembled NPs of glycol-chitosan

with 50β-cholanic loaded with paclitaxel showed sustained release behavior and

its intravenous administration to mice reduced the tumor volume. Antibodies can

be attached to the polymeric micelles as targeting moieties to target specific

tissues.

xi. Solid Lipid Nanoparticles (SLNs)

Recently, SLNs have been fabricated by numerous sophisticated techniques. SLNs as

drug delivery system is alternative to colloidal drug delivery systems like lipid

13


emulsion, polymeric nanoparticles and liposomes. SLN combines plusses of

numerous colloidal carriers but evades some of their major documented short-

comings. SLNs are nano-metric in size (50 to 1000nm) and in solid state at both room

and physiological temperature [53]. SLNs offer large surface area and sustained drug

release with quick cellular uptake [54]. In modern eras, excessive attention is focused

on SLNs to increase bioavailability of hydrophobic drugs [55]. Mostly BCS Class-II

and Class-IV drugs are incorporated in SLNs [56]. SLNs-based drug delivery system

has numerous benefits including enhanced solubility of hydrophobic drugs with

prolonged drug release which ultimately lower dose and frequency of administration.

Different types of nano-particulate drug delivery sysytems along their

respective applications are given below in Table 1.3.

S.No Drug Delivery Systems Applications1 Solid Lipid Nanoparticles

(SLN)Least toxic and most stable system carrier system as alternative system to polymer

2 Polymeric nanoparticles Controlled and targeted drug delivery3 Polymeric micelles Controlled and systemic delivery of water

insoluble drugs4 Nanocrystal and Nano-

suspensionStable system for controlled delivery of poorly water soluble drug

5 Carbon Nanotubes Controlled delivery of gene and DNA6 Liposomes Controlled and targeted drug delivery7 Quantom dots (QD’s) Imaging and diagnostic agent

Table 1.3: Roles of various drug delivery systems

1.5.2 APPROACHES FOR NANOPARTICLES PRODUCTION

The unique mentioned properties of Nanoparticulate Drug Delivery Systems can be

utilized only if the commercial production of Nanoparticles via feasible technologies

is economical and safe. There exist two approaches for production of nanoparticles

using different technologies: bottom up and top down [57].

14


i. Bottom-up Approach

Bottom up approach is also termed as Building-up approach. In Bottom-up approach,

nanoscale particles are build-up from molecular solutions. Different techniques used

for fabrication of nanoscale particles using bottom-up approach include

Emulsification-Diffusion and Supercritical Fluid Precipitation.

ii. Top-down Approach

In Top-down approach, mechanical forces are utilized to break-down macroscopic

particles to nanoscale size. High pressure homogenization and ball/pearl milling

techniques are employed for nanoparticles production via top-down approach. These

two approaches are envisioned in Figure 1.4.

Figure 1.4: Bottom-up and top-down approaches

1.5.3 MERITS OF NANOPARTICULATE DRUG DELIVERY SYSTEM

The major documented advantages of nano-particulate drug delivery systems

include;

i. Nanoparticles (NPs) increase the solubility of hydrophobic drugs in the body fluid.

Aqueous solubility of the hydrophobic drug increases by loading in lipophobic

polymer or in a carrier having an outer layer of lipophobic polymer. Chitosan and

polyethylene glycol are the mainly employed polymers for the solubility

enhancement of the encapsulated drugs [58, 59]. It has been reported that amorphous 15


nanoparticles have enhanced solubility due to having improved wet ability, increased

surface area and reduced diffusion path-length.

ii. Site-targeting potential is the major characteristic of NPs formulations. NPs have

been explored specially for targeting numerious pathological and physiological

tissues. Site-targeting may be passive or active. Passive site-targeting can be attained

by improved retention and penetration effects via leaky vasculature. Active site-

targeting can be attained by attaching ligand on the surface of NPs. monoclonal

antibodies are being used as site-targeting ligands, for tumor specific antigens or

over expressed receptors [60, 61]. This make sure best drugs delivery to the

preferred tissues though avoids healthy tissue from potentially toxic chemo-

therapeutic agents. Actually, this evades the side effects of the loaded agent.

iii. NPs have been effectively employed for achieving of sustained drug release profile.

Sustained release profile can be attained on the basis of slow degradation of the

carriers or slow diffusion following polymeric swelling [62].

iv. Nanoparticles (NPs) demonstrate improved cells penetration due to small size and

surface functionalisation with diverse polymers. NPs revealed improved penetration

in animal models and cell culture of diverse tissues. Additionally, NPs showed

increased drug delivery to the tumors due to having their vicinity of leaky

vasculature. Tumors vasculature becomes leaky because they are quickly

proliferating tissues due to boosted nutrition supply which provides the opportunity

of increased drug delivery via nanoparticles [63].

v. NPs demonstrate comparatively minimum side effects and improve therapeutic

effects of the encapsulated drug [64, 65]. NPs also combat the phenomenon of multi

drug resistance against cancers and also infectious organisms [66]. This can be

16


attaind by increased penetration, multiple drugs loading or complexing drugs with

specific ligands.

1.5.4 DE-MERITS OF NANOPARTICULATE DRUG DELIVERY SYSTEM

Research in the field of Nanoparticulate Drug Delivery System faced some critical

problems about the fate and toxicity of nanoparticles. These problems may arise due

to structural component like polymer and other employed reagents or from their

intrinsic capacity to cross physiological barriers and penetrate cells. Some of these are

listed as follow;

i. Biodistribution/tissue distribution of nanoparticles is extremely tricky to calculate

and also difficult to absolutely understand as a large number of variables are

involved in it like complex structures of newly synthesized materials and their

particles size [67].

ii. The desired characteristics can be achieved with surface modification of

nanoparticles but these surface functionalizing agents will also lead to some sort of

toxicity. Accordingly, the nano-scientists should carry out extensive work on the

toxicity associated problems as well as drug distribution at cellular level.

iii. Nanoparticles being very small in nature make their route of administration

extremely complex. Nanoparticles being formulated for oral route of

administration may be breath-in and those for topical purpose may cause skin

irritation or even cancer upon penetration [68, 69]. Nanoparticles toxicity has

diverse level of infectivity for every character being suffered from it, however the

available confirmations are still insufficient to make the final conclusion [70].

iv. Some of the reported techniques for nanoparticles production are extremely

complex and also require sophisticated equipments accompanied by their technical

characterization approaches.

17


v. The exclusion of toxic reagents and organic solvents is also challenging and very

costly. Therefore, the appliance of nanoparticles is limited to some sort of research

areas due to required sophisticated instruments, highly technical personnel and

high cost [71].

vi. Nanoparticles have extremely complex chemistry. Nel et al. (2006) reported that

despite of having the mentioned toxicities, some nanoparticles also have potential

of aggregation for making clumps whereas others may generate reactive oxygen

species.

1.6 DESCRIPTION OF SOLID LIPID NANOPARTICLES (SLNs)Recently developed SLNs have been fabricated by numerous sophisticated

techniques. SLNs are alternative to colloidal drug delivery systems like lipid

emulsion, polymeric nanoparticles and liposomes. SLN combines plusses of different

colloidal carriers but evades some of their major documented short-comings. SLNs

are nano-metric in size (50 to 1000nm) and in solid state at both room and

physiological temperature [53]. SLNs offer large surface area and sustained drug

release with quick cellular uptake [54]. In modern eras, excessive attention is focused

on SLNs to increase bioavailability of hydrophobic drugs [55]. Mostly BCS Class-II

and Class-IV drugs are incorporated in SLNs [56].

SLN is one of the novel nano-particulate drug delivery systems, which has

been developed by re-placing liquid lipids of the emulsions by solid lipids and is also

advantageous over the polymeric nanoparticles. Numerous advantages have been

reported for SLNs like low toxicity, physically stable, good bio-compatibility and

hydrophobic drugs are better delivered via SLNs.

18


Figure 1.5: Comparision of SLNs and Liquid Lipid Emulsions

1.6.1 COMPONENTS OF SLNs

The basic two components of SLNs are phospho-lipids and surfactants. Surfactants

should be carefully selected on the basis of required Hydrophile-Lipophile Balance

(HLB) values of the lipids [72]. Required HLB values for some common lipids/oils

are available in literature e.g for stearic acid required HLB value is 15, so, Tween 80

having HLB value 15 can be employed as surfactant. Sometimes, SLNs are fabricated

using surfactant/co-surfactant mixture for better results (lower particle size and better

stability) as compared to SLNs of unadded co-surfactants. Different constitute

elements of SLNs are effectively summarized in Table 1.4 [73, 74].

Table 1.4: Lipid and excipient employed for the fabrication of SLNs

Lip

ids

Triacylglycerols TricaprinTrimyristinTrilaurinTristearin Tripalmitin

Acylglycerols Glycerol monostearate Glycerol Palmitostearate Glycerol behenate

Fatty acids Stearic acid Decanoic acidPalmitic acid Behenic acid

19


Waxes CetylpalmitateCyclic complexes CyclodextrinHard fat types Witepsol H-35

Witepsol W-35

Su

rfac

tant

s

Phospholipids Phosphatidylcholine Soy lecithin Egg lecithin

Ethylene oxide/propylene oxide copolymers

Poloxamer-188 Poloxamer-407 Poloxamer-182 Poloxamine-908

Sorbitan ethylene oxide/propylene oxide copolymers

Polysorbate-20 Polysorbate-60 Polysorbate-80

Alkylaryl polyether alcohol polymers TyloxapolBile salts Sodium cholate

Sodium taurocholate Sodium glycocholate Sodium taurodeoxycholate

Alcohol Ethanol Butanol1.6.2 DRUG INCORPORATION MODELS OF SLNs

Solid Lipid Nanoparticles (SLNs) fabrications after cooling down have diverse sites

of drug distribution with-in their structures. Drug allocation/accumulation in the

specific site of SLNs structure is dependent on fabrication techniques. SLNs can be

divided into three different models on the basis of drug incorporation [75]:

i. Bioactive-Enriched Core Model

This model has drug-enriched core with drug-free lipid shell. Solid lipids being

melted above their melting point can incorporate high quantity of drug, which upon

cooling resultant in supersaturated solution causing drug crystallization prior to lipid.

In this fashion, the shell holds the quantity of drug corresponding to the saturation

level while core holds crystalline drug in addition to that of saturation level.

ii. Bioactive-Enriched Shell Model20


This model is usually formed when SLNs are fabricated by hot homogenization

technique as the drug partition into the shell upon cooling. This model has drug-free

core with drug-enriched shell.

iii. Solid Solution Model

Solid solution model is also called homogenous matrix model. This model is usually

formed when SLNs are fabricated using cold homogenization technique without

employing surfactant. In this model, there is strong interaction between drug and lipid

and the incorporated drug is molecularly dispersed in lipid matrix. Slow and

controlled drug release can be obtained via this model [76].

SLNs have undergone extensive research to realize diverse approaches for

enhancing drug loading efficiency and to optimize sustained drug release profile. One

approach to acquire sustained drug release is the shell modification of SLNs. Shell

modification of SLNs can be achieved via incorporation of materials such as lecithin,

modified phosphatidylethanolamine and non-lipid polymers [77-79]. These agents

stabilize the shells and decrease the quantity of drug being precipitating in the shell

upon cooling.

Figure 1.6: Drug incorporation models

21


Muhlen et al., (1998), evaluated the release of tetracaine, etomidate and

prednisolone from different drug models and found that tetracaine and etomidate

follow Bioactive-enriched shell model (100% drug release in 1 minute) while

prednisolone follows Bioactive-enriched core model (drug release over 5 weeks).

They also established that employment of low melting lipids exhibit prolonged drug

release compared to high melting lipids [53].

1.6.3 INFLUENCE OF DIVERSE PARAMETERS ON SLNs

During preparation process, different characteristics like zeta-size, PDI, zeta-potential,

drug release behavior and entrapment efficiency can be modified by various

parameters such as formulation composition, different production techniques and

conditions [80]. Formulation composition includes surfactant concentrations,

cosurfactant concentrations, properties of the selected solid lipid and incorporated

drug. Different production techniques include hot melt encapsulation, microemulsion,

solvent injection, solvent emulsification evaporation and solvent diffusion etc. While

different processing conditions include time, temperature, pressure, cycle number,

lyophilization and sterilization.

i. Influence of the Solid Lipid

By taking hot homogenization technique into consideration, it has been studied that

SLNs fabricated with higher melting lipids have increased average particle size. But

for other lipids, various parameters for nanoparticles production will be diverse.

Additional, in most cases it is studied that increase in the reported lipid content (5-

10%) causes production of larger size particles with increased polydispersity index

due to increased viscosity.

ii. Influence of the Surfactant

22


The concentration of surfactant influences the particle size of SLNs. In general, small

size particles were observed with high surfactant concentration. As, particle size can

be reduced by increasing surfactant concentration because higher concentration give

better stability to small lipid droplets which prevent them from coalescence [81].

During different storage studies, it is also found that particle size increases with

decreased surfactant concentration.

iii. Influence of the Co-surfactant

In different study models, it is also found that with addition of co-surfactant, z-

average particle size further reduce as SLNs fabricated with surfactant/co-surfactant

mixture have lower z-average particle size and better stability as compared to SLNs

formulaions fabricated by the use of surfactant only [81].

iv. Influence of Stirring Time

PDI can be controlled and reduced by increasing magnetic stirring time as it has

almost no effect on particle size reduction but reduces the polydispersity index (PDI)

[82].

v. Influence of Number of Cycles and Pressure

Particle size and polydispersity index (PDI) both are reported to be decreased with

increasing number of cycles (3-7 cycles) and homogenization pressure (up to 1500

bar).

vi. Influence of Temperature

23


Large size particles are obtained at low processing temperature. The hot

homogenization method yields small size particles, compared to cold homogenization

technique.

vii. Influence of Drug Pay-load

In different studies, it has been found that Increased pay-load of the drug result in

prolong drug release time and vice versa [83].

1.6.4 FATE OF SLNs

The in-vivo fate of SLNs mainly depends on the administration route as well as

distribution process. SLNs consist of physiological or physiologically related waxes

or solid lipids. Hence, path-ways for SLNs metabolism and transportation are present

inside the body which play key role in the in-vivo fate of the carrier materials. As for

as, the key enzymes inside the body responsible for SLNs degradation are lipases.

Lipases enzymes are found in numerious tissues and organs. Lipase acts by splitting

the ester linkage and make partial glycerol or glycerides and free fatty acids. Lipase

requires activation via oil/water interface, which open the catalytic center. In-

vitro experiments conductaed for SLNs indicate variable degradation velocities by the

lipolytic enzyme pancreatic lipase as a function of their composition.

1.6.5 FABRICATION TECHNIQUES OF SLNs

There are numerous techniques being employed for the fabrication of SLNs. Some of

these techniques are discussed below and shown in Figure 1.7:

24


Figure 1.7: Techniques for SLNs fabrication

i. High Shear Homogenization (HSH)

This technique is powerful and reliable for the fabrication of solid lipid nano-

emulsion. During this technique high pressure (100-2000 bar) is needed for pushing

the liquid via narrow gap of only few microns. With such a high pressure, the liquid

accelerates at extremely high viscosity of more than 1000 km/h but the covered

distance is very small. Thus, high cavitation forces and shear stress break-down the

particles to sub-micron range.

Two approaches are being used to attain HSH i.e. cold homogenization and

hot homogenization.

a. Cold Homogenization

Cold homogenization is basically used for to over come heat correlated degradation

issues, loss of drugs into the aqueous phase and partitioning connected with hot

homogenization technique. Here, drug is incorporated into the melted lipid and then it

25


is cooled down by dry ice or liquid nitrogen. Resultant solid material is grinded using

mortar mill. These prepared micro-particles of lipid are then added to the specified

cold emulsifier solution. The temperature of the solution should be thoroughly

checked and effectively regulated to guarantee the solid state of the lipid during

homogenization process. The resultant sample of cold homogenization method

possesses larger size particles with broader size distribution compared to hot

homogenization method [84].

b. Hot Homogenization

Hot homogenization technique is usually conducted at temperature above the melting

point of the selected solid lipid. Peviously heated pre-emulsion of the melted lipid

loaded with the drug is prepared followed by mixing with the aqueous phase of

emulsifier via high shear mixing instrument. As a result, hot o/w emulsion is

produced followed by cooling, causing solidification of the melted lipid as well as

fabrication of SLNs.

Higher processing temperature yields smaller size particles because of reduced

viscosity of the lipid phase. But, in case of heat liable carriers and drugs, elevated

temperature leads to their degradation.

Advantages Customary at laboratory scale.

Low capital cost.

Disadvantages Polydisperse distributions.

Energy intensive process.

Unproven scalability.

ii. Ultrasonic Homogenization

26


Ultrasonic homogenization is a combinative approach, using both sonication and

homogenization methods. In this technique, the selected drug is incorporated in the

melted lipid (10°C) followed by mixing with aqueous solution of surfactant with high

shear homogenization. Consequently, the emulsion is subjected to ultra-sonication,

which finally reduces the particle size and create cavities for better drug entrapment.

At last, the sonicated emulsion is rapidly cooled down to get the desired SLNs

dispersion [85].

Advantages Demonstrated at laboratory scale.

Reduced shear stress.

Low capital cost.

Disadvantages Extremely energy demanding process.

Biomolecule damage.

Metal contamination.

Unproven scalability.

Physical in-stability upon storage.

Polydisperse distributions.

iii. Hot Melt Encapsulation

During Hot Melt Encapsulation technique, specified amount of solid lipid is melted

above its melting point and drug is added to it which is then vortexed. Aqueous phase

containing surfactant and/or co-surfactant is also heated up to melted lipid’s

temperature. Both phases are mixed together on magnetic stirrer to produce hot

melted micro-emulsion. Stirring was continued without heating until it cooled down

to 25 OC to prepare the desired SLNs dispersion.

iv. Microemulsion

27


Fabrication of SLNs based on micro-emulsion dilution was developed by Gasco and

coworkers in 1997. During this technique, solid lipid is melted above its melting point

and drug is added to it which is then vortexed. Aqueous phase containing surfactant

and/or co-surfactant is also heated up to melted lipid’s temperature. Both phases are

mixed together on magnetic stirrer to produce hot melted micro-emulsion. This hot

melted microemulsion is dispersed in cold water (2-3°C) under magnetic stirring to

get the desired SLNs dispersion. Ratio in the range of 1:25 to 1:50 is generally used

for the dispersion of hot melt microemulsion in cold water. This SLNs dispersion can

be employed as granulating fluid during SLNs conversion to solid dosage. But, this

SLNs dispersion has low particle count as compared to water content. Thus, too much

of water content will be needed to remove.

Advantages Theoretically stabile.

Low mechanical energy input.

Disadvantages Labor intensive formulation work.

Extremely sensitive to change.

Low nanoparticle concentrations.

v. Supercritical Fluid Technology

This is a fresh and new method, being used for the fabrication of SLNs [86]. When

temperature and pressure of a fluid exceeds its respective critical values is considered

supercritical fluid. The dissolving capability of the fluid increases for compounds.

The idea of supercritical fluid technology provides several processing techniques for

nano-particles fabrication i.e. Rapid Expansion of Supercritical Solution (RESS),

Aerosol-Solvent Extraction-Solvent (ASES), Particles from Gas Saturated Solution

(PGSS), Supercritical Fluid Extraction of Emulsions (SFEE).

Advantages

28


Avoidance of organic solvent.

Obtained Particles as dry powder, despite of suspension.

Mild temperature and pressure conditions.

For this technique, the best choice of solvent is carbon dioxide solution [87].

vi. Solvent Emulsification Evaporation (SEE)

During SEE method, water immiscible organic solvent is being used to dissolve solid

lipid and drug. Surfactant and/or co-surfactant will be dissolved in aqueous solution.

Oily phase is emulsified by adding organic phase to the aqueous phase under stirring.

After mechanical evaporation of organic solvent, lipid start precipitating as SLNs in

the aqueous phase.

Advantages

Scaling up production.

Continuous process.

Grown-up technology.

Commercially demonstrated.

Disadvantages

Polydisperse distributions.

Biomolecule damage.

Extremely energy intensive process.

vii. Solvent Emulsification Diffusion

Solvent Emulsification Diffusion technique is also commonly employed for SLNs

production. This technique avoid the use of heat during processing, which is

considered to be its key advantage. During this technique, water immiscible organic

solvent is being employed for to dissolve the solid lipid as well as drug followed by

emulsification in an aqueous phase of surfactant and/or co-surfactant. Under reduced

pressure, the organic solvent evaporates which caused precipitation of the solid lipid

as SLNs dispersion [88, 89].

Advantage29


Avoidance of heat during fabrication process.

viii. Double Emulsion

During Double Emulsion technique, selected drug along with surfactant is

encapsulated to avoid drug partitioning to external water-phase during solvent

evaporation in the external water-phase of w/o/w Double Emulsion. Fabrication of

bovine serum albumin (BSA) loaded SLNs by Double Emulsion method has been

reported [90].

ix. Solvent Injection

In this method, organic phase is produced by dissolving solid lipid in water-miscible

organic solvent and warmed up. Aqueous-phase is produced by dissolving surfactant

and co-surfactant in de-ionized water. Hypodermic needle is being used for drop wise

addition of organic phase to the pre-warmed aqueous phasealong with continuous

stirring [91]. Magnetic stirring is continued till the organic solvent evaporates to yield

the desired nanoparticles dispersion.

x. Phase Inversion Temperature (PIT) Technique

Nano-emulsions can also be prepared using PIT idea. PIT ideas use the specific

capability of few poly-ethoxylated surfactants to amend their affinities for oil and

water as a function of the temperature. The employment of such kind of surfactant

causes emulsion inversion. Temperature is elevated above the PIT for inversion of

O/W emulsion to W/O emulsion, and for the production of O/W nano-emulsion the

temperature is reduced below the PIT [92].

Freshly it is tailored for SLNs fabrication. Two main constituents are

employed in this case: an oily phase, comprised of solid lipid with non-ionic

surfactant and an aqueous phase having NaCl. Both of these phases are heated

(~90°C) above the PIT; at invariable temperature, the aqueous-phase is then added 30


drop-wise to the oily-phase along with agitation to get W/O emulsion. Resultant

mixture is cooled down (25°C) under continuous and slow stirring. The turbid mixture

turns clearer at the PIT. Below the PIT, O/W nano-emulsion is shaped, which turns in

SLNs below the melting point of lipid [93].

1.6.6 SECONDARY STEPS IN SLNs FABRICATION

i. Freeze Drying

Lyophilization/freeze drying is used to enhance the physical and chemical stability of

SLNs for prolonged period of time. Lyophilization is basically meant for the

conversion of the sample into the dried solid powder which would avoid hydrolytic

reactions and prevent the Oswald ripening. Lyophilization is needed for to attain long

term stability for SLN product loaded with hydrolysable drugs. The removal of water

and freeze-drying conditions endorse the aggregation among SLNs. Specified quantity

of cryoprotectant can be added to evade the aggregation of SLNs during the freeze

drying process.

ii. Spray Drying

Spray drying is alternate process to lyophilization, carried out for conversion of SLN

dispersion to a dry powder product. This drying technique is hardly used for SLN

formulations, even though it is cheaper as compared to lyophilization.

Spray drying technique is recommended only for those solid lipids which have

melting points greater than 70°C (>70°C).

iii. Sterilization

Sterilization is especially desirable for those SLN formulations which are fabricated

for ocular and parenteral route of administration. During sterilization studies, it was

found that it causes distinct particle growth.

31


1.6.7 ANALYTICAL CHARACTERIZATION OF SLNs

For quality control purpose, SLNs required proper and adequate characterization. But,

different characterization parameters offer serious challenges because of colloidal size

of the particles and dynamic nature and complexity of the delivery systems. The key

characteristics analyzed for the SLNs are explained below;

Figure 1.8: Different characterization parameters of SLNs

i. Particle size analysis

The powerful techniques used for particle size determination includes Laser-

Diffraction (LD) and Photon-Correlation-Spectroscopy (PCS). PCS (also termed as

Dynamic-Light-Scattering) is a technique of physics which basically work by

measuring the variation in the intensity of the scattered light, caused by particles

random movement. PCS can detect small particles of suspensions in the range of 3 nm

to 3 μm while LD can detect size in range of 100 nm to 180 μm. Though, PCS is best

tool to measure particle size, but it has the capability for the detection of larger micro-

particles. The LD technique is based on diffraction angle on the particle size

32


(Fraunhofer spectra). Larger particle causes less intense scattering at low angle

compared to the smaller one.

Figure 1.9: DLS of Large particles (A) and small particles (B)

ii. Morphological Studies

The surface morphology of nanoparticles can be studied by numerous microscopic

methods such as, SEM, TEM, AFM and Confocal microscopy.

a. Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) is employed to evaluate the texture and

morphology of the sample particles. The sample is fixed on metallic stub with the

help of double ended carbon tape and then dried under vacuum. Platinum or gold are

used to coat sample particles before analysis. The sample surface is being focused by

electron beam and scanned over it. SEM is considered better for morphological study.

b. Transmission Electron Microscopy (TEM)

TEM has the ability to detect small size particles [94]. Grid is basically used for

sample loading. Negative staining of the sample is done to increase the contrast. For

33


staining purpose, normally eavy metals (phosphotungstic acid, uranyl acetate and lead

citrate) are employed [95]. During TEM analysis, beams of high electrons density

from atoms nuclei are disheaveled from the optical path and hence, on screen dark

image is produced. while the undeflected beams of electrons pass via low density

area of electrons, giving fluorescence after getting reached to the screen and hence,

the specimen appears to be white.

c. Confocal Microscopy

Confocal microscopy is used for making micrographs of three dimensions. As

compared to micrographs of conventional microscopes, confocal microscopy is

considered to give micrographs of high resolution.

d. Atomic Force Microscopy (AFM)

During AFM analysis, tip of the probe is rastered transversely to the sample in order

to make a topological map (scan pattern). It is based on the force between the surface

of samples and probe tip. During non-contacted mode, the probe is just allocated

above the sample. While in contacted mode, the probe moved across the sample, with

the correct nature of the particular force employed helping to differentiate among the

sub techniques. With this approach, ultra high-resolution is obtained. It has the ability

to scan pattern (map) according to the properties of the sample, in addition to particles

size e.g. colloidal resistance or attraction to de-formation. Hence, AFM is considered

as a valuable instrument [96].

iii. Fourier Transform Infra-red (FT-IR) Spectroscopy

FT-IR spectroscopy is used to detect any type of incompatibility between the drug-

excipients during nanoparticles fabrication. Particles size and FT-IR spectrum

have inverse relationship as with decrease in the particle size, peak width increases

34


and vice versa. Changes in FT-IR spectra of two formulations will be a sign for

incompatibility of the processed formulation components.

iv. Powered X-Ray Diffractometry (P-XRD)

P-XRD is a scientific mtechnique allowing rapid and non-destructive analysis without

the want of extensive sample preparation procedure. P-XRD analysis is carried out to

confirm phase identification and formation of new solid state [97]. P-XRD is used to

evaluate the crystalline, semi-crystalline and amorphous state of the unprocessed

drug, solid lipid and other excipients in comparison to the processed formulation.

v. Differential Scanning Calorimetry (DSC)

DSC is basically conducted for to know the crystalline nature of the sample.

Determination of crystalline nature via DSC is anticipated by comparing the melting

enthalpy/g of the unprocessed drug with that of the processed nanoformulation [98].

vi. Entrapment Efficiency (EE) and Drug Loading Capacity (DLC)

Percent Entrapment Efficiency (EE %) and Percent Drug Loading Capacity (DLC %)

is determined from the supernatant of the SLNs dispersion after centrifugation. The

quantity of un-entrapped drug in the supernatant is calculated using suitable

technique, which is further used for the calculation of both EE % and DLC %.

Entrapment Efficiency (EE%) is calculated by the following formula:

EE%=Totaldrug added−Unentrapped drug

Total drugadded ×100

Drug Loading Capacity (DLC%) is calculated by the following formula:

DLC%=Amount of drug loaded∈SLNs

Totalamount of drug added+ Amount of excipients ×100

vii. In-vitro Drug Release Study

35


In-vitro drug release is generally determined by diffusion via dialysis membrane. For

this purpose, specific volume of SLNs formulation is positioned in dialysis tube,

sealed both ends then suspended in dissolution apparatus having definite volume of

dissolution media at. The medium temperature is set at 37±1ºC with stirring speed of

50-100 rpm and samples are with-drawn at pre-determined time intervals. The

collected quantity is substituted with equal volume of fresh dissolution media and

analyzed for the quantity of drug released using suitable technique (UV-

Spectrophotometer or HPLC) [99-101].

Data got from in-vitro drug release can be putted in different mathematical

models to verify drug release kinetics of the SLNs drug delivery system. Different

kinetic models can be used in this study such as zero order, first order, higuchi and

kors-peppas models etc [102].

viii. Storage Stability Study of SLNs

The physical characteristics of SLN over extended period of time is basically

evaluated in terms of particles size, PDI, zeta potential, entrapment efficiency,

viscosity as well as appearance. Light and temperature considered as external factors,

emerged to be most effective for long term stability study.

4°C: Most favorable temperature for storage.

20°C: Long term storage of drug loaded SLNs cannot result in loss of drug or

aggregation.

50°C: A rapid particle growth can be observed at this high temperature [103, 104].

1.6.8 CONVERSION OF SLNs DISPERSION TO SOLID DOSAGE FORM

Scientifically, fabrication of nanoformulations is motivating art, however,

improvement in the prepared nanoformulations for oral administration is challenging

job. In most cases, the drug nano-suspensions for oral administration are not the final

36


products. For to improve the patients’ compliance rate, the drug loaded SLNs should

be designed in the conventional dry dosage forms, i.e. tablet, capsule, pellet and dry

powder for suspension. Solid-dosage forms are alway appealing compared to liquid-

dosage forms cause of ease of handling, convenience and accurate dosing. That’s why

solid dosage forms possess the highest percentage of the pharmaceutical market. Next

to this, water free formulations also avoid hydrolysis, thus, prevent deteriorations of

SLNs nanoformulation upon storage.

Accordingly, scientific community is now busy in the development of the "art"

of oral administration of SLNs. In this fashion, the issues connected with the dosage

form designing will be solved. A number of attempts have been made to develop dry

emulsions or other solid states. Previouly, dry emulsion has been produced via water

removal from aqueous emulsions by rotary lyophilization, evaporation, or spray

drying [105-107].

Most of the fabrication techniques give up drug loaded SLNs as dispersions

(suspensions). If the excipients comprising SLNs are stable in suspension form, then

after addition of appropriate additives (flavors, suspending agents, colors and

preservatives) they could be packaged as suspensions for oral administration. This

would be very trouble-free and simplest oral nanoformulation. Lyophilization of

SLNs suspension is advantageous as it evade the chances of degradation and also

reduce the quantity of used organic solvents. After lyophilization, the drug loaded

SLNs shall be in the form of free-flowing powder. For filling into hard gelatin capsule

shells, SLNs can be mixed directly with diluents (e.g. lactose). Additional attractive

approach for the formulation of SLNs is dry power formation for oral suspension. For

this purpose, excipients including sweetening agents, suspending agent, color, flavors

and preservative should be added to the SLNs power. Soft-gelatin capsule is much

37


more appealing oral dosage form due to its specialized needs (e.g. in-situ gelling or

sustained release systems). The drug-loaded SLNs can be filled into soft gelatin

capsules by suspending them in appropriate medium. The delivery module can also be

prepared in the tablet form [108]. For tablets production, aqueous nano-suspension as

a granulating fluid can be used as wetting agent for pellet production.

1.6.9 IN-VIVO STUDY OF SLNs

SLNs can be administrated via numerous routes, like peroral, intravenously, pulmonal

and dermal. Oral route of SLNs administration could increase the drug absorption as

well as also improve the absorption kinetics. SLNs have pluses due to increased

lymphatic up-take and increased bio-adhesion. Serious challenge for nanoparticulate

drug delivery system is the protection of the colloidal particle in the stomach, where

particle growth or agglomeration is favored by high ionic strengths and low pH

values. Stability study of SLNs in artificial gastric juice has been reported and its

findings confirmed preserved particle size of SLNs under acidic conditions [109].

Numerous animal model studies show enhanced absorption of hydrophobic drugs.

Mei et al., reported the efficacy of orally administrated Triptolide loaded SLNs and

unprocessed Triptolide in the carrageenan-induced rat paw edema, their results

proposed that SLNs can improve the anti-inflammatory effect of triptolide [110].

Gascos group examined the distribution and up-take of Tobramycin loaded SLNs in

rats, they investigated improved uptake via the lymph, which causes extended drug

residence time inside the animal [111, 112]. Stealth SLNs have been developed using

the stealth idea from polymeric nanoparticles and liposomes to evade the quick up-

take of the SLNs by the RES system. Reports show that Doxorubicin loaded stealth

SLNs circulate in the blood for prolonged period of time [113]. In the group of

Muller, human in-vivo results reveal that SLNs can increase skin visco-elasticity and

38


hydration [114]. Additional reports illustrate other applications of SLNs like gene

delivery, pulmonary delivery, delivery to the eye, and drug targeting of anti-cancer

drugs [115-118]. Reports of the numerous groups also put forward the use of SLNs to

deliver MRI contrast agents for brain targeting or anti-tumour drugs [113, 119, 120].

1.6.10 ROUTES OF ADMINISTRATION FOR SLNs

Nanoparticles can be administered via diverse routes like oral, subcutaneous topical,

inhalation, parenteral, vaginal and rectal routes [121-126]. Critical considerations

should be given to nanoparticles size while choosing the administration route.

Furthermore, nanoparticles size also defines their specific elimination route. Various

administration routes for SLNs are:

i. Parenteral Administration

Wissing et al. (2004) thoroughly studied parenteral use of SLNs. Most of the

nanoparticles are being employed for site-specific targeting via parenteral route as it

lacks the complex physiology of elimination and absorption. SLNs being consist of

physiologically well-tolerated components, extremely suitable for parenteral route of

administration. SLNs also have good storage capacity at tumor or inflammation sites

due to leaky vasculature. the nanoparticles having size less than 250 nm can pass via

these vasculature as they have pore size of 300 nm-700 [127]. When SLNs are

intravenously injected, they are so small enough to circulate in the micro-vascular

system as well as also stop macrophages up-take when there is lipophobic coating.

Hence, SLNs are recommended for both non-viral and viral gene delivery.

ii. Oral Administration

Sustained drug release profile with oral bioavailability enhancement has been reported

for SLNs. Avoidence of the gastric degradation of the drug being loaded into SLNs is

reported along with quick up-take via the intestinal mucosa.

39


iii. Rectal Administration

Rectal or parenteral administration is preferred for quick pharmacological effect.

These routes are especially preferred for pediatric or unconscious patients due to ease

of administration. SLNs fabricated with lipids which melt at body temperature will be

preferred for rectal route of administration.

iv. Nasal Administration

Nasal route is favored as it has fast onset and quick absorption of drugs. It also avoid

enzymatic degradation of labile drug in the gastro-intestinal tract and in-sufficient

transport across the layers of epithelial cells. SLNs are projected as alternative trans-

mucosal delivery system of macro-molecular therapeutic agent [128, 129].

v. Respiratory Delivery

Nebulisation of SLNs loaded with anti-tubercular drug, anti-cancer drug and anti-

asthmatic drug was studied and found thriving in enhancing drug bioavailability as

well as decreasing the administration frequency for improved management of

pulmonary diseases.

vi. Ocular Administration

Muco adhesive characteristics and bio-compatibility of SLNs increased corneal

residence time of drugs and also enhance their interaction with ocular mucosa with

the aims of targeted drug delivery.

vii. Topical Administration

SLNs are very significant and attractive carrier system for skin application due to

having different attractive effects on skin. Application of SLNs over inflamed or

damaged skins is reported as they are suitable due to composition of solid lipids

which are being non-irritant and non-toxic [130].40


1.6.11 ADVANTAGES OF SLNs

The risk of chronic as well as acute toxicity is reduced via the usage of

biodegradable and biocompatible solid lipids [131].

Enhanced bioavailability of hydrophobic drugs [132].

Controlled and targeted delivery of the incorporated drugs.

Possibility for commercial sterilization and scaling up production.

Protect drugs from gastric acid degradation.

Lyophilization is possible and can also be spray dried.

Improve physical stability of pharmaceutical ingredients [133].

Enhanced and high drug payload [134].

Conventional emulsion fabricating techniques can be applicable.

Same raw materials of emulsions can be used.

No toxic metabolites are formed.

Versatile Applications

1.6.12 DISADVANTAGES OF SLNs

Drug expulsion after polymeric transition during storage.

Poor drug loading ability.

Posses high water content (70-99.9%) [135].

1.7 ROXITHROMYCINAt the beginning of the 20th century, the central reason of death across the world was

infectious diseases [136]. From the last century, a lot of new antibacterial drugs are

synthesized and introduced into the market. These drugs lead to decrease in morbidity

and mortality due to infectious diseases. Roxithromycin is one among these newly

synthesized active pharmaceutical ingredients (APIs). Roxithromycin is the first new

generation, semi-synthetic and 14-membered ring macrolide.

41


In British pharmacopoeia, Roxithromycin is official drug. Its reported melting

point is 122 to 127℃. It is erythromycin’s derivative and more stable under low pH

and reveals enhanced clinical effects [137]. It is very similar to erythromycin in terms

of chemical structure, composition and mechanism of action. It has been synthesized

by the insertion of an etheroxime chain at the C-9 position of erythromycin.

Mechanism of Action:

The common location of the Roxithromycin binding site on the larger ribosomal

subunit (50S) has been mapped using a combination of genetic and bio-chemical

techniques. [2,5-9]. The conclusions of bio-chemical data have been verified by the

X-ray structures, which specified that RNA forms the chief part of the Roxithromycin

binding site [138].

Roxithromycin (RTM) prevents bacterial growth by inhibiting protein synthesis.

RTM binds to the 50S ribosomal sub-unit of the susceptible bacteria, thus inhibiting

the process of peptides synthesis which leads to inhibition of cell growth.

42

https://en.wikipedia.org/wiki/Peptide

https://en.wikipedia.org/wiki/Protein_synthesis

https://en.wikipedia.org/wiki/Bacterium


Figure 1.10: Mechanism of action of Roxithromycin

1.8 FAMOTIDINEFamotidine (FTD) is third-generation histamine H2-receptor antagonist; without

agonist or antagonist effects on histaminergic H1-, nicotinic, muscarinic, β- or

α - receptors. It is odorless, white to pale-yellow crystalline powder having a

moderately bitter taste. Its molecular formula is C8H15N7O2S3 and IUPAC Name is 3-

[[2-(diaminomethylideneamino)-1,3-thiazol-4-yl]methylsulfanyl]-N'

sulfamoylpropanimidamide.

During January 1994, Famotidine was re-classified in the United States (US) for

prescription only to pharmacy status rendering it accessible over the counter for

specific indications. Subsequently, Centra Healthcare launched Pepcid AC in the UK 43


[139]. After that, in the United States (US) the department of Food and Drug

Administration (FDA) granted agreement for Pepcid AC to be marketed in the US by

Merck and Co [139]. During January 1995, in South Africa, the Medicines Control

Council (MCC) point out, that it had resolved to advise the re-scheduling of

Famotidine to Schedule-2, "when intended for the symptomatic aid of heart-burn

cause of excessive gastric acid, having maximum dose of 10 mg and the highest daily

dose (for 24 hours) is 20 mg, for a time period of maximum of two weeks".

Famotidine is official drug in IP, USP, BP, JP and EP. It is the most widely

used prokinetic drug in Japan [140]. Estimated solubility of Famotidine at 20°C is

80% m/v in di-methyl formamide, 0.3% m/v in methanol, 50% m/v in acetic acid, 0.l

% m/v in water, and 0.01% m/v in ethanol, ethyl acetate and chloroform [139].

Famotidine is freely soluble in glacial acetic acid, and highly soluble in diluted

mineral acids.

Mechanism of Action:

Famotidine (FTD) is highly selective H2-receptors antagonist, thus having no effect

on H1 and H3 receptors and has almost no effect on motility. It is competitive

antagonist of histamine and is fully reversible.

FTD binds to H2-receptor positioned on basolateral-membrane of the gastric

parietal cells, thus blocking the binding of histamine to it. As a result reduction in

intracellular concentration of cyclic adenosine monophosphate (cAMP) occur which

cause decrease in gastric acid secretion. This inhibition result in reduced nocturnal

and basal gastric acid secretion and also gastric acid released in response to stimuli

including, betazole, insulin, pentagastrin food or caffeine.

44


Figure 1.11: Mechanism of action of Famotidine

45


1.9 MOTIVATION TO DO THIS WORKAs Roxithromycin and Famotidine are most profoundly prescribed by physician during

treatment and management of numerous diseases e.g. different infectious diseases and

gastric acid suppression. But generally different drawbacks are associated with their use.

In this project Roxithromycin and Famotidine loaded Solid Lipid Nanoparticles have

been fabricated and its characters are thoroughly studied. Motivation to do this work has

been divided into the following sub-headings:

1.9.1 STATEMENT OF THE PROBLEM

Famotidine and Roxithromycin, being members of Class-IV of the

Biopharmaceutical Classification System, have low solubility and low

permeability. This leads to limit their absorption which in-turn affect their oral

bioavailability. Hence, the use of their available dosage forms in the market is

limited because of low oral bioavailability. Conventional or even advanced oral

drug delivery systems are not appropriate for these drugs administration especially

via oral route.

After oral administration, these drugs exhibit extensive first pass effect, so very

small fraction of the drug reaches the systemic circulation. After oral

administration, drug first form solution with-in the gastric medium, then absorb

into the blood stream. These drugs are then directly carried to the liver by hepatic

portal vein being metabolized there. This metabolism is termed as first pass effect.

First pass effect is being followed by the selected drugs, so just a minute fraction

of drug reaches systemic circulation for their pharmacological effect.

Roxithromycin originate few other problems when taken orally like irritation of

the GI tract and are also degradation by the enzymatic activity and acidic

environment of the stomach. This drugs, when given orally, can also induce a

46


number of reactions or diverse problems like nausea, vomiting, cramps and

diarrhea which limit patients’ acceptability. Some of these problems arise due to

antibiotics influence on the normal flora of the bowel.

These drugs may require frequent oral administration due to short half life which

probably may leads to be inconvenient to the patients.

Between the two dosing intervals, blood level of these drugs may remain below

the minimum effective concentration, which may result in treatment failure.

1.9.2 AIMS AND OBJECTIVES OF THE STUDY

To fabricate economical, easy, safe and efficient sustained release Solid Lipid

Nanoparticles of the selected lipophilic drugs i.e. Famotidine and Roxithromycin.

Enhance solubility which in turn boosted the oral bioavailability of the selected

BCS-IV drugs.

Ensure increased entrapment efficiency and drug loading capacity of the selected

BCS-IV drugs.

Ensure improved physical stability of SLNs loaded with the selected BCS-IV

drugs.

To develop pharmaceutical dosage form having comparatively increased oral

bioavailability.

Improve the patients’ medication compliance rate via sustained release with

decreased frequency of administration.

Selection of biocompatible and biodegradable solid lipids and other chemicals not

to be toxic to the human body.

47

CHAPTER No. 2 LITERATURE REVIEW

Chapter 2 LITERATURE REVIEW

Conventional dosage forms like solution, emulsion or suspension for drug delivery

purpose has numerous boundaries such as low availability, high dose, intolerance,

instability, faster reach effect and poor oral bioavailability. Conventional dosage

forms aslo do not provide sustained drug release effect. Due to presence of numerous

basic and acidic mediums inside our body, it is important that each and every drug

should reach to its targeted site without any alteration in its chemical and physical

properties. Therefore, need for development of some novel drug carriers was felt,

which could reach to its targeted side without making any un-wanted effect to body

and can target the drug safely and easily to its desired site.

Recently, Solid Lipid Nanoparticles (SLNs) are developed and being

fabricated by different techniques. SLNs are alternative to colloidal drug delivery

systems like lipid emulsion, polymeric nanoparticles and liposomes. SLN combines

plusses of different colloidal carriers but evades some of their major documented

short-comings. SLNs are nano-metric in size (50 to 1000nm) and in solid state at both

room and physiological temperature [53]. SLNs offer large surface area and sustained

drug release with quick cellular uptake [54]. In modern eras, excessive attention is

focused on SLNs to increase bioavailability of hydrophobic drugs [55]. Mostly BCS

Class-II and Class-IV drugs are incorporated in SLNs [56]. SLNs-based drug delivery

system has numerous benefits including enhanced solubility of hydrophobic drugs

with prolonged drug release which ultimately lower dose and frequency of

administration.

48


2.1 SLNs FABRICATION TECHNIQUESIn the literature, numerous diverse methods for the fabrication of SLNs have been

reported. These techniques include Solvent Injection (or Solvent Displacement),

Solvent Emulsification Evaporation, Hot Melt Encapsulation, Solvent Emulsification

Diffusion, Microemulsion, Multiple Emulsion Technique, High Pressure

Homogenization, Phase Inversion, Membrane Contractor And Ultrasonication

technique [85, 88, 141-153].

Each and every process has its own plusses compared to other techniques, e.g.

easy scale up, short production time and avoidance of organic solvents.

However, during the presented research work the mentioned three techniques have

been employed.

2.1.1 SOLVENT INJECTION (SI) TECHNIQUE

Schubert, M., et. Al., (2003) prepared lipid nanoparticles (LNPs) via injecting a

mixture of melted solid lipid and organic solvent (water miscible) into de-ionized

water. Purpose of the presented work is to assess potential of the SI technique being

employed for LNPs fabrication. Outcomes of SI technique, presented versatile as well

as potent approach for LNPs fabrication. Approximately 96.5% of the lipid being

employed during this technique was directly transformed into LNPs [148].

Wang, W., et al., (2004) reported Solvent Injection technique for the fabrication

of curcumin loaded SLNs (curcumin-SLNs). Improvement in curcumin therapeutic

efficacy has been reported in ovalbumin (OVA) induced allergic rat-model of asthma.

In-vitr drug release experiments have been performed for curcumin loaded SLNs. The

in-vivo pharmacokinetic study was conducted, using mice as animal model. During

in-vivo studies, therapeutic effect of the curcumin loaded SLNs formulation and

tissues drug distribution was thoroughly evaluated in the mice model. The

49


concentrations of curcumin in plasma suspension were considerably elevated

compared unprocessed curcumin. Following oral administration of curcumin-SLNs,

its concentration was elevated in tissue, specially in liver and lung. In the animal-

model of asthma, curcumin-SLNs successfully suppressed air-way hyper-

responsiveness as well as inflammatory cell infiltration. Curcumin loaded SLNs

considerably inhibited the expression of T-helper-2-type cytokines, i.e. interleukin-13

and interleukin-4, in broncho-alveolar lavage fluid compared to asthma-group and

curcumin treated group. Results showed that curcumin loaded SLNs is a significant

way for treatment of asthma [154].

Arıca Yegin, B., et.al., (2006) reported Solvent Injection technique for

fabrication of paclitaxel loaded solid lipid nanoparticles. During this study,

phospholipids were employed as the lipid matrix while sucrose fatty acid ester was

employed as emulsifier. Paclitaxel loaded SLNs showed particle size of 187.6 nm. In-

vitro release profile showed slow drug release; only 12.5–16.5% of paclitaxel released

from loaded SLNs with-in 14 days. SLNs showed itself as a significant

pharmaceutical nanoformulation of paclitaxel [155].

Shah, M., et. Al., (2010) reported the development of simvastatin loaded SLNs

and optimize it on basis of different variables (concentration of glycerol monostearate,

volume of isopropyl alcohol and concentration of poloxamer). The fabricated eight

nanoformulations (F1–F8) were optimized via 23 full factorial design. Validation of

the design was done by extra design checkpoint formulation (F9) and probable

interaction was studied between in-dependent variables. The response of the design

was evaluated via Design Expert 7.1.6. (Stat-Ease, Inc, USA) and the analytical tool

of software was employd to draw Pareto chart and response surface plot. Optimized

nanoformulation F10 with desired factor (0.611) was checked for independent

50


parameters having particle size of 258.5 nm, EE% of 75.81%, with of 82.67 CDR%

after 55 hr. Drug release kinetics of the optimized nanoformulation fitted Higuchi

model having re-crystallization index of 65.51% [156].

Rawat, M., et al., (2010) reported both Solvent Injection (modified) and Ultra-

sonication techniques, employing stearic acid as solid lipid, soya lecithin and pluronic

F68 (PLF68) as stabilizer. Repaglinide loaded Solid Lipid Nanoparticles (RG-SLNs)

were successfully fabricated via Solvent Injection (modified) and Ultra-sonication

method. RG-SLNs fabricated with Solvent Injection (modified) technique showed

particle size of 360±2.5 nm while with Ultra-sonication technique 281± 5.3 nm.

While zeta potential for these two techniques varied from - 23.10±1.23 to -26.01±0.89

mV. Maximum entrapment efficiency attained with modified Solvent Injection

technique was 62.14 ± 1.29%. Total drug content was almost similar (98% ) in both of

the techniques. In-vitro release study was conducted in phosphate buffer (pH 6.8) with

0.5% sodium lauryl sulphate (SLS) using dialysis-bag diffusion method. Solvent

Injection (modified) and Ultra-sonication technique showed cumulative drug release

of 30% and 50% respectively, within 2 hrs study. This specified that RG-SLNs

fabricated by Solvent Injection technique (modified) released RG more slowly than

nanoformulation prepared with Ultra-sonication technique. These results proposed

that Solvent Injection technique (modified) is appropriate for fabrication of RG-SLNs

[157].

Luo, C. F., et al., (2011) reported Solvent Injection method for fabrication of

Solid Lipid Nanoparticles loaded with Puerarin (Pue-SLNs). During in-vivo study,

being conducted on rats, different pharmacokinetic parameters of puerarin in rats was

investigated. The value of Cmax for puerarin after the oral administration of Pue-SLNs

was extensively elevated compared to puerarin suspension. The value of Tmax after

51


oral administration of Pue-SLNs was extensively smaller compared to puerarin

suspension. The values of AUC0→t after oral administration of the Pue-SLNs and

puerarin suspension were 2.48±0.30 mg h/L and 0.80±0.23 mg h/L respectively.

Following oral administration of Pue-SLNs, concentrations of puerarin in different

tissues was also augmentd, specially in the targeted organs like brain and heart. This

research project suggested that SLNs as druh delivery system is significant to boost

the oral bioavailability of puerarin [158].

Nair, R., et al., (2011) reported Solvent Injection method for the fabrication of

Solid Lipid Nanoparticles loaded with Rizatriptan (RZT-SLNs). RZT-SLNs were

successfully fabricated by Solvent Injection technique (modified) and thoroughly

characterized. RZT-SLNs were spherical shaped with smooth-surfaces having

particles size in the range of 141.1 to 185.7 nm. The drug release from Rizatriptan

loaded SLNs formulation showed the sustained release behavior. The drug release

kinetics were analysed and the best fitted model was ascertained. In conclusion,

rizatriptan loaded SLNs with small particle size and sustained drug release profile can

be obtained by Solvent Injection technique [91].

Kaushik, M., et al., (2012) prepared Solid Lipid Nanoparticles (SLNs) of

aceclofenac via Solvent Injection method. Glyceryl behenate (Compritol 888 ATO)

was employd as solid lipid and Poloxamer 188 as the surfactant. Isopropyl alcohol

(IPA) was employd as organic solvent for to dissolve both drug and lipid. This

research project suggested that SLNs are a promising delivery system to boost the oral

bioavailability of aceclofenac [159].

Shah, S., et al., (2015) carried out a research project on Solid Lipid

Nanoparticles (SLNs). The aim of his work was to fabricate SLNs of hydrophobic

drug i.e. Irbesartan (IRB) to boost its oral bioavailability. SLNs were fabricated using

52


stearic acid as a solid lipid. Two variable factors i.e. concentration of polyvinyl

alcohol and quantity of lipid were found to have major effects on the entrapment

efficiency (EE), drug loading (DL) and in-vitro drug release. The optimized

formulation was evaluated for particles size, zeta potential and surface morphology.

Additionally, optimized nanoformulation was also compared for in-vitro drug release

with drug solution. The results showed the sustained release properties of the

fabricated SLNs. Therefore, the IRB-SLNs would be helpful for delivering

hydrophobic IRB which may useful in improving oral bioavailability and anti-

hypertensive efficacy [160].

2.1.2 SOLVENT EVAPORATION TECHNIQUE

Sjöström, B., et. Al., (1992) prepared nanoparticles of a model drug, viz., cholesteryl

acetate. Cholesteryl acetate was dissolved in cyclohexane having lecithin. Organic

solution was emulsified in aqueous solution having a co-surfactant. The resulted o/w

emulsion was stable. Cholesteryl acetate started precipitation in the emulsion droplets

as solvent evaporated. Particles size showed minor variation with the concentration of

cholesteryl acetate in cyclohexane. Additionally, increased in particles size, as a result

of an increased oil/water ratio was negligible. With a blend of sodium glycocholate

and phosphatidylcholine as emulsifiers, particles size down to 25 nm was achieved.

The ratio between sodium glycocholate and phosphatidylcholine appeared critical.

The suspension became un-stable with the increased ratio of above 9:1, as particles

size increased during storage of the sample. optimized condition coincide with those

given an extensively swelling lamellar liquid crystalline phase having sodium

glycocholate and phosphatidylcholine [146].

Lemos-Senna, E., et al., (1998) verified the feasibility of fabricating nano-

spheres from amphiphilic 2,3-di-O-hexanoyl-γ-cyclodextrin (γCDC6) via solvent

53


evaporation technique. This fabrication technique consists in emulsifying an organic-

phase having cyclodextrin in an aqueous-phase and Pluronic F68 as surfactant. Effect

of different process parameters, i.e. initial γCDC6 content as well as surfactant

concentrations, on the properties of nanospheres fabrication as well as on the

nanospheres loading of a lipophilic drug was analysed. Cyclodextrin nanosphere

showing a mean diameter in the range of 50 to 200 nm was attained, even with low

concentration of surfactant. Fabrication of colloidal particles in the mentioned

conditions was associated with the amphiphilic characteristics of the derivative of

cyclodextrin. However, the partitioning of the γCDC6 molecule between the aqueous

and organic phases was seen as being a function of concentration of surfactant in the

continuous-phase. This partitioning was related to the production of aggregates of the

order 10-20 nm, probably Pluronic F68/γCDC6 mixed micelles as evidenced by the

micro-graphs attained by TEM. In the case of the progesterone loaded nanospheres,

the partitioning of the drug between the dispersed-phase having the cyclodextrin and

the continuous-aqueous-phase having Pluronic F68/γCDC6 aggregates was also

demonstrated. The content of drug in the final nanospheres was in the range of 4-5%

(w/w) of the carrier. Finally, dilution experiment was carried-out to evaluate the

stability of the drug particles association [161].

Desgouilles, S., et al., (2003) reported that fabrication of nanoparticles by

extremely popular technique i.e. Solvent Emulsification Evaporation. Aim of this

work was to clarify the mechanism via which nanoparticles of poly-lactic-acid (PLA)

and ethyl-cellulose (EC) are formed during Solvent Emulsification Evaporation

procedure. From the attained data as well as dependence on the employd polymer

PLA or EC, two models are presented to clarify nanoparticles fabrication mechanism.

In the EC model, after shrinkage of the emulsion drop-lets as the direct result of

54


solvent evaporation, coalescence happened prior to formation of stable and solvent

free nanoparticles. In the PLA model, limited or no coalescence occured hence, the

picture is that one PLA nanoparticle originated from one (or only a few) PLA

emulsion droplet after its shrinkage [162].

Sarmento, B., et al., (2007) produced and characterized cetyl-palmitate based

Solid Lipid Nanoparticles (SLNs) loaded with insulin (In-SLNs). In-SLNs were

produced by Solvent Emulsification Evaporation (modified) technique, thoroughly

analysed the potential of these colloidal carriers for oral administration. The In-SLNs

were characterized having particles size of around 350 nm and negative charge with

insulin association efficiency of almost 43%. After oral administration of In-SLNs to

diabetic rats, a considerable hypo-glycemic effects were seen with-in 24 hrs. These

results revealed that SLNs authenticate the oral absorption of insulin [163].

Liu, D., et al., (2011) prepared Solid Lipid Nanoparticles (SLNs) via Solvent

Emulsification Evaporation technique. Aim of this work was to enhance the

incorporation of diclofenac sodium (DS) into SLNs. Results confirmed that almost

100% of the incorporated DS can be entraped by decreasing the pH of dispersed-

phase. Increment in concentrations of PVA and existence of co-surfactant had been

enhanced EE of DS-SLNs. In dispersed-phase, combination of PEG-400 with

stabilizers also resulted in elevated EE and DLC. DLC decreased and EE improved as

the phospholipid/DS ratio became greater, whereas, quantity of DS had converse

effect. Moreover, the viscosity of aqueous and dispersed phase as well as the stirring

speed had almost no effect on DLC and EE of DS-SLNs. According to the reported

investigation, improvement in EE is highly favoured by drug solubility in the

dispersion-medium [164].

55


2.1.3 HOT MELT ENCAPSULATION TECHNIQUE

Hot Melt Encapsulation method being employed for SLNs fabrication is very easy

and acceptable due the avoidance of organic solvents. This method has been reported

by researchers but very limited literature is available for this technique.

Rehman, M., et al., (2015) prepared SLNs by Hot Melt Encapsulation technique

for diacerein delivery, without and with simultaneously loaded gold nanoparticles

(GNPs). High encapsulation of diacerein was attained with GNPs-diacerein-loaded

and diacerein-loaded SLNs. In-vitro dissolution study showed sustained release

profile of 12 hrs for GNPs-diacerein-loaded SLNs and 72 hrs for diacerein-loaded

SLNs. Kinetic models showed that drug release followed Higuchi and zero-order

models however, Korsmeyer-Peppas model predicted diffusion-release-mechanism

[83].

List of different drugs loaded into SLNs via numerous methods are given below

in Table 2.1.

Table 2.1: List of drugs loaded into SLNs via different techniquesS.No Drug Fabrication Method Reference

1 Olanzapine Modified high pressure homogenization [165] 2 Rizatriptan Modified Solvent Injection method [91] 3 Alendronate NP Double emulsion solvent diffusion [166] 4 Gatifloxacin Modified Coacervation [167] 5 Insulin Ionic gelation [168] 6 Paclitaxel Microemulsion [169] 7 Vinpocetine Ultrasonic solvent emulsification [170] 8 Insulin Solvent Emulsification Evaporation [163] 9 Methotrexate Microemulsion congealing technique [171]10 Gatifloxacin Modified coacervation [167]11 Melatonin Microemulsion technique [145]12 Cholesteryl acetate Solvent Emulsification Evaporation [146]13 Clobetasol propionate solvent diffusion [147]

56


2.2 FORMULATIONS OF ROXITHROMYCIN2.2.1 CONVENTIONAL FORMULATIONS

Several dosage formulations of Roxithromycin and their available strengths i.e.

suspensions (50 mg/5 ml) , tablets (50 mg , 100 mg , 150 mg , 300 mg) and

capsules (150 mg , 300 mg) are available in the market. A lot of pharmaceutical

industries i.e. Alliance Pharmaceuticals (PVT) Limited, Peshawar (Pakistan),

Medicraft Pharmaceuticals (PVT) Limited, Peshawar (Pakistan), Pharmevo (PVT)

Limited, Karachi (Pakistan) and Sami Pharmaceuticals (PVT) Limited, Karachi

(Pakistan) etc are engaged in the production of these dosage forms. Being an efficient

drug and having a lot of applications in human and veterinary medicines, other

competitors are also involved in its production [172]. It has a lot of clinical

applications i.e. the safe treatment of respiratory tract, urinary tract and soft tissue

infections and also for bacterial infections associated with stomach as well as

intestinal ulcers [172]. It has anti-cancer activity and also inherent anti-malarial

activity [173, 174]. Some reports have designated that Roxithromycin might act as

anti-inflammatory and antitumor agent [175, 176]. For example, Roxithromycin has

been reported to prevent tumor cell growth and tumor induced angiogenesis in a

mouse model and in human hepatoma cells [177, 178]. It is the drug of choice for

treatment of acute diarrheal-conditions of Cryptosporidiosis in AIDS patients [179].

Thus, it is cleared that there are a lot of tasks for Roxithromycin but unluckily it

belongs to Class-IV of Biopharmaceutical Classification System (BCS) and drugs of

this class having low solubility and low permeability [180]. That’s why the

mentioned oral formulations have not been successful due to poor physico-chemical

properties (solubility 0.0189 mg/L at 25 °C) and an unfavorable pharmacokinetic

57

http://druginfosys.com/AvailableBrands.aspx?query=300%20mg&form=Caps&drugCode=1001&drugName=Roxithromycin&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=150%20mg&form=Caps&drugCode=1001&drugName=Roxithromycin&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=300%20mg&form=Tabs&drugCode=1001&drugName=Roxithromycin&type=1&Ing==1




http://druginfosys.com/AvailableBrands.aspx?query=50%20mg/5ml&form=Susp&drugCode=1001&drugName=Roxithromycin&type=1&Ing==1


profile, including oral bioavailability of only 50% [181, 182]. Additionally, it also

causes gastric irritation which limits the clinical uses of Roxithromycin.

2.2.2 MODIFIED FORMULATIONS

Some efforts were being made by numerous pharmaceutical scientists to solve the

mentioned problems of Roxithromycin regarding oral bioavailability. Literature also

shows that a lot of work has also been performed on Roxithromycin in different

aspects i.e. solubility enhancement, sustained release formulations, taste masking and

for targeting hair-follicle etc.

Patro, S., et al., (2005) studied dissolution rate enhancement of Roxithromycin

via combination of different hydrophilic polymers. They prepared solid dispersion

formulations by both coprecipitate methods and physical mixing and evaluated the

significant increase in their dissolution rates compared to unprocessed Roxithromycin

(pure). The dissolution rate of Roxithromycin was proportional to the increment in the

drug to polymer ratios in the solid dispersions. Dispersions prepared by coprecipitate

method have shown faster dissolution rate compared to physical mixing techniques.

The dissolution efficiency of the formulation polyethylene glycol 6000:CP5 was

found to be highest compared to other formulations. It was concluded that lipophobic

polymer can be employed to produce solid dispersions to enhance the solubility of

Roxithromycin [183].

Biradar, S.V., et al., (2006) reported a study for solubility enhancement, which

in turn boosted the oral bioavailability of Roxithromycin. The reported work deals

with exploring the homogenization effects, homogenization followed by

lyophilization and homogenization followed by spray-drying in the presence of

solubilizer on drug dissolution rate and solubility. Homogenization and subsequent

lyophilization showed 3–4 folds improvement in saturation solubility as well as 18-

58


times faster dissolution rate compared to unprocessed drug. During this work, a more

eminent aqueous system was developed for improvement of the saturation-solubility

as well as dissolution-rate and compared with the non aqueous system. The resultant

aqueous-system was comparatively more stable and equally effective. Improvement in

the dissolution-rate and saturation-solubility was due to reduction in particles size and

enhanced hydrophilicity. This work is based on utilization of solubilizer to

synergistically increase the efficiency of the processing methods. The effects of

formulation and processing variables on the stability, antimicrobial activity, physical

interaction, drug crystallinity, in-vitro dissolution and saturation-solubility was also

studed [182].

Gao, Y., et al., (2006) reported preparation of Roxithromycin microspheres

using silica and eudragit S-100 to mask its bitter-taste. They studied effect on taste

masking due to numerous polymers as well as drug polymer ratio and they also

investigated different characteristics of microspheres. It was seen that among the six

types of polymers, Eudragit S100 was the best for masking the un-pleasant taste of

Roxithromycin. In conclusion, the present work will be help-ful for the production of

oral formulations of Roxithromycin having acceptable taste.

[184].

Koopaei, M.N., et al., (2012) reported fabrication of Roxithromycin (RTM)

loaded polymeric nanoparticles via Solvent Evaporation technique. They used

pegylated poly-lactide-co-glycolide (PEG-PLGA) as a polymer for drug

encapsulation. They reported particles in the size range of 150 to 200 nm with

entrapment efficiency 80.0±6.5% and drug loading capacity 13.0±1.0%. In-vitro drug

release studies demonstrated burst-release effect, as, almost 50.03±0.99% at 6.5 hrs

followed by steady and slow release of RTM. In-vitro anti-bacterial effect

59


demonstrated that the minimum inhibitory concentration (MIC) of RTM loaded PEG-

PLGA NPs was 4.5 times lower on S. Epidermidis, 4.5 times lower on B. subtilis and

9 times lower on S. aureus compared to RTM solution. In conclusion, it was shown

that polymeric NPs substantially improved the anti-bacterial efficacy of RTM [137].

Główka, E., et al., (2014) reported fabrication and evaluation of pluronic

lecithin organogel (PLO) loaded Roxithromycin (RTM) nanoparticles for follicular

targeting. They un-covered that Roxithromycin delivery with nanoparticles into the

hair follicles is much more striking idea. As, RTM loaded nanoparticles can

accumulate in the openings of hair follicles. The fate of nanoparticles was traced in

the skin via incorporation of a fluorescent dye into the prepared nanoformulation.

Results showed that it was possible to attain preferential targeting to the

pilosebaceous unit employing polymeric nanoparticles formulated either into the

semisolid topical formulations or aqueous suspensions [180].

Wosicka-Frąckowiak, H., et al., (2015) also reported fabrication of

Roxithromycin (RTM) loaded Solid Lipid Nanoparticles for better penetration and

accumulation in skin hair-follicles. They preferentially penetrated in the skin hair-

follicles and created high local concentration of RTM [185].

Rocas, P., et al., (2015) worked on bio-functionalisation of titanium with

Roxithromycin loaded RGD-decorated polyurethane-polyurea nanoparticles (PUUa

NPs) for improving osteoblast adhesion and also bacterial attachment suppression,

they symbolize this approach to increase the osseointegration of implant materials

[186].

Masood, F., et al., (2016) designed formulations by loading Roxithromycin in

the cavity of each of the hydroxypropyl-β-cyclodextrin and β-cyclodextrin. Then,

each of the resulting inclusion complexes were separately loaded into poly-lactic-co-

60


glycolic-acid to synthesize βCD-RTM/PLGA and HPβCD-RTM/PLGA nanoparticles,

then they studied their anti-bacterial activity against the selected multi drug resistant

(MDR) bacterial strains [187].

Methods Employed for Roxithromycin Analysis

In all the above mentioned literature review, different techniques have been employed

for the determinaion of Roxithromycin in samples. Several methods such as HPLC

with various detectors [188], electrochemical detectors [189], mass spectroscopy

[190], flow injection analysis coupled with chemi-luminescence [191], Fluorimetry

[192] spectrophotometric detection [193], liquid chromatography with different

detections, such as amperometric detection [194, 195], fluorescence detection [196]

and mass spectrometric detection have been employed [197, 198].

2.3 FORMULATIONS OF FAMOTIDINE

2.3.1 CONVENTIONAL FORMULATIONS

Commercially available dosage forms of Famotidine with their respective strengths

are injection (20 mg, 40 mg), syrup (10 mg, 10 mg/5 ml), suspension (10 mg, 10

mg /5 ml, 40 mg/5 ml), tablets (10 mg, 20 mg, 40 mg, 250 mg), capsules (20 mg, 40

mg). Beside injections, capsules and tablets, also chewable tablets in 10 mg strength

are available for adults. Moreover, oral suspension powder was also prepared but its

stability was limited to 30 days after reconstitution and also had extremely bitter taste

[199]. Hence, researchers also tried numerous techniques to mask its bitter taste [200].

A lot of pharmaceutical industries i.e. Alliance Pharmaceuticals (PVT) Limited,

Peshawar (Pakistan), Medicraft Pharmaceuticals (PVT) Limited, Peshawar (Pakistan),

Zafa Pharmaceutical Laboratories (PVT) Limited, Karachi (Pakistan) and Novartis

Pharma (PVT) Limited, Karachi (Pakistan) etc are engaged in the production of these

61

http://druginfosys.com/AvailableBrands.aspx?query=40%20mg&form=Caps&drugCode=298&drugName=Famotidine&type=1&Ing==1



http://druginfosys.com/AvailableBrands.aspx?query=250%20mg&form=Tabs&drugCode=298&drugName=Famotidine&type=1&Ing==1




http://druginfosys.com/AvailableBrands.aspx?query=40%20mg/5ml&form=Susp&drugCode=298&drugName=Famotidine&type=1&Ing==1



http://druginfosys.com/AvailableBrands.aspx?query=10%20mg&form=Susp&drugCode=298&drugName=Famotidine&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=10%20mg/5ml&form=Syrup&drugCode=298&drugName=Famotidine&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=10%20mg&form=Syrup&drugCode=298&drugName=Famotidine&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=40%20mg&form=Inj&drugCode=298&drugName=Famotidine&type=1&Ing==1

http://druginfosys.com/AvailableBrands.aspx?query=20%20mg&form=Inj&drugCode=298&drugName=Famotidine&type=1&Ing==1


dosage forms. Being an efficient drug and much more potent than other compounds of

its class (ranitidine and cimetidine), other competitors are also involved in its

production [201]. It is mainly used to suppresses the volume of gastric secretion and

acid concentration [202, 203]. It gives protection against gastric injury in normal

subjects receiving short courses of non-steroidal ant-inflammatory drugs (NSAIDs)

[204]. It is also widely recommended in duodenal ulcers, gastric ulcers, gastro

esophageal reflux disease and Zollinger-Ellison syndrome. It decreases stomach acid

production upto almost 90% when given in oral doses of 20 or 40 mg and improves

duodenal ulcer healing [199]. It has been used in children and was found to be of

having no serious side effects [205].

Thus, it is clearly indicated that there are a lot of tasks for Famotidine but

unluckily its hydrophobic nature has been reported, which reduced its aqueous-

solubility and exposure to gastric-degradation also contribute to its variable and low

oral bioavailability [206]. Along with poor aqueous solubility, intestinal permeability

and gastric emptying also effect its overall bioavailability [207]. It belongs to BCS

Class-IV drugs, since, drugs of this class show poor aqueous solubility and low

permeability [207]. Due to which its oral formulations have not been successful

because of poor physico-chemical characteristics (solubility 1.1 mg/mL) and un-

favorable pharmacokinetics, including poor oral bioavailability (43%) and a short

plasma half-life (2.59 hours) [208-210].

2.3.2 MODIFIED FORMULATIONS

Some efforts were being made by numerous pharmaceutical scientists to solve the

mentioned problems of Famotidine regarding oral bioavailability. Literature also

shows that a lot of work has also been performed on Famotidine in different aspects

i.e. solubility enhancement and taste masking etc.

62


Before selecting Famotidine as drug model for research work, the available

limited literature for addressing the oral bioavailability issues has been studied.

Patel, D.J., et al., has reported Famotidine nano-suspension having minimum

particle size of only 566 nm and also lacking stability study. Also, there has not been

reported any in-vivo pharmacokinetic study.

Mummaneni, V., et al., (1990) reported fusion method for the enhancement of

solubility and dissolution-rate of Famotidine from solid glass dispersions of xylitol.

They revealed a noticeable boost in the dissolution-rate of Famotidine from solid

glass dispersion as compared to the unprocessed Famotidine [211].

Hassan, M.A., et al., (1990) prepared inclusion complex of Famotidine and β-

cyclodextrin by mixing them together in distilled water and heating under reflux for

one hour followed by stirring for five days at room temperature, they revealed

noticeable increased in the in-vitro dissolution rate of the complex from constant

surface-area discs which was about six times and twice high than that of the pure drug

and physical mixture, respectively [212].

Islam, M.S., et al., (1991) reported the formation of inclusion complex of

Famotidine with 2-hydroxypropyl-ß-cyclodextrin (HPCD) having significantly greater

dissolution rate than that of the unprocessed drug (pure) [213].

Gupta, U., et al, (2007) reported dendrimers mediated solubility enhancement

of Famotidine, they evaluated that Polypropylene Imine (PPI) dendrimers have a key

role in solubility enhancement of hydrophobes of different chemical nature while

hydrophobic and electrostatic interactions also have chief role in boosting solubility.

Their work also lack stability study, conversion to solid dosage form and in-vivo

pharmacokinetic profile of the processed formulation [214].

63


Mady, F.M., et al., (2010) used freeze-drying and kneading techniques for

preparation of binary (drug-β-CyDs) and ternary (drug-β-CyDs-Povidone K30)

systems with much faster dissolution rates of Famotidine and also masked its taste

[206].

Methods Employed for Famotidine Analysis

Numerous analysis methods for determining Famotidine in pharmaceutical dosage

forms have been developed and are being used including HPLC, HPTLC,

polarography, capillary electrophoresis, potentiometry, differential pulse voltametry,

spectrofluorometry and chemiluminescence spectrometry [215-222]. Some of these

techniques have sufficient sensitivity to determine low concentration of drug;

however, some of these methods are deficient in simplicity, accessibility and cost-

effectiveness. Spectrophotometry is characterized by its speed and accuracy,

simplicity and inexpensive instrument rrquired. Therefore, it is mostly used and an

important alternate method to the other available analytical techniques having clear

advantage in terms of cost of analysis.

2.4 DEFICIENCES IN ROXITHROMYCIN AND FAMOTIDINE

FORMULATIONSThere are some deficiences in the mentioned different reported studies i.e.

somewhere stability studies have not been conducted for the processed

formulations to realize the time for which they would be stable. The processed

formulation should be converted into the suitable solid dosage form for to ensure

maximum physical and chemical stability and to give the acceptable final shape

to the processed material, which would be convenient to the patients.

Everywhere, in-vitro studies of the processed formulations have been conducted

64


and the in-vivo theoretical boosted oral bioavailability has been supported with

it. In-vivo studies should be conducted using animal model (rabbits/mice) to

confirm the desired enhanced oral bioavailability of the processed material,

which is the aim of every solubility enhancement study.

Beside all these, the mentioned formulation studies of Roxithromycin and

Famotidine might have toxicity to some extent as toxicity has been associated

somewhere with polymeric nanoparticles. The polymeric nanoparticles

developed till now also have huge problems of consistent and gradual drug

release. Stability of NPs might be another big issue with the already available

nanoformulations as this stability study is also not being conducted somewhere

in the reported literature. In-vivo studies of the processed drugs have also not

been conducted in all places which is the last, most critical and significant step

in conducting oral bioavailability enhancement studies, which shows the fold

increase in the oral bioavailability compared to the marketed product.

2.5 PROPOSED APPROACH FOR ROXITHROMYCIN AND

FAMOTIDINENone of the reported techniques appeared to be complete in all respects and also not

sufficient for the effective replacement of non-aqueous based vehicle. Pharmaceutical

scientists should work a lot for the final formulation of Famotidine for use in humans

which is still far-away. Due to this reason, great attention is being focused to

developed well tolerable (biocompatible and biodegradable) carriers which give best

efficacy of Famotidine in clinical therapy.

Among the newly developed nano-particulate drug delivery systems, solid

lipid based nanoparticles have gained much more significance and attention owing to

65


their ability of high drug loading capacity and targeted drug delivery with sustained

release profile [343, 344]. SLNs offer large surface area and sustained drug release

with quick cellular uptake [54]. In modern eras, excessive attention is focused on

SLNs to increase bioavailability of hydrophobic drugs [55]. Mostly BCS Class-II and

Class-IV drugs are incorporated in SLNs [56]. SLNs-based drug delivery system has

numerous benefits including enhanced solubility of hydrophobic drugs with

prolonged drug release which ultimately lower dose and frequency of administration.

SLN is one of the novel nano-particulate drug delivery systems, which has been

developed by replacing the liquid lipids of the emulsions by solid lipids and is also

advantageous over the polymeric nanoparticles. SLN has numerous plusses like low

toxicity, physically stable, good bio-compatibility and hydrophobic drugs are better

delivered via SLNs.

Numerous successful studies of drugs loaded Solid Lipid Nanoparticles have

been reported regarding boosting oral bioavailability. Furthermore, they also possess

better stability during storage and also in biological fluids. Nanoparticles of

biocompatible and biodegradable lipids represent a striking engineering solution

to oral bioavailability enhancement. Nanoparticles composed of biodegradable

and biocompatible lipids improved different in-vivo parameters.

66

CHAPTER No. 3 MATERIALS AND METHODS

Chapter 3 MATERIALS AND METHODS

3.1 MATERIALSAll Active Pharmaceutical Ingredients (APIs), chemicals and formulation

excipients used in this study were of analytical grade and highest purity, obtained

from the following sources.

3.1.1 CHEMICALS

Active Pharmaceutical Ingredients (APIs)

Roxithromycin was supplied by Alliance Pharmaceuticals (Pvt) Ltd, Peshawar,

Pakistan.

Famotidine was procured as generous gift from Polyfine Chempharma (Pvt) Ltd

(Peshawar-Pakistan).

Formulation Excipients

Stearic Acid, polysorbate 80 (Tween® 80) and Polyethylene Glycol-400 were got

from Acros Organics Thermo Fisher Scientific, New Jersey-USA.

Polyvinylpyrrolidine (PVP-K30) was got from Crescent Chemical Company,

Islandia, New York-USA.

Organic Solvents and Water

Ethanol (absolute) was got from Sigma-Aldrich, Germany.

Chloroform was purchased from Alfa Aesar, Ward Hill, Massachusetts.

Ultra-pure deionized water was prepared with Millipore ultra-pure water

system (Milford, USA)

Other Required Materials

Dialysis bags were obtained from Spectrum lab Canada.

67


3.1.2 INSTRUMENTS USED

i. Photon Correlation Spectroscopy

Zeta sizer Nano ZS-90, Malvern Instrument (UK) is a perfect system for measuring z-

average particle size, PDI and zeta potential (ζ) determination. Prior to particle size

and zeta potential measurement, SLN formulations were diluted with de-ionized water

to yield proper scattering intensity. Particle size and zeta potential of all formulations

were obtained by calculating the average of 3 measurements at an angle of 90°.

ii. Scanning Electron Microscopy (SEM)

SEM (JSM5910, JEOL, Japan)was employed for studying the morphological

characteristics and texture of SLNs by. Varied accelerating voltage and magnification

power was used to get the desired clear micrographs. Few sample drops were fixed on

metallic stub of microscope with the help of double ended adhesive carbon tape,

followed by drying under vacuum for analysis.

iii. Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR spectra of different samples were obtained in the range of 400–

4500 cm−1, using FT-IR spectrophotometer (IR Prestige 21Shimadzu, Japan). SLN

sample was diluted with KBr mixing powder at 1% and pressed to obtain self-

supporting disks. For the compatibility of the formulation components, the peaks and

patterns shaped by unprocessed compound were compared with the processed

formulation [223].

iv. Powder X-Ray Diffractometer (P-XRD)

P-XRD technique is commonly employed to evaluate the production of new solid-

state in the processing samples [97]. Thus, P-XRD analysis was conducted via X-Ray

Diffractometer JDX-3532 JEOL (Japan) to study the variations in the crystalline

nature and physical state of different samples. P-XRD study was performed having

68


scan range of 2θ = 5°– 80°, with Cu Kα radiation. Tube was operated at 40 kV,

30 mA, step size 0.05°, step time 1.0 sec, receiving slit 0.2 mm, scattering slit 1.0

degree and divergence slit 1.0 degree.

v. Differential Scanning Calorimetry (DSC)

Phase transition of different processed and unprocessed materials was studied via

Differential Scanning Calorimetry (Perkin Elmer, Diamond Series DSC Equipment,

US). Sample was investigated in aluminum pan at a temperature range of 40-300℃ and heating rate of 10°C/min [224].

vi. Nano-drop Spectrophotometer

The supernatant was analyzed for un-entrapped drug by Nano-drop Spectro-

photometer (Thermo scientific 2000c/2000 UV-VIS Spectrophotometer) and then

used for calculation of percent entrapment efficiency and percent drug loading

capacity.

vii. High Performance Liquid Chromatography (HPLC)

HPLC (Perkin Elmer 200 series, Perkin Elmer, USA) was used to measure plasma

drug concentrations [195]. Prior to processing, blood plasma samples containing drug

were diluted with the mobile phase of HPLC followed by centrifugation. After

centrifugaion the resultant supernatent was seperated. For analysis via HPLC, the

supernatent (20 µl) was injected in to the stream of mobile-phase for quantification of

drug plasma concentration. Reversed phase column (Supelco C18, 5 µm particles size,

4.6 mm width and 25 cm in length), generally used for hydrophobic drugs and

precolumn (Supelco Cl8) were used at 37oC.

69


viii. Digital Weighing Balance

Electronic Digital Weighing Balance (Shimadzu, Japan) was used during this research

work for to measure the accurate weight of chemicals and other standard materials.

ix. pH-Meter

During this research work pH-meter (Inolab, Germany) was used to measure pH of

numerous formulations and also various phosphate buffers.

x. Vortex Mixer

Vortex Mixer (Fisher Scientific®, USA) also called Vortexer or Touch mixer was

used for mixing of various samples, especially for mixing melted lipid with the

selected hydrophobic drugs. It was also used for vortexing different samples prior

to the zeta sizer analysis.

xi. Micro Ultra-centrifuge

Ultra-centrifugation (Himac CS150GXL, Hitachi, Japan) of numerous freshly

prepared SLNs samples was accomplished for 10 minutes at 30,000 rpm to separate

SLNs as pellet at the bottom. The supernatant was also separated for to analyze the

un-entrapped drug in it.

xii. Magnetic Stirrer

Magnetic stirrer also called magnetic mixer, utilizes a rotating magnetic field for

spinning a stir bar at very high speeds. During SLNs fabrication magnetic stirrer

(Benchmark, USA) was continuously used for the vigorous mixing of numerous

formulations along with heating.

xiii. Freeze Dryer

Lyophilization of different processed formulations of SLNs samples were carried out

using Freeze Dryer (Heto Power Dry LL1500- Thermo Electron Corporation, USA).

70


xiv. Bath Sonicator

Prior to partricle size analysis, different SLNs formulations were sonicated using bath

sonicator (Soniprep, 150 instruments, Sanyo, UK.). The key purpose of sonication

was to disperse the particles properly and evade aggregation.

Bath Sonicator works by applying sound energy to the liquid sample to agitate its

particles, for numerous purposes. Bath sonicator usually used ultrasonic frequency

(>20 kHz).

xv. Tray dryer

Tray dryer was used for drying wet pellets of loaded SLNs to get dried powder in bulk

quantity for conversion to solid dosage form. The SLNs samples to be dried were

spread on trays inside the tray dryer. The flow of uniform air is retained for drying

purpose while heating was switched-off to maintain the quality of fabricated SLNs.

xvi. Moisture Analyzer Balance

Moisture analyzer balance being used (Sartorius AG Germany) is having heat source

in its lid which cover-up the weighing pan. Sample is uniformly spread over weighing

pan and is placed inside the balance. The sample is heated until the differentiation in

weight noted by the balance has reached the ascribed set point. The loss in moisture

content (in terms of percent weight loss) is then displayed on a LED screen.

3.2 METHODS3.2.1 METHODS FOR SLNS FABRICATION

3.2.1.1 Method-I (Solvent Injection Method)

i. Fabrication of Blank SLNs

71


Twelve different blank SLN formulations were fabricated by Solvent Injection

method using varied surfactant (Tween® 80) concentrations, co-surfactant (PVP)

concentrations and stirring time (Table 3.1).

During fabrication process, organic phase was prepared by dissolving stearic

acid in ethanol and warmed upto 79°C. Aqueous-phase was prepared by dissolving

Tween® 80 and PVP in phosphate buffer solution (pH 7.4). Organic-phase was

added drop wise to the pre-warmed aqueous-phase (79°C) with the help of a

hypodermic needle under continuous magnetic stirring [91]. Magnetic stirring was

continued till the organic solvent evaporates to yield the desired nanoparticles

dispersion followed by centrifugation for 10 minutes at 30,000 rpm [83]. Particles size

and PDI of these nanoformulations were figured out using Zeta sizer Nano ZS-90,

Malvern Instrument (UK) [225].

Table 3.1: Formulations of blank SLNs (Solvent Injection method)Formulation Stearic

Acid (g)Tween® 80

(ml)PVP(g)

Stirring Time(min)

BFSi-1 1.00 0.5 0 5BFSi-2 1.00 1 0 5BFSi-3 1.00 1.5 0 5BFSi-4 1.00 2 0 5BFSi-5 1.00 1.9 0.1 5BFSi-6 1.00 1.8 0.2 5BFSi-7 1.00 1.7 0.3 5BFSi-8 1.00 1.6 0.4 5BFSi-9 1.00 1.5 0.5 5BFSi-10 1.00 1.6 0.4 10BFSi-11 1.00 1.6 0.4 15BFSi-12 1.00 1.6 0.4 20

Abbreviation: PVP, Polyvinylpyrrolidone.

ii. Fabrication of Roxithromycin Loaded SLNs

72


Best processing conditions of blank BFSi-11 nanoformulation i.e. stearic acid (1.00

g), Tween® 80 (1.6 ml) and PVP (0.4 g) were used for fabricating RTM loaded

SLNs. Five different blank nanoformulations of RTM loaded SLNs were fabricated

on basis of lipid drug ratio (Table 3.2). Specified quantitiesof RTM and stearic acid

were dissolved in ethanol. The rest of process followed was similar as adopted for

unloaded SLNs.

Table 3.2: Formulations of RTM-SLNs (Solvent Injection method)Formulation Stearic Acid

(g)RTM (mg)

Tween® 80 (ml)

PVP(g)

Stirring Time (min)

RFSi-1 1.00 40 1.6 0.4 15RFSi-2 1.00 50 1.6 0.4 15RFSi-3 1.00 66.6 1.6 0.4 15RFSi-4 1.00 100 1.6 0.4 15RFSi-5 1.00 200 1.6 0.4 15

Abbreviations: RTM;Roxithromycin, PVP; polyvinylpyrrolidone

iii. Fabrication of Famotidine Loaded SLNs

Best processing conditions of blank BFSi-11 nanoformulation i.e. stearic acid (1.00

g), Tween® 80 (1.6 ml) and PVP (0.4 g) were used for fabricating FTD loaded SLNs.

Five different formulations of FTD loaded SLNs were fabricated on basis of lipid

drug ratio (Table 3.3). Specified quantitiesof FTD and stearic acid were dissolved in

ethanol. The rest of process followed was similar as adopted for blank SLNs .

Schematic diagram for preparation of SLNs by Solvent Injection method is shown in

Figure 3.1.

Table 3.3: Formulations of FTD-SLNs (Solvent Injection method)Formulatio

nStearic Acid

(g)FTD (mg)

Tween® 80 (ml)

PVP(g)

Stirring Time (min)

FFSi-1 1.00 40 1.6 0.4 15FFSi-2 1.00 50 1.6 0.4 15FFSi-3 1.00 66.6 1.6 0.4 15FFSi-4 1.00 100 1.6 0.4 15

73


FFSi-5 1.00 200 1.6 0.4 15Abbreviations: FTD;Famotidine, PVP; polyvinylpyrrolidone

Figure 3.1: Schematic diagram of Solvent Injection technique

3.2.1.2 Method-II (Solvent Evaporation Method)

i. Fabrications of Blank SLNs

Blank SLNs were fabricated by Solvent-Emulsification-Evaporation (SEE) technique

using diverse surfactant (Tween® 80) concentrations, co-surfactant (PVP)

concentrations and stirring time. Twelve different blank nanoformulations (BFSe-1 to

BFSe-12) were fabricated (Table 3.4).

Specified amount of stearic acid was dissolved in chloroform. PVP and

Tween® 80 in definite quantities were dissolved in phosphate buffer solution. Oily

phase was emulsified by adding it to aqueous phase under magnetic stirring (1000

rpm) for various time intervals. After mechanical evaporation of chloroform, lipid

start precipitating as SLNs in aqueous phase followed by centrifugation for 10

74


minutes at 30,000 rpm [83]. Particles size and PDI of these nanoformulations were

figured out using Zetasizer Nano ZS-90, Malvern Instrument(UK) [225].

Table 3.4: Formulations of blank SLNs (Solvent evaporation method)Formulation Stearic Acid

(g)Tween® 80

(ml)PVP(g)

Stirring Time(min)

BFSe-1 1.00 0.5 0 5BFSe-2 1.00 1 0 5BFSe-3 1.00 1.5 0 5BFSe-4 1.00 2 0 5BFSe-5 1.00 1.9 0.1 5BFSe-6 1.00 1.8 0.2 5BFSe-7 1.00 1.7 0.3 5BFSe-8 1.00 1.6 0.4 5BFSe-9 1.00 1.5 0.5 5BFSe-10 1.00 1.6 0.4 10BFSe-11 1.00 1.6 0.4 15BFSe-12 1.00 1.6 0.4 20

Abbreviation: PVP, Polyvinylpyrrolidone.

ii. Fabrications of Roxithromycin Loaded SLNs

Using best conditions of blank BFSe-11 nanoformulation, different five

nanoformulations (RFSe-1 to RFSe-5) of RTM-SLNs were fabricated (Table 3.5).

They were differentiated on the basis of different lipid drug ratios. During fabrication

process, specified quantities of RTM and stearic acid were dissolved in chloroform.

The rest of process followed was similar as adopted for blank SLNs.

Table 3.5: Formulations of RTM-SLNs (Solvent evaporation method)Formulatio

nStearic Acid

(g)RTM (mg)

Tween® 80 (ml)

PVP(g)

Stirring Time (min)

RFSe-1 1.00 40 1.6 0.4 15RFSe-2 1.00 50 1.6 0.4 15RFSe-3 1.00 66.6 1.6 0.4 15RFSe-4 1.00 100 1.6 0.4 15RFSe-5 1.00 200 1.6 0.4 15

Abbreviations: RTM, Roxithromycin, PVP; polyvinylpyrrolidone

75



Best conditions of UFSe-11 formulation i.e. stearic acid (1.0 g), Tween® 80 (1.6 ml),

PVP (0.4 g) were further used for fabricating FTD loaded SLNs (FTD-SLNs).

Different formulations of FTD-SLNs were fabricated on basis of lipid drug ratio

(Table 3.6). Specified quantities of FTD and stearic acid was dissolved in chloroform.

The rest of process followed was similar as adopted for unloaded SLNs. Schematic

diagram for preparation of SLNs by Solvent evaporation method is shown in Figure

3.2.

Table 3.6: Formulations of FTD-SLNs (Solvent evaporation method)Formulatio

nStearic Acid

(g)FTD(mg)

Tween® 80 (ml)

PVP (g)

Stirring Time (min)

FFSe-1 1.00 40 1.6 0.4 15FFSe-2 1.00 50 1.6 0.4 15FFSe-3 1.00 66.6 1.6 0.4 15FFSe-4 1.00 100 1.6 0.4 15FFSe-5 1.00 200 1.6 0.4 15

Abbreviations: FTD, Famotidine; PVP, Polyvinylpyrrolidone

76


Figure 3.2: Schematic diagram of Solvent Evaporation technique

3.2.1.3 Method-III (Hot Melt Encapsulation Method)

i. Fabrications of Blank SLNs

Blank SLNs were fabricated by Hot Melt Encapsulation (HME) technique and

optimized via surfactant (Tween® 80) concentrations, co-surfactant (PEG)

concentrations and stirring time. Twelve different blank nanoformulations (BFH-1 to

BFH-12) were prepared (Table 3.7).

Specified amount of stearic acid was melted 10℃ above melting point.

Aqueous phase containing Tween® 80 and PEG was also heated up to melted lipid’s

temperature. Both phases were mixed on magnetic stirrer (1200 rpm) at similar

temperature for specified time intervals to prepare hot melt micro-emulsion.

Continued magnetic stirring, without heating until it cool-down to 25OC followed by

centrifugation for 10 minutes at 30,000 rpm [83]. Zeta-size and PDI of these

formulations were figured out using Zeta sizer Nano ZS-90, Malvern Instrument (UK)

[225].

Table 3.7: Formulations of blank SLNs (Hot Melt Encapsulation method)Formulatio

nStearic Acid

(g)Tween® 80

(ml)PEG-400

(g)Stirring Time

(min)BFH-1 1.00 0.5 0 5BFH-2 1.00 1 0 5BFH-3 1.00 1.5 0 5BFH-4 1.00 2 0 5BFH-5 1.00 1.9 0.1 5BFH-6 1.00 1.8 0.2 5BFH-7 1.00 1.7 0.3 5BFH-8 1.00 1.6 0.4 5BFH-9 1.00 1.5 0.5 5BFH-10 1.00 1.6 0.4 10BFH-11 1.00 1.6 0.4 15BFH-12 1.00 1.6 0.4 20

Abbreviations: PEG, Polyethylene Glycol

ii. Fabrication of Roxithromycin loaded SLNs

77


Optimized conditions of blank BFH-11 nanoformulation were used for fabrication of

of RTM-SLNs. Five different nanoformulations (RFH-1 to RFH-5) of RTM-SLNs

were fabricated, differentiated in terms of different lipid drug ratios (Table 3.8).

During fabrication process, specified quantities of RTM were added to melted lipid

and vortexed for 5 minutes. Rest of process followed was similar as adopted for blank

SLNs.

Table 3.8: Formulations of RTM-SLNs (Hot Melt Encapsulation method)Formulation Stearic Acid

(g)Drug (mg)

Tween® 80 (ml)

PEG-400 (g)

Stirring Time (min)

RFH-1 1.00 40 1.6 0.4 15RFH-2 1.00 50 1.6 0.4 15RFH-3 1.00 66.6 1.6 0.4 15RFH-4 1.00 100 1.6 0.4 15RFH-5 1.00 200 1.6 0.4 15

Abbreviations: PEG: Polyethylene Glycol


Optimized conditions of blank BFH-11 nanoformulation were used to fabricate FTD

loaded SLNs. Five different nanoformulations (FFH-1 to FFH-5) of FTD-SLNs were

fabricated, differentiated on lipid drug ratios (Table 3.9). During preparation process,

specified quantity of FTD was added to melted lipid and vortexed for 5 minutes. Rest

of process followed was similar as adopted for blank SLNs. Schematic diagram for

preparation of SLNs by Hot Melt Encapsulation technique is shown in Figure 3.3.

Table 3.9: Formulations of FTD-SLNs (Hot Melt Encapsulation method)Formulatio

nStearic Acid (g)

FD(mg)

Tween® 80 (ml)

PEG(g)

Stirring Time(min)

FFH-1 1.00 40 1.6 0.4 15FFH-2 1.00 50 1.6 0.4 15FFH-3 1.00 66.6 1.6 0.4 15FFH-4 1.00 100 1.6 0.4 15FFH-5 1.00 200 1.6 0.4 15

Abbreviations: FD, Famotidine; PEG, Polyethylene glycol

78


Figure 3.3: Schematic diagram of Hot Melt Encapsulation technique

3.3 LYOPHILIZATIONLyophilization is commonly used as promising way to increase physical and chemical

stability of SLNs for prolonged period of time. Hence, the optimized formulations of

SLNs, were freeze dried using freeze dryer (Heto Power Dry LL1500- Thermo

Electron Corporation, USA), needed for further characterization like DSC, P.XRD

and FT-IR. Addition of cryoprotectant (e.g. Glucose, Polyvinylpyrrolidon, Sorbitol,

Trehalose, and Mannose) is used to reduce particles aggregation and to get better re-

dispersion of dry product. Hence, 10% glucose solution was added before drying as

cryoprotectant [80, 226]. The optimized formulations of SLNs were kept overnight at

-20 C and then shifted to freeze dryer (-75 C). The samples were lyophilized for 48⁰ ⁰

hrs till room temperature at increasing rate of 5 C/h ⁰ [227].

3.3.1 TRAY DRYING AS ALTERNATIVE TO

LYOPHILIZATION

The SLNs dispersion prepared via hot melt encapsulation technique was tray dried in

order to get dried powder material. Tray drying technique is employed as alternative

79


to lyophilization in order to get bulk quantity of dried powder, sufficient for

conversion to solid dosage form for conducting in-vivo study. Tray dryier as

alternative to lyophilization was investigated via SEM to compare with the

lyophilized sample. The conditions being employed for tray drying technique are

mentioned under heading "3.5.2. TRAY DRIER".

3.4 CHARACTERIZATIONi. Particle Size Analysis

Zeta sizer Nano ZS-90, Malvern Instrument (UK) was used for particle size, PDI and

zeta potential (ζ) determination. Prior to measurement, de-ionized water was

employed to dilute nanoformulations followed by analysis for particle size and PDI.

In case of zeta potential, proper concentration of nanoformulations (single

particle suspension) was made to get good zeta potential. Prepared sample of SLNs

was then filled into the specially designed cuvette for zeta potential with the help of

micropeppite for analysis. The particles size, PDI and zeta potential of

nanoformulations were calculated by taking the average of three results.

ii. Scanning Electron Microscopy (SEM)

Surface morphology of the optimized nanoformulations were evaluated via SEM

(JSM5910, JEOL, Japan) [228]. Magnification power in the range of 15000-60000X

have been used with varied voltage. Prior to conducting SEM analysis, deionized

water was used to dilute all nanoformulations to form clear and visible samples.

Double ended adhesive carbon tape was employed to fixed sample drops of on

metallic stub of microscope followed by drying under vacuum for further analysis.

iii. Fourier Transform Infrared (FT-IR) Spectroscopy

80


FT-IR spectra of unprocessed drug and processed formulations of SLNs were

obtained in the range of 400–4500 cm−1, using FT-IR spectrophotometer (IR Prestige

21Shimadzu, Japan). Samples were diluted with KBr mixing powder at 1% and

pressed to obtain self-supporting disks. For the compatibility of the formulation

components, the peaks and patterns shaped by the unprocessed drug were compared

with their processed formulations of SLNs [223].

iv. Powder X-Ray Diffraction (P-XRD) Analysis

P-XRD technique is commonly employed to evaluate the production of new solid-

state in the processing samples [97]. Thus, P-XRD analysis was conducted via X-Ray

Diffractometer JDX-3532 JEOL (Japan) to study the variations in the crystalline

nature and physical state of different samples. P-XRD study was performed using

plain plastic holder for sample in the scan range of 2θ = 5°– 80° with Cu Kα radiation.

Tube was operated at 40 kV, 30 mA, step size 0.05°, step time 1.0 sec, receiving slit

0.2 mm, scattering slit 1.0 degree and divergence slit 1.0 degree.

v. Differential Scanning Calorimetric (DSC) Studies

Phase transition of different processed and unprocessed materials was studied via

Differential Scanning Calorimetry (Perkin Elmer, Diamond Series DSC Equipment,

US). Samples were investegated in aluminum pans at a temperature range of 40-

300℃ and heating rate of 10°C/min [224].

vi. Entrapment Efficiency and Drug Loading Capacity

The optimized formulations of SLNs, fabricated by the mentioned three techniques

samples were centrifuged. The supernatant was analyzed for un-entrapped drug by

Nano-drop Spectro-photometer (Thermo scientific 2000c/2000 UV-VIS Spectro-

81


photometer) and then used for calculation of percent entrapment efficiency using

equation (1):

EE %=Total amount of drugadded –Unloaded Drug× 100Total amount of drug (1)

Drug loading capacity (DLC) was calculated by using equation (2);

DLC %= Totalquantity of drugi nSLNs X 100Quantity of Drug added+Quantity of Excipientsadded

(2)

vii. In-vitro Drug Release Studies

Dialysis bag technique was employed to study in-vitro drug release kinetics [229].

Dialysis bags were soaked in de-ionized water for 12 hrs prior to use. The optimized

SLN dispersions (1 ml) were transferred and placed in dialysis bag, both ends were

fixed with thread and kept in phosphate buffered (pH 7.4) solution (250 ml).

Conditions were set at 37±0.5°C and 50 rpm. After specific interval of time (1_12

hours), sample (1 ml) was taken for analysis and similar volume of phosphate buffer

was replaced to make up the volume.

viii. Release KineticsData from in-vitro drug release was fitted into different release kinetic models (zero

order, first order, Higuchi and Korsmeyer-Peppas model) to figure out both release

rate and mechanism followed.

Zero-order Kinetic Model

Zero-order kinetic model demonstrates a system in which rate of the drugs release is

not dependent on their concentrations. Thus, the dissolution of drug molecules is only

a function of time. In this model, the in-vitro drug release data is plotted as percent

cumulative drug release verse time.

First-order Kinetic Model

82


Relationship of log cumulative percent drug release and time is being explained by

First-order kinetic model. Drug release kinetics will be first order if the graph between

these mentioned two parameters is linear. Basically, this model explains that drug

release is concentration dependent.

This model has also been employed to explain elimination and/or absorption

of some drugs, even though it is complicated to conceptualize this mechanism on

theoretical basis.

Higuchi Model

In 1961, Huguchi for the very first time proposed a mathematical kinetic model for to

explain the release of drug from a matrix-system [102]. Higuchi model explains drug

release as a diffusion mechanism based on Fick's law. This model is based on the

hypotheses that (i) initial concentration of the drug in the matrix is higher compared to

drug solubility; (ii) particles of the drug are smaller in comparison to system

thickness; (iii) drug diffusion arises in one dimension only; (iv) matrix dissolution and

swelling are negligible; (vi) perfect sink circumstances are always achieved in the

release environment and (v) drug diffusivity is constant.

Here, in this model, the in-vitro drug release data was plotted as cumulative

drug release versus square root of time.

Kormeyer Peppas model

Korsmeyer et al., (1983) presented drug release mechanism from a polymeric system.

Here, in this model, the drug release mechanism is described by the value of n.

As,

n > 0.89 describes super case-II transport

n = 0.89 describes Case-II transport

0.89 > n> 0.45 describes non-Fickian transport

n ≥ 0.45 describes Fickian diffusion mechanism,

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Here in this study, in-vitro drug release data was plotted as log cumulative

percentage drug release versus log time.

ix. Stability Study

Physical stability study was conducted for optimized formulations of SLNs [230]. The

freshly fabricated sample was divided into two parts. Each was then put in two

different vials and stored at different temperatures (5±2°C and 25±3°C). Change in

zeta size and PDI was observed for 3 months. Data was analyzed statistically by two-

tailed t-test. Probability ˂0.05 was considered significant.

3.5 CONVERSION OF NANO-SUSPENSION TO CAPSULESLarge amount of de-ionized water was removed from the optimized SLNs dispersion

via ultra-centrifugation. Pellets of SLNs obtained as a result of ultra-centrifugation

were converted into dried powder using tray dryer.

3.5.1 TRAY DRYING

During drying process, the wet pellets of SLNs were properly spread on trays, inside

the tray dryer. In tray dryer, the use of heating in favor of drying, for formulations

having Tween® 80 as constituent, causes leakage of Tween® 80 . Only air was

switched-on, fresh air introduced via in-let and passed over trays having wet pellets

material. The water molecules present on the surface of wet pellets were evaporated

by air flow. As a result, the water from the interior of the wet pellets diffused by

capillary action. These events occurred repeatedly until the desired moisture content is

attained in the resultant dried SLNs powder.

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3.5.2 MOISTURE LEVEL DETERMINATION

Samples obtained after the drying process were analyzed with moisture analyser

balance to find-out the “loss on drying” (LOD). The Loss on Drying Test basically

measured the quantity of water in the samples when dried under particular conditions.

The sample putted in the weighing pan was uniformly spread over its surface.

The weighing pan was placed in the moisture balance and the lid was closed-up.

Heating source was adjusted to a particular temperature. The sample was heated until

the differentiation in weight noted by the balance has reached the ascribed set point.

The loss in moisture content (in terms of percent weight loss) was then displayed on a

LED screen which was less than 0.50 %.

After performing the “loss on drying” test, the resultant dried SLNs powder was

subjected to various pharmaceutical techniques to be converted in to the granules for

filling into capsule shells.

3.5.3 GRANULATION

Two different techniques termed as wet granulation and dry granulation can be used

for preparation of granules from dried powder materials. Different excipients are used

in these two techniques. In this research work, wet granulation technique has been

used for preparation of granules from the dried powder material. Excipients used for

granulation purpose are enlisted below in Table 3.10;

Table 3.10: Excipients employed during wet granulationS. No Material Quantity

1 Aerocil (200) 3 mg2 Starch 18 mg3 Polyvinylpyrrolidone (K-30) 3.91 mg4 Isopropyl alcohol (IPA) 0.03 ml5 Primogel 5.38 mg6 Magnesium stearate 1.5 mg7 Lactose monohydrate 60.41 mg

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In the first step, aerocil (200), starch, lactose monohydrate and dried-powder of

SLNs nanoformulation were thoroughly mixed for 15 minutes. To ensure the

avoidance of any contamination, mask and gloves were used during mixing process.

The resultant mixture was passed through a mesh (size 16) to get uniform sized

particles.

In the second step, isopropyl alcohol (IPA) was used for dissolving

polyvinylpyrrolidone (K-30). The resultant product of first step was thoroughly mixed

with this clear solution of isopropyl alcohol (IPA) and polyvinylpyrrolidone (K-30).

After proper mixing, drying (tray drier) was carried-out followed by passing through

mesh (size 12) to get uniform size granules.

In the last step, aerocil (200) and magnesium stearate were added to the final

granules and followed by mixing for 15 minutes to ensure high flow property.

i. Coating of Granules

The final granules were further subjected to coating process. Materials used for

coating purpose are enlisted in Table 3.11;

Table 3.11: Excipients employed for granules coatingS.No Material Quantity

1 Methocil (E5) 26 mg2 Titanium dioxide (TiO2) 18 mg3 Methylene chloride 325 mg4 Isopropyl alcohol (IPA) 265 mg

In the first step, isopropyl alcohol (IPA) was used for dissolving methocil.

Methylene chloride was added to this clear solution of isopropyl alcohol (IPA) and

methocil followed by proper mixing.

86


In the second step, isopropyl alcohol (IPA) was aslo used for dissoving titanium

dioxide (TiO2). Its solution is then mixed with the resultant product of the first step,

followed by proper mixing to produce the desired white spraying solution.

The prepared white spraying solution was further used for applying on the

produced granules via spray-gun. After its application, the resultant wet granules were

subjected to drying to get the desired single-layer coated granules.

3.5.4 CAPSULE SHELLS FILLING

Capsule shells filling is a technical process and the dried granules to be encapsulated

should be well build-up to make sure mass uniformity. The selection of capsule size

and fill quantity was stated by the type of formulation and unit dose required.

Capsules being filled for research purpose have diverse dimensions or geometries

from the capsules used in commercial production. Usually, the batch size of capsules

for research purpose is smaller than for commercial production, therefore, specific

machine is not required for filling purpose. So in this case, it is possible to fill capsule

shells manually. Two types of granules (uncoated & coated), filled in different ratios

in capsules shells to get capsules of the following two types:

Type-I Capsules

Type-I Capsules contain 100% uncoated granules, which were used for conducting

comparative in-vivo study with the marketed product to confirm the desired boosted

oral bioavailability. In-vitro study has also been conducted on Type-I Capsules for

Dissimilarity (f1) and Similarity (f2) factors determination.

Type-II Capsules

Type-II Capsules contain 40% uncoated granules and 60 % coated granules. In-vitro

study has been conducted on Type-II Capsules for determination of Dissimilarity (f1)

87


and Similarity (f2) factors to confirm the remarked difference with the marketed

product.

Standard operating procedure (SOP) was followed for filling the prepared

granules in hard gelatine capsule shells (size 5; Capsugel, North Peapack, NJ, USA).

3.6 COMPARATIVE IN-VIVO STUDY OF PREPARED

NANOFORMULATIONS WITH MARKETED PRODUCTSFor conducting in-vivo pharmacokinetic study, healthy rabbits (2±0.3 kg) were used.

All experimental animals (rabbits) were screened and apccepted for experimental

purpose by the Ethical Committee (Department of Pharmacy, University of

Malakand). All the experimental animals (rabbits) were kept in fasted state (12 hours)

before dosing but access to water was given. Any experimental animal having dis-

comfort was expelled from studies.

3.6.1 ORAL DRUG ADMINISTRATION

Prior to oral drug administration (Famotidine 10 mg, Roxithromycin 20 mg), three

groups of animals were made, each having six rabbits. SLNs nano-suspension was

administered to Group-I, prepared capsules to Group-II while marketed product to

Group-III, via feeding tube (stainless steel tube).

3.6.2 COLLECTION OF BLOOD SAMPLES

At various time interval (0_24 hours), sample of blood (0.5 ml) was taken from

marginal ear vein of rabbits. Blood samples were kept in 3 ml tubes (heparinized),

plasma was separated through centrifugation and stored (-20°C) for further analysis.

3.6.3 ANALYSIS OF PLASMA DRUG CONCENTRATION VIA

HPLC

High performance liquid chromatography (HPLC) was used to measure plasma drug

concentrations [195]. Reversed phase column (Supelco C18, 5 µm particles size, 4.6 88


mm width and 25 cm in length), generally used for hydrophobic drugs and precolumn

(Supelco Cl8) were used at 37oC.

a. Analysis of Roxithromycin by HPLC

For Roxithromycin, acetonitrile and phosphate buffer (0.067 M and pH 4) in 45:55

v/v were used as mobile phase (flow-rate: 1 ml.min-1, retention-time: 5 minutes). Prior

to HPLC analysis, plasma samples were mixed with acetonitrile and then centrifuged

for 10 minutes. After centrifugation, proteins precipitated and from the resultant

supernatant aliquot of 20 µl was introduced into the mobile phase of HPLC

instrument, to determine concentration of RTM using UV detector at λmax 210 nm

[195]. Concentration of RTM was calculated by using the calibration curve.

b. Analysis of Famotidine by HPLC

For Famotidine, acetonitrile: Methanol: (0.016 mol/l) Phosphoric Acid (10:10:80)

were used as mobile phase (Retention time: 3 min, Flow rate: 1 ml.min-1). Prior to

HPLC analysis, plasma samples were mixed with acetonitrile and then centrifuged for

10 minutes. After centrifugation, proteins precipitated and from the resultant

supernatant aliquot of 20 µl was introduced into the mobile phase of HPLC

instrument, to determine concentration of FTD using UV detector at λmax 254 nm.

Concentration of Famotidine was determined from the area of chromatographic peak

using the calibration curve.

3.6.4 ANALYSIS OF DRUG DATA

Different pharmacokinetic parameters were determined for non−compartmental

model. From concentration−time curve, Area Under Curve (AUC0→t) was determined

via trapezoidal rule. From the individual plasma concentration−time curve, peak

plasma concentration (Cmax) and peak plasma concentration time (Tmax) were

calculated. Total area under the curve (AUC0→24) was determined by equation (3):

89


AUC 0→ 24 =AUC 0 → 24+ C tK e (3)

Ct is drug concentration at 24th hour and Ke is apparent elimination rate constant.

Relative bioavailability after 24 hours for equal dose was determined by equation (4):

Fr= AUC -SLNs Formulation 0→24 AUC - Marketed product 0→24 (4)

One−way analysis of variance and t−test (p<0.05) were used for statistical

analysis of data.

3.7 DISSIMILARITY (f1) AND SIMILARITY (f2) FACTORS Capsules prepared from the optimized nanoformulations of SLNs loaded with

Roxithromycin and Famotidine (Hot Melt Encapsulation Technique) as well as their

respective marketed products were processed under similar conditions.

Dissimilarity (f1) and similarity (f2) factors for the prepared solid dosage form

of the drug loaded SLNs and its respective marketed product were determined from

in-vitro release data using the following formulae;

f1¿{[∑t=1

n

R t−T t ]∑t=1

n

R t }f2=50× log {[1+( 1

n )∑t=1

n

.|Rt−T t|.2] .−0.5 ×100}Whereas,

Rt = % of Reference drug released at each time point t

Tt = % of Test drug released at each time point t

n = Number of withdrawal points

90

CHAPTER No. 4 RESULTS AND DISCUSSION

Chapter 4 RESULTS AND DISCUSSION

4.1 SOLVENT INJECTION METHOD

4.1.1 PARTICLE SIZE ANALYSIS

i. Particle Size Analysis of Blank SLNs

The first technique employed for preparation of blank SLNs was Solvent Injection

method. For fabrication of blank SLNs, stearic acid was used as solid lipid, Tween®

80 as surfactant and PVP as co-surfactant. During fabrication of blank SLNs,

optimization was carried out using three different variable factors i.e. concentration of

surfactant (Tween® 80), concentration of co-surfactant (PVP) and magnetic stirring

time.

a. Concentration of Surfactant

During Solvent Injection method, blank SLNs of stearic acid were fabricated using

Tween® 80 as surfactant. The quantity of stearic acid was kept contant at 1 g, while

the concentration of Tween® 80 was increased from 0.5-2 g, which caused abrupt

reduction in particles size upto 227.4±2.3 nm. It was noticed that particle size reduced

upto the maximium level with 2 g of Tween® 80. As, further increase in

concentration of Tween® 80 showed almost no effect on particle size. It has been

reported in literature that higher concentration of surfactant showed lower particle

size and also offer better stability to small lipid droplets as it prevent them from

coalescence [81].

b. Concentration of co-surfactant

Further decrease in particle size was achieved with addition of co-surfactant. In this

case, the quantity of stearic acid (1 g) was kept constant while the concentration of

surfactant (Tween® 80) was divergently decreased with the increased in 91


concentration of co-surfactant (Table 3.1). PVP being employed as co-surfactant in

the concentration range of 0.1-0.5 ml, further reduced particle size upto 196.8±3.1

nm. As, SLNs fabricated with surfactant/co-surfactant mixture have lower particle

size and better stability as compared to SLNs of unadded co-surfactants.

c. Stirring Time

During variation in magnetic stirring time, the concentration of stearic acid, Tween®

80 and PVP were kept constant. By increasing the magnetic stirring time from 5 to 15

min, PDI reduced to the desired acceptable range. During increase in the magnetic

stirring time, it has been noticed that particle size also reduced to some extent but it

mainly controlled the PDI. Thus, PDI was controlled and reduced by increasing

stirring time which has shown almost little bit effect on particle size reduction [82].

Important variations in terms of particle size and PDI were seen by changing the

mentioned three variable parameters (Figure 4.1).

During optimization process of blank SLNs, desired particles size and

acceptable PDI were produced with stearic acid (1 g), Tween® 80 (1.6 ml), PVP (0.4

g) and magnetic stirring time (15 minutes). Further increase in the concentration of

surfactant and co-surfactant was avoided to prevent decrease in the entrapment

efficiency and also toxic effects associated with them. The optimized blank SLNs

formulation (BFSi-11 nanoformulation) being fabricated with stearic acid (1 g),

Tween® 80 (1.6 ml), PVP (0.4 g) and stirring time (15 min) showed best results of

particle size 184.9±2.2 nm and PDI 0.456±0.09.

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Figure 4.1: Particle size and PDI of blank SLNs formulations (SI)

ii. Particle Size Analysis of Roxithromycin and Famotidine

The optimized conditions of blank SLNs formulation (BFSi-11 nanoformulation) was

further used for drug loading. Based on drug lipid ratio, different five

nanoformulations of SLNs loaded with RTM (RFSi-1 to RFSi-5) as well as FTD

(FFSi-1 to FFSi-5) were prepared.

The optimized nanoformulations of Roxithromycin and Famotidine were RFSi-

4 and FFSi-4, presenting excellent results in terms of particle size 169.6±2.3 nm &

162.7±2.3 nm, PDI 0.462±0.02 & 0.352±0.03, zeta potential -32.6±1.9 mV & -

34.35±2 mV respectively (Figure 4.2 & Figure 4.3).

After drug loading the particle size reduced to 169.6±2.3 nm for RTM (RFSi-4

nanoformulation) and to 162.7±2.3 nm for FTD (FFSi-4 nanoformulation) as

compared to optimized blank SLNs formulation (BFSi-11). As after drug pay load,

particle size reduced due to decrease in free lipid content [231].

The PDI <0.5 and ZP in the range of ±30 revealed that the fabricated nano-

formulations would be stable in nature [232]. For our prepared nanoformulations both

PDI and ZP were within the acceptable range, which exhibit electrostatic stabilization

to avoid aggregation thus preventing particles growth and Ostwald ripening [233].

93


As the fabricated loaded SLNs are for oral administration, so, the produced

average particles size is less than 400 nm having the ability of easily crossing the

linings of gastro-intestinal cells to achieve the desired boosted oral bioavailability

[234]. Moreover, the fabricated particles comprised of 100-200 nm size range, since

particles having size less than 200 nm are undetectable to the Reticulo-Endothelial

System (RES) and remain in circulatory system for a prolonged time period [235].

Figure 4.2: Particle size (A) & Zeta Potential (B) of RFSi-4 nanoformulation

Figure 4.3: Particle size (A) & Zeta Potential (B) of FFSi-4 nanoformulation

4.1.2 SCANNING ELECTRON MICROSCOPY (SEM)

Scanning Electron Microscopy confirmed nano-metric size particles of SLNs loaded

with RTM and FTD. Particle size estimated from SEM micrographs was

approximately in the range of 150 nm to 180 nm in diameter (Figure 4.4 & Figure

4.5). White patches in micrograph showed solid, identical and fairly spherical shaped

94


nanoparticles with a well-defined periphery. Most of the SLNs were present in

dispersed form with homogeneous distribution which exhibit amorphous nature of the

produced nanoparticles.

SEM representing nanometric size particles confirmed the results of zeta sizer

analysis. Furthermore, the blunt and non spiky white patches in the micrographs

revealed amorphous nature nanoparticles, which plays a vital role in the solubility

enhancement of the drugs being a successful outcome of pharmaceutical nano-

engineering.

Figure 4.4: SEM micrograph of RFSi-4 nanoformulation

Figure 4.5: SEM micrograph of FFSi-4 nanoformulation

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4.1.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY

(FT-IR)

Fourier Transform Infrared (FT-IR) spectroscopy is a reliable way of assessing drug-

excipients interaction in pharmaceutical formulations [236].

FT-IR spectrum of unprocessed RTM offered peaks at 2968 cm-1 (C–H

stretching vibration of alkane) and 1726 cm-1 corresponding to carbonyl stretching of

the lactone ring containing more than six carbon atoms. These characteristic peaks

and others were also observed in RFSi-4 nanoformulation (Figure 4.6).

Whereas, the major peaks of C=C stretch at 1639 cm-1, SO2 stretch peak at 1147

cm-1, C-H bend at 1284 cm-1, C=S stretch at 1146 and N-H bend at 984 cm-1 were

present in both unprocessed FTD as well as in FFSi-4 nanoformulation (Figure 4.7)

This clearly indicated that the unprocessed samples and their respective

prepared loaded SLNs have similar chemical structure. Thus, no interaction of RTM

as well as FTD and excipients was proved by FTIR spectra of unprocessed drugs and

processed nanoformulations. This analysis exposed that the formation of a new

complex has not been observed among the formulation components, which confirm

the compatibility of the drugs with the formulation components. Thus, on the basis of

FT-IR analysis, representing no chemical interactions, the prepared loaded

nanoparticles can be further processed to ahieve the desired boosted oral

bioavailability results.

96


Figure 4.6: FT-IR Spectra of (A) unprocessed RTM (B) RFSi-4 Nanoformulation

Figure 4.7: FT-IR Spectra of (A) unprocessed FTD (B) FFSi-4 Nanoformulation

4.1.4 POWDERED X-RAY DIFFRACTOMETRY (P-XRD)

According to Powdered X-Ray Diffraction patterns, unprocessed RTM and FTD

showed enormous number of sharp peaks as well as possesing larger peak counts at

high intensity, while some of these peaks were disappeared and reduced in the

diffractograms of RFSi-4 and FFSi-4 nanoformulations (Figure 4.8 & Figure 4.9).

Disappearance and reduction in intensities of the peaks in the diffractograms of

RFSi-4 and FFSi-4 nanoformulations is indicative for reduction in the crystalline

nature [232, 237]. Reduction in the crystalline nature to semi-crystalline form or

97


conversion to amorphous form favors increased solubility which in-turn boosted the

the oral bioavailability [238]. Semi-crystalline and amorphous drugs have greater free

energy compared to crystalline form, so, easily solubilized favoring enhanced oral

bioavailability [239-242]. Thus, modification in the crystalline nature via nano-sizing

approach being confirmed by P-XRD studies is highly appreciated and reported in

literature [242].

Figure 4.8: P-XRD of unprocessed RTM and RFSi-4 nanoformulation

Figure 4.9: P-XRD of unprocessed FTD and FFSi-4 nanoformulation

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4.1.5 DIFFERENTIAL SCANNING CALORIMETRY (DSC)

DSC thermograms for unprocessed drugs, processed nanoformulations and

formulation components were recorded separately. Their respective melting points are

shown in Table 4.1 & Table 4.2.

Sharp melting point peak appeared on 128°C for the unprocessed RTM which

disappeared for the RFSi-4 nanoformulation at this scale. However for RFSi-4

nanoformulation, a small diffused peak appeared at lower scale of 122.1°C.

Sharp melting point peak appeared on 166.9°C for the unprocessed FTD

which disappeared for the FFH-4 nanoformulation at this scale. However for FFH-4

nanoformulation, a small diffused peak appeared at lower scale of 161°C.

The mentioned results for both drugs indicating reduction in particles size,

increased surface area as well as closed contact of solid lipid (stearic acid) with the

drugs. This change could be considered as a proof for the reduction in the crystallinity

of nanoformulations. The mentioned results also showed the dispersion of the drugs in

lipid layers as the level of melting point lowered along with fading of the peaks of

other formulation components.

In the literature of SLNs, the shifting of the melting point peak of drugs to the

decreased level has been previously reported [243, 244].

S. No Sample Melting peak1 Roxithromycin (RTM) 128°C2 Stearic Acid (SA) 69°C3 Physical Mixture (SA & RTM) 68.6°C & 128.5°C 4 RFSi-4 Nanoformulation 122.1°C

Table 4.1: Melting point of RTM, Stearic Acid and RFSi-4 nanoformulation

99


Table 4.2: Melting point of FTD, Stearic Acid and FFSi-4 nanoformulationS. No Sample Melting peak

1 Famotidine (FTD) 166.9°C2 Stearic Acid (SA) 69°C3 Physical Mixture (SA & FTD) 68.6°C & 166.5°C 4 FFSi-4 Nanoformulation 161°C

4.1.6 ENTRAPMENT EFFICIENCY AND DRUG LOADING

CAPACITY

Based on drug-lipid ratio, different five nanoformulations of SLNs loaded with RTM

and FTD were fabricated and evaluated for Percent Entrapment Efficiency (EE%) and

Percent Drug Loading Capacity (DLC%).

During this study, it was found that by increasing the quantity of RTM from

40 mg to 50 mg, 66.6 mg and 100 mg, EE% slightly decreased from 97% to 95%,

94% and 84% respectively. While further increased in the quantity of RTM (100 mg

to 200 mg) caused quick fall in EE% i.e. upto 57%. Therefore, RFSi-4

nanoformulation having EE% 84% and DLC% 2.709% was optimized (Figure 4.10).

In case of FTD, FFSi-1 nanoformulation having only 40 mg of FTD, showed

entrapment efficiency (EE) 96±1.4% and drug loading capacity (DLC) 1.26±0.14%.

While FFSi-5 nanoformulation having 200 mg of FTD showed reduced EE

55±2.31%. The optimized FFSi-4 nanoformulation having 100 mg of FTD, showed

EE 85±2.7% and DLC 2.74±0.33% (Figure 4.11).

In both cases of RTM and FTD, it was found that by increasing the quantity of

drugs from 40 mg to 100 mg, EE% decreased upto some extent only. While further

increased in the quantity of drugs from 100 mg to 200 mg, causes sudden fall in EE%.

This sudden fall might be due to loading of drugs beyond saturation level of lipid

[83]. Lipophilic drugs can gain super-saturation in melted lipids; on cooling, this

100


saturation level of lipophilic drugs reduces and excessive quantity tends to partition in

outer shell or external solvent [89].

The combination and specified concentrations of RTM (100 mg) and stearic

acid (1.00 g) were found effective to demonstrate maximum encapsulation of the

drug. There has been reported in literature that in polymer and lipid based nano-

particulate drug delivery systems, high binding energy of the drugs with the polymers

and lipids is required for the successfull encapsulation drugs in polymers as well as

lipid layers [245]. In the reported work, maximum entrapment efficacy and drug

loading capacity can be credited to the higher binding energy of the drugs with stearic

acid [245].

Figure 4.10: EE% and DLC% of RTM-SLNs formulations (SI)

Figure 4.11: EE (%) and DLC (%) of FTD-SLNs formulations

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4.1.7 IN-VITRO STUDY

In-vitro release of drugs from SLNs can be altered by appropriate selection of the

lipid type, surfactant/co-surfactant concentrations as well as fabrication variables

[246].

This study revealed that RTM loaded SLNs formulations initially showed

maximum (burst) release. Almost 13% to 28% of the total RTM was released during

the first hour followed by sustained release. RFSi-1 nanoformulation containing 40

mg of RTM, released almost 99% of RTM in 12 hours. Whereas, RFSi-5

nanoformulation containing 200 mg of RTM, released only 77.87% of RTM in 12

hours.

While in case of FTD, FFSi-1 nanoformulation containing 40 mg of FTD, also

released almost 99% of FTD in 12 hours. Whereas, FFSi-5 nanoformulation

containing 200 mg of FTD, released only 75.87% of FTD in 12 hours.

This clearly indicated that when drug pay-load increased, commulative percent

drug release decreased and vice versa. Thus, it is concluded that increased payload of

drugs resulted in prolonged drug release time [83].

Figure 4.12: Drug Release from RTM-SLNs Formulations

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Figure 4.13: Drug Release from different FTD-SLN formulations

4.1.8 KINETIC MODELING

Kinetic modeling study was conducted for the produced nanoformulations to predict

the rate and mechanism of drug release. In-vitro drug release data being putted in

mathematical kinetic models, exposed that it best fitted into zero-order kinetic model

(i.e. drug release from SLNs is not dependent on the amount of drug still existing in

SLNs) with R2 values in the range of 0.927-0.992 for RTM and 0.934-0.994 for FTD

(Table 4.3 & Table 4.4) [247].

Korsmeyer-Peppas model presented drug release in very appropriate way. In

this model, the value of n (release exponent) was exceeding 0.5 (n˃0.5). This

confirmed non-Fickian diffusion kinetics (anomalous transport), i.e. the drug release

followed both erosion/dissolution of the lipid matrix as well as diffusion of the drug

from SLNs [248, 249]. This showed that the release mechanism of druds from SLNs

has been changed to anomalous transport (non-Fickian diffusion kinetics) from

diffusion-controlled. In non-Fickian diffusion kinetics, both erosion/dissolution as

well as diffusion is controlling drugs release from SLNs.

103


Table 4.3: R2 value of different kinetic models for RTM-SLNs formulations

Formulation Zero order (R2)

First order (R2)

Higuchi model(R2)

Korsmeyer-Peppas model

Release exponent (n)

(R2)

RFSi-1 0.927 0.402 0.595 0.6522831 0.947RFSi-2 0.951 0.445 0.690 0.7125320 0.948RFSi-3 0.967 0.460 0.482 0.7709249 0.941RFSi-4 0.984 0.367 0.367 0.8417757 0.935RFSi-5 0.992 0.455 0.546 0.9094167 0.909


First order (R2)

Higuchi model (R2)


Release exponent(n)

(R2)

FFSi-1 0.934 0.405 0.493 0.67783598 0.961FFSi-2 0.953 0.348 0.329 0.71886479 0.950FFSi-3 0.970 0.566 0.478 0.77771697 0.941FFSi-4 0.985 0.448 0.563 0.84339715 0.920FFSi-5 0.994 0.343 0.428 0.97661015 0.895Table 4.4: R2 value of different kinetic models for FTD-SLNs formulations

4.1.9 STABILITY STUDY

The optimized drug loaded nanoformulations were selected for stability studies for the

period of three months.

In terms of selecting the best drug loaded SLNs formulation; RFSi-4 and

FFSi-4 nanoformulations were selected and their stability was evaluated for the period

of 90 days. Each sample was divided and stored in two plain glass vials at both room

and refregerated temperature. Samples stored at 5±2°C showed no significant

particles growth. As in case of RTM, the particles size increased from 169.6±2.3 nm

to 172.4±2.1 nm only, while in case of FTD the particles size increased from 162.7

nm to 173.9 nm (Table 4.5 & Table 4.6). However, at 25±3°C, some rapid growth

was observed for the initial 30 days which was because of the amorphous nature of

the drug loaded SLNs followed by stabilization for rest of the period. This might be

104


attributed to the dissolution of the small particles while depositing onto the surface of

the large particles which is common in amorphous particles [232, 237]. Additionally,

at room temperature, amorphous solids have increased free energy due to which

chemical and physical stability is decreased [250, 251].

For RTM, two-tailed t-test for particle size and PDI showed p-value of 0.047

and 0.024 respectively.

For FTD, two tailed t-test conducted for particle size and PDI showed p-value

of 0.049 and 0.033 respectively.

Day Size (nm)5±2OC

Size (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 169.6 169.6 0.462 0.46215th 171.1 179.3 0.463 0.52230th 172.9 181.6 0.472 0.59160th 173.3 187.1 0.482 0.61390th 175.1 195.2 0.485 0.655

Mean 172.4 182.56 0.4728 0.5686SD 2.114 9.485 0.010 0.076

P-Value 0.047 0.024Table 4.5: Stability study of RTM Loaded SLNs (RFSi-4 nanoformulation)

Day Size (nm)5±2OC

Size (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 162.7 162.7 0.352 0.35215th 169.4 176.2 0.357 0.39930th 171.1 179.7 0.374 0.47960th 172.2 185.1 0.378 0.48290th 173.9 185.6 0.381 0.489

Mean 170.86 178.86 0.3684 0.4402SD 2.408 7.360 0.013 0.061

P-Value 0.049 0.033Table 4.6: Stability study of FTD Loaded SLNs (FFSi-4 nanoformulation)

105


4.1.10 COMPARATIVE IN-VIVO STUDY OF PREPARED NANO-

SUSPENSION WITH MARKETED PRODUCT

The graph of drug plasma concentration vs time for prepared nanoformulations and

marketed product is envisioned in Figure 4.14 & Figure 4.15. Numerous parameters

of pharmacokinetics are also shown in Table 4.7 & Table 4.8. Drug blood plasma

concentration was significantly increased in rabbits orally administered with

nanoformulations compared to marketed product.

In case of RTM, Cmax (Peak blood plasma concentration) for marketed product

(Roxyrex® 150 mg, Biorex Pharmaceuticals, (PVT) LTD, Pakistan) and processed

RFSi-4 nanoformulation was 2.11±0.14 μg.ml-1 and 4.84±0.105 μg.ml-1 respectively.

For marketed product and RFSi-4 nanoformulation, AUC0→24 was 1.11 μg.hr.ml-1 and

23.50 μg.hr.ml-1 respectively. As compared to marketed product, RFSi-4

nanoformulation showed increase of 2.29-fold in Cmax and 21.17-fold in AUC0→24.

In case of FTD, Cmax (Peak blood plasma concentration) for marketed product

(Kamcid® 20 mg, Bloom Pharmaceuticals, (PVT) LTD, Pakistan) and processed

FFSi-4 nanoformulation was 0.498±0.14 μg.ml-1 and 1.03±0.204 μg.ml-1 respectively.

For marketed product and FFSi-4 nanoformulation, AUC0→24 was 4.39 μg.hr.ml-1 and

31.81 μg.hr.ml-1 respectively. As compared to marketed product, FFSi-4


Interesting results obtained from statistically analyzed data of in-vivo

pharmacokinetics, confirmed boosted oral bioavailability with sustained release

profile of prepared nanoformulation compared to marketed product. SLNs as drug

delivery system open angles to formulate available drugs (BCS-II and BCS-IV) in the

market for to boost their oral bioavailability and attain sustained release behavior.

SLNs are not only responsible for improvement of oral absorption but can

106


correspondingly be formulated for parenteral administration, which needs additional

studies [252].

Parameters RFSi-4 Formulation Marketed Product (Roxyrex®)Cmax (μg ml−1) 4.84±0.105 2.11±0.14Tmax (h) 12±0.2 2±0.3AUC (μg.hr.ml−1) 23.50 1.11Relative Bioavailability (Fr)

21.17

Table 4.7: Comparative in-vivo pharmacokinetic parameters of RTM (±SD, n=6)

Table 4.8: Comparative in-vivo pharmacokinetic parameters of FTDParameters FFSi-4 Formulation Marketed Product (Kamcid®)

Cmax (μg ml−1) 1.03±0.204 0.498±0.14Tmax (h) 12±0.2 2±0.3AUC (μg.hr.ml−1) 31.81±0.003 4.39±0.21Relative Bioavailability (Fr)

7.24

(±SD, n=6)

Figure 4.14: Comparative in-vivo drug release study of RTM

107


Figure 4.15: Comparative in-vivo drug release study of FTD

108


4.2 SOLVENT EMULSIFICATION EVAPORATION METHOD4.2.1 PARTICLE SIZE ANALYSIS

i. Particle Size Analysis of Blank SLNs

Prior to fabrication of drug loaded SLNs, blank SLNs were prepared by Solvent

Emulsification Evaporation method. During fabrication process, stearic acid was used

as solid lipid, Tween® 80 as surfactant and PVP as co-surfactant. Three different

variable factors were considered for optimization purpose i.e. concentration of

Tween® 80, concentration of PVP and stirring time. By changing these three

variables, twelve SLNs formulations with different particle sizes and PDI were

fabricated.

During blank SLNs fabrication, size reduction was achieved by increasing

Tween® 80 concentration. By increasing Tween® 80 concentration from 0.5 g to 2 g,

particles size reduced from 210.5 nm to 190.8 nm. As, higher concentration of

surfactant (Tween® 80) showed lower particle size and better stability to small lipid

droplets which prevent them from coalescence [81]. However, increase in

concentration of Tween® 80 beyond 2 g, did not reduce the particle size. Further

reduction in particle size occurred because of Polyvinylpyrrolidine addition, being

employed as co-surfactant. As, SLNs fabricated with surfactant/co-surfactant mixture

have lower particle size and better stability as compared to SLNs of unadded co-

surfactants. That’s why PVP (co-surfactant) was used in combination of Tween® 80,

which further reduced particle size from 190.8 nm to 143.4 nm. Further increase in

concentration of surfactant and co-surfactant was avoided to prevent decrease in the

entrapment efficiency and also toxic effects associated with them. Moreover, PDI was

controlled and reduced by stirring time as it has almost no effect or little bit effect on

particle size reduction but reduces the PDI [82]. PDI was reduced from 0.679 to 0.485

by increasing magnetic stirring time from 5 min to 20 min.

109


During optimization process of blank SLNs, BFSe-11 nanoformulation was

considered best in terms of desired particle size and PDI. The optimized BFSe-11

nanoformulation was considered for drug loading. The optimized BFSe-11

nanoformulation showed best results of particle size 127.8±2.3 nm and PDI

0.485±0.001.

Twelve different blank SLNs formulations with their respective particle size

and PDI are shown below in Figure 4.16.

Figure 4.16: Particle size and PDI of blank SLNs formulations


Optimized blank nanoformulation fabricated by Solvent Emulsification Evaporation

method was BFSe-11 being designated for drug loading. Optimized blank BFSe-11

nanoformulation showed average particle size 127.8±2.3 nm and PDI 0.485±0.001.

After drugs loading, RFSe-4 and FFSe-4 nanoformulations were optimized for

Roxithromycin and Famotidine respectively. The optimized RFSe-4 and FFSe-4

nanoformulations showed average particle size 126.27±2.1 nm & 111.9±1.3 nm, PDI

0.435±0.01 & 0.464±0.03, zeta potential -36.72±2 mV & -33.46±2 mV respectively

(Figure 4.17 & Figure 4.18).

110


In the results, it is clearly shown that the particle size of unloaded SLNs

formulation has been reduced after drugs loading. This reult of reduction in particle

size with drug pay-load is being supported by literature, saying that after drug pay

load, particle size reduced due to decrease in free lipid content [231].

The PDI <0.5 and ZP in the range of ±30 revealed that the fabricated nano-

dispersions would be stable in nature [232]. For our prepared nanoformulations both

PDI and ZP were within the acceptable range, which exhibit electrostatic stabilization

to avoid aggregation thus preventing particles growth and Ostwald ripening [233].

The first aim of this technique was to fabricate drugs loaded SLNs of desired

size at room temperature using stearic acid as solid lipid. The greatest advantage of

this technique is the easily production of drugs loaded SLNs without employing

sophisticated instruments. Tween® 80 was employed as surfactant and PVP as co-

surfactant, avoiding droplets of emulsion from becoming closed to each other and

thus preventing coalescence and flocculation. As the fabricated loaded SLNs are for

oral administration, so, great attention has been focused on getting the desired particle

size. The prepared average particles size is less than 400 nm having the ability of

easily crossing the linings of the gastro-intestinal cells for to achieve the desired

boosted oral bioavailability [234]. Moreover, the fabricated particles comprised of

100-200 nm size range, since particle having size less than 200 nm is undetectable to

the Reticulo-Endothelial System (RES) and remain in circulatory system for a

prolonged time period [235].

111


Figure 4.17: Particle size (A) and Zeta Potential (B) of RFSe-4 nanoformulation

Figure 4.18: Particle size (A) and Zeta Potential (B) of FFSe-4 nanoformulation


Scanning Electron Microscopy (SEM) was done to study the texture and surface

morphology of drug loaded SLNs.

The surface morphology of RFSe-4 and FFSe-4 nanoformulations was studied

by SEM. The white patches in the micrographs showed the formation of solid and

fairly spherical shaped nanoparticles with a well-defined periphery. The particles size

was also in nano-metric range. Most of SLNs were identical in shape and present in

dispersed form (Figure 4.19 & Figure 4.20).

SEM being a key characterization step in the field of pharmaceutical nano-

engineering, represented nanometric size particles which further confirmed the results

of zeta sizer analysis. Furthermore, the amorphous nature of the nanoparticles was

resulted from the blunt and non spiky white patches in the micrographs. This

112


amorphous nature of the drug loaded nanoparticles, plays a vital role in the solubility

enhancement of the drugs, being a successful outcome of pharmaceutical nano-

engineering.

Figure 4.19: SEM micrographs of RFSe-4 nanoformulation

Figure 4.20: SEM micrograph of FFSe-4 nanoformulation

4.2.3 FOURIER TRANSFORM INFRARED MICROSCOPY

(FT-IR)


excipients interaction in pharmaceutical formulations [236].

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FT-IR spectrum of unprocessed RTM offered peak at 1726 cm-1 corresponding

to carbonyl stretching of the lactone ring containing more than six carbon atoms. This

characteristic peak and others were also observed in RFSe-4 nanoformulation (Figure

4.21).

The major peaks of C=C stretch at 1639 cm-1, SO2 stretch peak at 1147 cm-1, C-

H bend at 1284 cm-1, C=S stretch at 1146 and N-H bend at 984 cm-1 were present in

both unprocessed FTD as well as in FFSe-4 nanoformulation (Figure 4.22).

This clearly indicated that the prepared loaded SLNs of RTM and FTD, being

prepared by Solvent Emulsification Evaporation technique have similar chemical

structure to their respective unprocessed drugs. Thus, no interaction of RTM as well

as FTD with the excipients was proved by FTIR spectra. The formation of a new

complex among the formulation components has not been reported by the FT-IR

studies, which confirm the compatibility of the selected drugs with the formulation

components. Thus, on the basis of FT-IR analysis, representing no chemical

interactions, the prepared loaded nanoparticles can be further processed to ahieve the

desired results of boosted oral bioavailability.

Figure 4.21: FT-IR Spectra of (A) unprocessed RTM (B) RFSe-4 nanoformulation

114


Figure 4.22: FT-IR Spectra of (A) unprocessed FTD (B) FFSe-4 nanoformulation


Powdered X-Ray Diffractometric (P-XRD) analysis is used to study the formation of

new solid-state in the processed samples [97].

Diffractograms of unprocessed RTM and FTD showed a series of sharp peaks

indicating their crystalline nature. While in diffractograms of RFSe-4 and FFSe-4

nanoformulations, most of these peaks were suppressed but few minor one

disappeared, indicating reduction in crystalline nature (Figure 4.23 & Figure 4.24)

[232, 237].

Disappearance and reduction in intensities of the peaks in the diffractograms

of RFSe-4 and FFSe-4 nanoformulations is indicative for reduction in the crystalline

nature, which favors increased solubility which in-turn boosted the the oral

bioavailability [238]. Semi-crystalline and amorphous drugs have greater free energy

compared to crystalline form, so, easily solubilized favoring enhanced oral

bioavailability [239-242]. Thus, modification in the crystalline nature via nano-sizing

approach being confirmed by P-XRD studies is highly appreciated and reported in

literature [242].

115


Figure 4.23: Diffractogram of unprocessed RTM and RFSe-4 nanoformulation

Figure 4.24: Diffractogram of unprocessed FTD and FFSe-4 nanoformulation


DSC thermograms of unprocessed drugs (RTM & FTD), stearic acid (SA), physical

mixture and prepared nanoformulations (RFSe-4 & FFSe-4) were recorded separately

(Table 4.9 & Table 4.10).

Sharp melting point peak appeared on 128°C for the unprocessed RTM which

disappeared for the RFSe-4 nanoformulation at this scale. However for RFSe-4

nanoformulation, the appearance of a small diffused peak at lower scale of 122.4°C

indicating reduction in particles size, increased surface area as well as closed contact

of stearic acid with drug RTM. The same case was with FTD, as for unprocessed FTD 116


the melting point appeared at 166.9°C which was lowered to 160°C for FFSe-4

nanoformulation.

Thus, this change could be considered as a proof for the reduction in the

crystallinity of prepared nanoformulations, which approved the enhanced solubility of

the hydrophobic drugs. The mentioned result also showed the dispersion of the drugs

in the lipid layers as the level of melting point of drugs lowered with fading of the

peaks of other formulation components.

In the literature of SLNs, the shifting of the melting point peak of drugs to the

decreased level has been previously reported [243, 244].

S. No Sample Melting peak1 Roxithromycin (RTM) 128°C2 Stearic Acid (SA) 69°C3 Physical Mixture (SA & RTM) 68.6°C & 128.5°C 4 RFSe-4 Nanoformulation 122.4°C

Table 4.9: Melting point of RTM, Stearic Acid and RFSe-4 nanoformulation

S. No Sample Melting peak1. Famotidine (FTD) 166.9°C2. Stearic Acid (SA) 69°C3. Physical Mixture (SA & FTD) 68.6°C & 166.5°C 4. FFSe-4 Nanoformulation 160°C

Table 4.10: Melting point of FTD, Stearic Acid and FFSe-4 nanoformulation

4.2.6 ENTRAPMENT EFFICIENCY AND DRUG LOADING

CAPACITY

Based on drug-lipid ratio, five nanoformulations of drug loaded SLNs were fabricated

followed by characterization for percent entrapment efficiency (EE%) and drug

loading capacity (DLC%).

RFSe-1 nanoformulation having only 40 mg of RTM, showed entrapment

efficiency (EE) 94±2.9% and drug loading capacity (DLC) 1.236±0.03%. While

117


FFSe-5 nanoformulation having 200 mg of RTM showed reduced EE 51±2.03%. The

optimized RFSe-4 nanoformulation having 100 mg of RTM, showed EE 83.61±2.3%

and DLC 2.677±0.13% (Figure 4.25).

FFSe-1 nanoformulation having only 40 mg of FTD, showed entrapment

efficiency (EE) 96±2.9% and drug loading capacity (DLC) 1.263±0.13%. While

FFSe-5 nanoformulation having 200 mg of FTD showed reduced EE 59±3.17%. The

optimized FFSe-4 nanoformulation having 100 mg of FTD, showed EE 84±2.7% and

DLC 2.709±0.13% (Figure 4.26).

During this study, it was found that by increasing the quantity of drug from 40

mg to 100 mg, EE% decreased gradually. While further increased in the quantity of

drug from 100 mg to 200 mg, causes sudden fall in EE%. This sudden fall in EE%

may be because of drug loading beyond saturation level of lipid [83]. Lipophilic drugs

can gain super-saturation in melted lipids; on cooling, this saturation level of

lipophilic drugs reduces and excessive quantity tends to partition in outer shell or

external solvent [89].

The combination and specified concentrations of drug (100 mg) and stearic acid

(1.00 g) were found effective to demonstrate maximum encapsulation of the drug.

There has been reported in literature that in polymer and lipid based nano-particulate

drug delivery systems, high binding energy of the drugs with the polymers and lipids

is required for the successfull encapsulation of drugs in polymers as well as lipid

layers [245]. In the reported work, maximum entrapment efficacy and drug loading

capacity can be credited to the high binding energy of the drugs with stearic acid.

118


Figure 4.25: EE (%) and DLC (%) of RTM-SLNs formulations

Figure 4.26: EE (%) and DLC (%) of FTD-SLNs formulations


During in-vitro drug release investigations, cumulative percent release of RTM from

RFSe-1 to RFSe-5 nanoformulations were 98.43%, 94.55%, 89.37%, 79.34% and

71.35% respectively (Figure 4.27).

During 12 hrs in-vitro drug release study of FTD, cumulative percent drug

release from FFSe-1 to FFSe-5 nanoformulations were 99.21%, 94.12%, 88.31%,

78.87% and 71.94% respectively (Figure 4.28).

119


Nanoformulation of RTM & FTD containing 40 mg of drug showed

commulative percent drug release greater then 98% in 12 hours. Whereas,

nanoformulations containing 200 mg of drug, released almost 71-72 % of drug only in

12 hours.

This clearly indicated that when drug pay-load increased from 40 mg to 200

mg, commulative percent drug release decreased almost 30% in 12 hours study. Thus,

it is shown that increased drug payload caused prolonged drug release time [83].

Figure 4.27: Drug Release from RTM-SLNs formulations

Figure 4.28: Drug Release from FTD-SLNs formulations

120



In-vitro drug release data being putted in mathematical kinetic models, exposed that it

best fitted into zero-order model (i.e. drug release from SLNs is not dependent on the

amount of drug still existing in SLNs) with R2 values in the range of 0.958-0.996 for

RTM and 0.958-0.993 for FTD (Table 4.11 & 4.12) [247].

Korsmeyer-Peppas model presented drug release in very appropriate way. In

this model, the value of n (release exponent) was exceeding 0.5 (n˃0.5). This

confirmed non-Fickian diffusion kinetics (anomalous transport), i.e. the drug release

followed both erosion/dissolutionof the lipid matrix as well as diffusion of the drug

from SLNs [248, 249]. This showed that the release mechanism of drug from SLNs

has been changed to non-Fickian diffusion kinetics (anomalous transport) from

diffusion-controlled. In non-Fickian diffusion kinetics, both erosion/dissolution and

diffusion is controlling the drug release.


First order (R2)

Higuchi model(R2)



(R2)

RFSe-1 0.958 0.412 0.469 0.80696337 0.978RFSe-2 0.974 0.356 0.554 0.90638848 0.963RFSe-3 0.981 0.472 0.257 0.88271668 0.962RFSe-4 0.994 0.577 0.545 0.97277989 0.943RFSe-5 0.996 0.282 0.441 0.97109871 0.935Table 4.11: R2 value of different kinetic models for RTM-SLNs formulations


First order (R2)

Higuchi model (R2)


Release exponent(n)

(R2)

FFSe-1 0.958 0.866 0.566 0.82067309 0.978FFSe-2 0.973 0.460 0.455 0.89488353 0.965FFSe-3 0.981 0.376 0.656 0.89479258 0.962FFSe-4 0.991 0.485 0.350 0.93489612 0.947FFSe-5 0.993 0.389 0.449 0.94189608 0.940

121


Table 4.12: R2 value of different kinetic models for FTD-SLNs formulations


The selected RFSe-4 & FFSe-4 nanoformulations showed no significant change in

particle size and PDI stored at refrigerated temperature (Table 4.13 & Table 4.14). At

refrigerated temperature, increase in particle size was less than 5% but at room

temperature it was greater than 12%. At both temperatures, the particles growth was

in acceptable range but was little bit more at room temperature. This might be

attributed that at room temperature, amorphous solids have increased free energy

which resulted in decreased physical and chemical stability [250, 251]. Additionally,

dissolution of the small particles while depositing onto the surface of large particles is

common in amorphous particles [232, 237].

For RTM, two-tailed t-test for particle size and PDI showed p-value of 0.047


For FTD, two-tailed t-test for particle size and PDI showed p-value of 0.044 and

0.046 respectively.

Day Size (nm)5±2OC

Size (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 126 126 0.435 0.43515th 129.5 140.3 0.453 0.57530th 131.5 144.6 0.462 0.58760th 133.8 147.7 0.476 0.65390th 134.7 149.8 0.479 0.678

Mean 131.1 141.68 0.461 0.5856SD 3.499 9.466 0.017 0.094

P-Value 0.047 0.020Table 4.13: Stability study of RTM Loaded SLNs (RFSe-4 nanoformulation)

122


Day Size (nm)5±2OC

Size (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 111.9 111.9 0.464 0.46415th 113.6 124.2 0.465 0.48430th 116.3 135.7 0.474 0.56560th 119.5 137.1 0.482 0.57890th 121.9 139.6 0.488 0.586

Mean 116.64 129.7 0.4746 0.5354SD 4.113 11.574 0.010 0.056

P-Value 0.044 0.046Table 4.14: Stability study of FTD Loaded SLNs (FFSe-4 nanoformulation)

4.2.10 COMPARATIVE IN-VIVO STUDY OF PREPARED NANO-


Comarative in-vivo study has been conducted using rabbits as animal model. Drug

plasma concentration was significantly higher in rabbits treated with

nanoformulations compared to those treated with Marketed product.

The graph of plasma concentration vs time for prepared nanoformulations and

marketed products is envisioned in Figure 4.29 & Figure 4.30. Numerous parameters

of pharmacokinetics are also shown in Table 4.15 & Table 4.16. Drug plasma

concentration was significantly increased in rabbits orally administered with prepared

nanoformulations compared to marketed products.

In case of RTM, Cmax (Peak blood plasma concentration) for marketed product

(Roxyrex® 150 mg, Biorex Pharmaceuticals, (PVT) LTD, Pakistan) and processed

RFSe-4 nanoformulation was 2.11±0.14 μg.ml-1 and 4.66±0.204 μg.ml-1 respectively.

For marketed product and RFSe-4 nanoformulation, AUC0→24 was 1.11 μg.hr.ml-1 and

24.28 μg.hr.ml-1 respectively. As compared to marketed product, RFSe-4


123


In case of FTD, Cmax (Peak blood plasma concentration) for marketed product

(Kamcid® 20 mg, Bloom Pharmaceuticals, (PVT) LTD, Pakistan) and processed

FFSe-4 nanoformulation was 0.498±0.14 μg.ml-1 and 1.03±0.204 μg.ml-1respectively.

For marketed product and FFSe-4 nanoformulation, AUC0→24 was 4.39 μg.hr.ml-1 and

23.122 μg.hr.ml-1 respectively. As compared to marketed product, FFSe-4


Interesting results obtained from statistically analyzed data of in-vivo


profile of prepared nanoformulations compared to marketed products. SLNs as drug





studies [252].

Table 4.15: Comparative in-vivo pharmacokinetic parameters of RTMParameters RFSe-4 Formulation Marketed Product (Roxyrex®)Cmax (μg ml−1) 4.66±0.204 2.11±0.14Tmax (h) 12±0.2 2±0.3AUC (μg.hr.ml−1) 24.28 1.11Relative Bioavailability (Fr)

21.21

(±SD, n=6)

Table 4.16: Comparative in-vivo pharmacokinetic parameters of FTDParameters FFSe-4 Formulation Marketed Product (Kamcid®)

Cmax (μg ml−1) 1.03±0.204 0.498±0.14Tmax (h) 12±0.2 2±0.3AUC (μg.hr.ml−1) 23.122±0.003 4.39±0.21Relative Bioavailability (Fr)

5.25

(±SD, n=6)

124


Figure 4.29: Comparative in-vivo drug release study of RTM

Figure 4.30: Comparative in-vivo drug release study of FTD

125


4.3 HOT MELT ENCAPSULATION METHOD4.3.1 PARTICLE SIZE ANALYSIS

i. Particle Size Analysis of blank SLNs

Blank SLNs prepared by Hot Melt Encapsulation method was the most suitable

technique as it avoids the use of organic solvent. The nano-suspension prepared via

this technique was further converted into solid dosage form (capsules) followed by

conducting in-vivo study. Blank SLNs were optimized using following three variable

factors.

a. Concentration of Surfactant

During Hot Melt Encapsulation method, Tween® 80 was employed as surfactant. The

concentration of Tween® 80 was increased from 0.5-2 g, which caused reduction in

particles size upto 300.2±3.1. As, higher concentration of surfactant showed lower

particle size and better stability to small lipid droplets which prevent them from

coalescence [81]. Further increase in Tween® 80 concentration showed almost no

effect on particle size reduction.

b. Concentration of co-surfactant

Further decrease in particle size was achieved with addition of co-surfactant.

Polyethylene glycol (PEG) being employed as co-surfactant in the concentration

range of 0.1-0.5 ml, further reduced particle size upto 203.8±2.5. As, SLNs fabricated

with surfactant/co-surfactant mixture have lower particle size and better stability as

compared to SLNs of unadded co-surfactants.

c. Stirring Time

By increasing the magnetic stirring time from 5 to 15 min, PDI reduced to the

acceptable range. During increase in the magnetic stirring time, it has been noticed

that particle size also reduced to some extent but it mainly controlled the PDI. Thus,

126


PDI was controlled and reduced by increasing stirring time which has shown almost

little bit effect on particle size reduction [82].

Important variations in terms of particle size and PDI were noticed by changing

the mentioned three variable parameters, which are envisioned in Figure 4.31.

During optimization process of blank SLNs, desired particles size and

acceptable PDI were produced via stearic acid (1 g), Tween® 80 (1.6 ml), PEG (0.4

g) and magnetic stirring time (15 minutes). During optimization process of blank

SLNs, BFSe-11 nanoformulation was considered best in terms of desired particle size

and PDI. The optimized BFH-11 nanoformulation showed best results of particle size

182.7±3.2 nm and PDI 0.481±0.03.

Figure 4.31: Particle size and PDI of unloaded SLNs (HME)


The optimized conditions of BFH-11 nanoformulation (blank) i.e. stearic acid (1 g),

Tween® 80 (1.6 ml), PEG (0.4 g) and magnetic stirring time (15 minutes), were

further used for drug loading. Different five nanoformulations of RTM (RFH-1 to

RFH-5) and FTD (FFH-1 to FFH-5) were fabricated, based on drug lipid ratio.

Among these five nanoformulations, the RFH-4 and FFH-4 nanoformulations were

optimized.

127


The optimized RFH-4 and FFH-4 nanoformulations showed particle size

size179.7±2.3 nm & 174.8±2.1 nm , PDI 0.424±0.03 & 0.419±0.03 , zeta potential -

38.16±1.6 mV & -36.35±2 mV (Figure 4.32 & Figure 4.33).

After drug loading the particle size of loaded SLNs was reduced as compared

to optimized blank SLNs formulation (BFH-11). As after drug pay load, particle size

reduced due to decrease in free lipid content [231]. The PDI <0.5 and ZP in the range

of ±30 revealed that the fabricated nano-dispersions would be stable in nature [232].

For our prepared nanoformulations both PDI and ZP were within the acceptable

range, which exhibit electrostatic stabilization to avoid aggregation thus preventing

particles growth and Ostwald ripening [233].

As the fabricated RTM loaded SLNs are for oral administration, so, the

fabricated average particles size is smaller than 400 nm having the ability to easily

cross the linings of the gastro-intestinal cells for to achieve the desired boosted oral

bioavailability [234]. Moreover, the fabricated particles comprised of 100-200 nm

size range, since particle size less than 200 nm is undetectable to the Reticulo-

Endothelial System (RES) and remain in circulatory system for a prolonged time

period [235].

Figure 4.32: Particle size (A) and Zeta potential (B) of RFH-4 nanoformulation

128


Figure 4.33: Particle size (A) and Zeta potential (B) of FFH-4 nanoformulation


The dried powder of SLNs formulation obtained with tray drying technique

(alternative to lyophilization) was investigated via SEM. SEM micrographs of

samples (tray dried sample and lyophilized sample) showed white patches,

representing SLNs which were almost similar in size and shape. SEM results showed

tray drying technique can also be employed for SLNs drying purpose.

SEM being conducted for RFH-4 and FFH-4 nanodispersions also confirmed

nano-metric size particles. The white patches in the micrographs representing SLNs,

seemed solid, identical and fairly spherical shaped with a well-defined periphery

(Figure 4.34 & Figure 4.35). Most of the SLNs were present in dispersed form with

homogeneous distribution which exhibit amorphous nature of the produced

nanoparticles.

SEM being a key characterization step in the field of pharmaceutical nano-

engineering, represents the texture and surface morphology of of the particles in SLNs

dispersion. SEM represented nanometric size particles which further confirmed the

results of zeta sizer analysis. Furthermore, the amorphous nature of the nanoparticles

was resulted from the blunt and non spiky white patches in the micrographs. This

amorphous nature of the drug loaded nanoparticles, plays a vital role in the solubility

129


enhancement of the drugs, being a successful outcome of pharmaceutical nano-

engineering.

Figure 4.34: SEM micrograph of RFH-4 nanoformulation

Figure 4.35: SEM micrograph of FFH-4 nanoformulation

4.3.3 FOURIER TRANSFORM INFRARED SPECTROSCOPY

(FT-IR)


excipients interaction in pharmaceutical formulations [236]. FTIR spectra of

unprocessed drug and nanoparticles fabricated by HME technique showed

characteristic peaks with numerous intensities, as different functional groups vibrate

at distinct frequencies.

130


FT-IR spectra of unprocessed RTM as well as RFH-4 nanoformulation being

fabricated by HME technique offered peaks at 2968 cm-1 (C–H stretching vibration of

alkane) and 1726 cm-1 corresponding to carbonyl stretching of the lactone ring

containing more than six carbon atoms. These characteristic peaks and also others

were observed as almoost similar (Figure 4.36).

Unprocessed FTD as well as in FFH-4 nanoformulation being fabricated by

HME technique also presented almost similar peaks. The major peaks of C=C stretch

at 1639 cm-1, SO2 stretch peak at 1147 cm-1, C-H bend at 1284 cm-1, C=S stretch at

1146 and N-H bend at 984 cm-1 were present in both unprocessed FTD as well as in

FFH-4 nanoformulation (Figure 4.37).

Thus, FT-IR analysis of both unprocessed drugs as well as prepared

nanoformulations clearly indicated that the unprocessed samples and their respective

prepared loaded SLNs have similar chemical structure. This analysis exposed that the

formation of a new complex has not been observed among the formulation

components.

Figure 4.36: FT-IR Spectra of (A) unprocessed RTM (B) RFH-4 nanoformulation

131


Figure 4.37: FT-IR Spectra of (A) Unprocessed FTD (B) FFH-4 nanoformulation


Powdered X-Ray Diffractometric (P-XRD) analysis is used to study the formation of

new solid-state in the processed samples [97].

Diffractograms of unprocessed RTM and FTD showed diffraction peaks of

sharp and intense nature, which indicated their crystalline nature. While in the

diffractograms of RFH-4 and FFH-4 nanoformulations, most of these diffraction

peaks were suppressed but few minor one disappeared, indicating reduction in

crystalline nature to the semi-crystalline form (Figure 4.38 & Figure 4.39).

In P-XRD analysis, disappearance and reduction in intensities of the peaks in

the diffractograms of prepared nanoformulations is indicative for reduction in its

crystalline nature. This reduction in crystalline nature via nano sizing is an ideal

approach for increasing the solubility which ultimately boosted the oral bioavailability

[232, 237].

132


Figure 4.38: P-XRD of unprocessed RTM and RFH-4 nanoformulation

Figure 4.39: P-XRD of unprocessed FTD and FFH-4 nanoformulation


DSC studies of unprocessed drugs and prepared nanoformulations by HME technique

were conducted to evaluate their respective melting points. DSC thermograms for

unprocessed drugs and prepared nanoformulations were recorded separately (Table

4.17 & Table 4.18).

A sharp endothermic peak was observed at 128.8°C for the unprocessed RTM

which disappeared for the RFH-4 nanoformulation at this scale. However for RFH-4

133


nanoformulation, a small diffused peak appearanced at lower scale of 120.2°C.

Similar case was with FTD, as for unprocessed FTD sharp melting point peak

appeared on 166.9°C which was transferred to the lower scale of 159°C. Thus, this

change could be considered as a proof for the reduction in the crystallinity of prepared

nanoformulations. Additionally, the particle size of the drug as well as the exposed

surface area of the particle has great effect on melting point. Decrease in melting

point peaks of the processed nanoformulations was observed which was due to

particle size, increased exposed surface area and low packing density of the

nanoparticles.

The mentioned result also showed the dispersion of the drugs in lipid layers as

the level of melting point lowered with fading of the peaks of other formulation

components. In the literature of SLNs, the shifting of the melting point peak of drugs

to the decreased level has been previously reported [243, 244].

S. No Sample Melting peak1 Roxithromycin (RTM) 128.8°C2 Stearic Acid (SA) 69°C3 Physical Mixture (SA & RTM) 68.6°C & 128.5°C 4 RFH-4 Nanoformulation 120.2°C

Table 4.17: Melting point of RTM, Stearic Acid and RFH-4 nanoformulation

134


Table 4.18: Melting point of FTD, Stearic Acid and FFH-4 nanoformulation

4.3.1 ENTRAPMENT EFFICIENCY AND DRUG LOADING CAPACITY

Based on drug-lipid ratio, five nanoformulations of drug loaded SLNs were fabricated

followed by characterization for percent entrapment efficiency (EE%) and drug

loading capacity (DLC%).

RFH-1 nanoformulation having only 40 mg of RTM, showed entrapment

efficiency (EE) 98% and drug loading capacity (DLC) 1.289%. While FFSi-5

nanoformulation having 200 mg of RTM showed reduced EE 59%. The optimized

RFH-4 nanoformulation having 100 mg of RTM, showed EE 86% and DLC 2.774%

(Figure 4.40).

FFH-1 nanoformulation having only 40 mg of FTD, showed entrapment

efficiency (EE) 97% and drug loading capacity (DLC) 1.28%. While FFH-5

nanoformulation having 200 mg of FTD showed reduced EE 54%. The optimized

FFH-4 nanoformulation having 100 mg of FTD, showed EE 87% and DLC 2.81%

(Figure 4.41).

During this study, it was found that by increasing the quantity of drugs from

40 mg to 100 mg, EE% decreased gradually. While further increased in the quantity

of drugs from 100 mg to 200 mg, causes sudden fall in EE%. This sudden fall in EE%

may be because of drug loading beyond saturation level of lipid [83]. Lipophilic drugs

can gain super-saturation in melted lipids; on cooling, this saturation level of

lipophilic drugs reduces and excessive quantity tends to partition in outer shell or

external solvent [89].

135

S. No Sample Melting peak1 Famotidine (FTD) 166.9°C2 Stearic Acid (SA) 69°C3 Physical Mixture (SA & FTD) 68.6°C & 166.5°C 4 FFH-4 Nanoformulation 159°C


The combination and specified concentrations of drug (100 mg) and stearic acid

(1.00 g) were found effective to demonstrate maximum encapsulation. There has been

reported in literature that in polymer and lipid based nano-particulate drug delivery

systems, high binding energy of the drugs with the polymers and lipids is required for

the successfull encapsulation of drugs in polymers as well as lipid layers [245]. In the

reported work, maximum entrapment efficacy and drug loading capacity can be

credited to the high binding energy of the drugs with stearic acid.

Figure 4.40: EE% and DLC% of RTM-SLNs formulations

Figure 4.41: EE% and DLC% of FD-SLNs formulations


In-vitro release profile showed that all RTM loaded SLNs nanoformulations exhibited

burst drug release. During first hour, almost 14% to 26% of RTM was released 136


followed by sustained release. RFH-1 nanoformulation containing 40 mg of RTM

showed commulative percent drug release of 98% in 12 hours. Whereas, RFH-5

nanoformulation containing 200 mg of RTM, released only 78% of RTM in 12 hours

(Figure 4.42). This clearly indicated that when RTM pay-load increased from 40 mg

to 200 mg, commulative percent drug release decreased from 99% to 78%.

In case of FTD, cumulative percent drug release from FFH-1 to FFH-5

nanoformulations were 98.97%, 95.98%, 90.88%, 83.73% and 74.76% respectively

during 12 hrs in-vitro drug release study (Figure 4.43). FFH-1 nanoformulation

containing 40 mg of FTD, released almost 99% of FTD in 12 hours. Whereas, FFSe-5

nanoformulation containing 200 mg of FTD, released only 74.76% of FTD in 12

hours. This clearly indicated that when FTD pay-load increased from 40 mg to 200

mg, commulative percent drug release decreased from 99% to 74.76%.

Thus, from in-vitro drug release study, it was concluded that increased drug

payload resulted in prolonged release time [83].

Figure 4.42: Drug release from RTM-SLNs Formulations

137


Figure 4.43: Drug release from FTD-SLNs nanoformulations


By putting the in-vitro drug release data into different mathematical kinetic models, it

was seen that prepared nanoformulations followed Zero order release kinetics with R2

values in the range of 0.946-0.987 for RTM and 0.931-0.989 for FTD (Table 4.19 &

Table 4.20). However, Korsmeyer-Peppas kinetic model presented RTM release from

SLNs in more appropriate way. In this model, the value of n was higher than 0.5,

which confirmed anomalous transport (non-Fickian diffusion kinetics) for the

presented nanoformulations [248, 249].

Table 4.19: R2 value of different kinetic models for RTM-SLNs formulations

Formulation Zero order(R2)

First order(R2)

Higuchi model(R2)


Release exponent(n)

(R2)

RFH-1 0.946 0.682 0.491 0.703665 0.953RFH-2 0.958 0.345 0.383 0.747878 0.948RFH-3 0.970 0.551 0.575 0.803012 0.942RFH-4 0.985 0.459 0.456 0.877147 0.924RFH-5 0.987 0.438 0.343 0.903406 0.898

138


Table 4.20: R2 value of different kinetic models for FTD-SLNs formulations

Formulations Zero order (R2)

First order (R2)

Higuchi model (R2)



(R2)

FFH-1 0.931 0.383 0.594 0.65925794 0.948FFH-2 0.954 0.442 0.489 0.72295143 0.950FFH-3 0.969 0.564 0.480 0.77670613 0.940FFH-4 0.989 0.365 0.361 0.86766069 0.932FFH-5 0.989 0.561 0.448 0.88261066 0.908


The physical stability of the optimized drug loaded nanoformulations was monitered

at two different temperatures for the period of three months. The parameters studied

during stability study were particles size and polydispersity index (PDI). The stability

study of RFH-4 and FFH-4 nanoformulations was carried out at refrigerated and

room temperature. At refrigerated condition, prepared nanoformulations showed

maximim stability as increase in particle size and PDI was in acceptable range. But at

room temperature, increase in particle size was also in the acceptable range, however,

the values of PDI exceed (Table 4.21 & Table 4.22).

For RTM, two tailed t-test for particle size and PDI showed p-value of 0.028


For FTD, two tailed t-test for particle size and PDI showed p-value of 0.027 and

0.026 respectively.

The increase in particle size at room temperature might be attributed to the

phenomenon that amorphous solids have increased free energy which results in

decreased physical and chemical stability [250, 251]. Additionally, dissolution of the

small particles while depositing onto the surface of large particles is common in

amorphous particles [232, 237]. Nanoparticles have high interfacial tension and

increased free energies due to large surface area. Therefore, it is challenging issue to 139


control the particle growth especially in nano-suspensions with subsequent

thermodynamically stabilized system [253, 254]. Some major issues has been reported

with NPs fabricated by bottom-up approach which includes uncontrolled particle

growth. [255].

Day Size (nm)5±2OC

Size (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 179.7 179.7 0.424 0.42415th 181.2 189.1 0.425 0.51230th 182.3 191.9 0.434 0.58960th 183.7 193.1 0.444 0.62190th 184.1 196.3 0.467 0.699

Mean 182.2 190.02 0.4388 0.569SD 1.811 6.320 0.017 0.105

P-Value 0.028 0.025Table 4.21: Stability study of RTM Loaded SLNs (RFH-4 nanoformulation)

Table 4.22: Stability study of FTD Loaded SLNs (FFH-4 nanoformulation)Day Size (nm)

5±2OCSize (nm)25±3OC

PDI5±2OC

PDI25±3OC

1st 174.8 174.8 0.419 0.41915th 175.2 184.2 0.423 0.53730th 176.3 187.7 0.425 0.57160th 179.1 191.1 0.445 0.60190th 179.9 192.6 0.491 0.675

Mean 177.06 186.08 0.4406 0.5606SD 2.311 7.090 0.029 0.094

P-Value 0.027 0.026

4.3.5 COMPARATIVE IN-VIVO STUDY OF SLNs NANO-


Comparative in-vivo study has been conducted using rabbits as animal model. Drug

plasma concentration was significantly higher in rabbits treated with prepared

formulations of nano-suspensions compared to those treated with Marketed product.

During conducting comparative in-vivo study, the rabbits used as animal model have

140


been divided into two groups i.e. Group-I and Group-II. Group-I was orally

administered with the prepared nano-suspension while Group-II was orally

administered with the marketed product.

i. Comparative In-vivo Study of Roxithromycin Loaded SLNs Nano-suspension

With Marketed Product

Comparative in-vivo studies being conducted using rabbit as animal model, showed

that peak plasma concentration (Cmax) for marketed product (Roxyrex® 150 mg,

Biorex Pharmaceuticals, (PVT) LTD, Pakistan) and prepared nano-suspension (RFH-

4 nanoformulation) was 2.11±0.14 μg.ml-1 and 4.944±0.204 μg.ml-1 respectively.

AUC0→24 for Marketed product was 1.11 μg.hr.ml-1 whereas, for prepared nano-

suspension (RFH-4 nanoformulation) was 23.05 μg.hr.ml-1. Prepared nano-suspension

(RFH-4 nanoformulation) demonstrated 2.34-folds increase in Cmax as well as 20.76-

folds increase in AUC0→24 compared to Marketed product.

The graph of blood plasma concentration-time curve of prepared RTM nano-

suspension and marketed product is envisioned in Figure 4.44. Numerous parameters

of pharmacokinetics are also shown in Table 4.23.

Parameters Marketed Product (Roxyrex®)

RFH-4 Nano-suspension

Prepared Dosage Form

(Capsules)Cmax (μg ml−1) 2.11±0.14 4.944±0.204 4.314±0.316Tmax (h) 2±0.3 12±0.1 12±0.1AUC (μg.hr.ml−1) 1.11 23.05 21.32Relative Bioavailability (Fr)

20.76 19.20

Table 4.23: Comparative in-vivo pharmacokinetic parameters for RTM(±SD, n=6)

The mentioned results clearly indicated that RTM blood plasma concentration

was significantly increased in rabbits orally administered with RFH-4 nano-

suspension compared to marketed product.

141


ii. Comparative In-vivo Study of Famotidine Loaded SLNs Nano-suspension With

Marketed Product

Comparative in-vivo studies being conducted using rabbit as animal model, showed

that peak plasma concentration (Cmax) for marketed product (Kamcid® 20 mg, Bloom

Pharmaceuticals, (PVT) LTD, Pakistan) and prepared nano-suspension (FFH-4

nanoformulation) was 0.498±0.14 μg.ml-1 and 1.03±0.204 μg.ml-1respectively.

AUC0→24 for marketed product was 4.39 μg.hr.ml-1 whereas, for prepared nano-

suspension (FFH-4 nanoformulation) was 27.3 μg.hr.ml-1. Prepared nano-suspension

(FFH-4 nanoformulation) demonstrated 2.07-folds increase in Cmax as well as 6.22-

folds increase in AUC0→24 compared to marketed product.

The graph of blood plasma concentration-time curve of prepared FTD

formulation of nano-suspensions and marketed product is envisioned in Figure 4.45.

Numerous parameters of pharmacokinetics are also shown in Table 4.24.

Table 4.24: Comparative in-vivo pharmacokinetic parameters for FTDParameters Marketed

Product (Kamcid®)

FFH-4 Nano-suspension

Prepared Dosage Form (Capsules)

Cmax (μg ml−1) 0.498±0.14 1.03±0.204 0.93±0.14Tmax (h) 2±0.3 12±0.2 12±0.2AUC (μg.hr.ml−1) 4.39±0.21 27.3±0.003 25.12±0.20Relative Bioavailability (Fr)

6.22 5.72

(±SD, n=6)

FTD blood plasma concentration was significantly increased in rabbits orally

administered with FFH-4 nano-suspension as well as its solid dosage form compared

to marketed product.

142


4.3.6 COMPARATIVE IN-VIVO STUDY OF PREPARED

CAPSULES WITH MARKETED PRODUCT

In-vivo study has been conducted for comparing the oral bioavailability of the

prepared capsules with the marketed product. For this study, the rabbits used as

animal model have been divided into two groups i.e. Group-I and Group-II. Group-I

was orally administered with the prepared capsules while Group-II was orally

administered with the marketed product. Drug plasma concentration was significantly

higher in rabbits treated with prepared solid dosage form (capsules) compared to those

treated with Marketed product.

In-vivo study showed significant increase in the peak plasma concentrations

(Cmax) and AUC0→24 for the prepared capsules compared to marketed products, which

can be envisioned in Table 4.25 & 4.26. Graphically, the increased Cmax and

AUC0→24 for the prepared capsules compared to marketed product can also be

envisioned in Figure 4.44 and 4.45.

Figure 4.44: Comparative in-vivo drug release of RTM

143


Figure 4.45: Comparative in-vivo drug release of FTD

The interesting results obtained from statistically analyzed data of in-vivo


profile for the prepared capsules of RTM and FTD compared to marketed products.

The prepared capsules of RTM and FTD showed comparatively enhanced oral

bioavailability, as, the average particle size of the prepared nanoformulations was less

than 400 nm which can easily cross the gastro-intestinal cells linings for to achieve

the desired boosted oral bioavailability [234].

As compared to marketed product the prepared capsules of RTM and FTD

have particles of decreased size with increased surface area, thus, having much more

exposed surface molecules for to react with the medium which play a vital role in

enhancing the solubility as well as oral bioavailability ”[238].

The conducted DCS and P-XRD studies have confirmed the reduction in the

crystalline nature of drug loaded SLNs via nano-technological engineering, which is

reported to be a perfect approach for enhancing solvency which additionally played a

vital role in boosting oral bioavailability [242].

144


SLNs also have adhesive properties that could increase the residence time for

drug loaded SLNs in its administered area and hence lead to enhance oral

bioavailability [256].

The use of Tween® 80 as surfactant and PEG as co-surfactant may also

improve oral bioavailability of RTM and FTD as they contribute to enhance

permeability as well as affinity between lipids and intestinal membrane [257, 258].

Moreover, sustained drug release profile has been exhibited by drug loaded

SLNs, its might be due to the reason that the fabricated particles comprised of 100-

200 nm size range, since particles size less than 200 nm is undetectable to the

Reticulo-Endothelial System (RES) and remain in circulatory system for a prolonged

time period [235].

The mentioned results for RTM and FTD clearly indicates that SLNs as drug





studies [252].

4.3.7 DISSIMILARITY FACTOR (f1) AND SIMILARITY

FACTOR (f2)

Dissimilarity (f1) and similarity (f2) factors were determined for the prepared Type-I

Capsules (100% uncoated granules) and Type-II Capsules (40% uncoated & 60%

coated granules) compared to marketed products.

i. f1 and f2 Values for Prepared Capsules of Roxithromycin

f1 and f2 factors for the prepared Type-I Capsules (100% uncoated granules) compared

to marketed product (Roxyrex® 150 mg, Biorex Pharmaceuticals, Pakistan) were

145


calculated, f1 was found to be 69 and f2 was 21. While, f1 was 72 and f2 was 19 for the

prepared Type-II Capsules (40% uncoated & 60% coated granules) compared to

marketed product. Dissimilarity factor (f2) for both Type-I Capsules and Type-II

Capsules has a desired high value of 69 and 72 respectively. The data analysis clearly

showed that RFH-4 nanoformulation (Type-I Capsules and Type-II Capsules) have

the desired remarked difference in comparison to the marketed product.

0

17.6423.33

29.6336.73

43.63

51.83

59.28

0

8.1514.54

22.2627.73

33.6338.64

44.63

0

27.11

41.78

53.51

64.51

73.79

88.12

100

0

20

40

60

80

100

120

0 40 80 120 160 200

Cum

ulat

ive

Dru

g R

elea

sed

(%)

Sampling Time (Minutes)

Type-I Capsule of RoxithromycinType-II Capsule of RoxithromycinMarketed Drug

Figure 4.46: Drug release profile of RFH-4 nanoformulation and marketed drug

During the conducted study of 180 minutes, marketed product released almost

100% of RTM. While, only 59% of the entraped RTM is released from prepared

Type-I Capsules (100% uncoated granules) and 44% from the prepared Type-II

Capsules (40% uncoated & 60% coated granules). The prepared Type-II Capsules

released the drug much more slowly and for prolonged period of time as compared to

Type-I Capsules. The advantageous nature of Type-II Capsules over the Type-I

Capsules is the little bit abrupt release of drug from the 40% uncoated granules to

obtain the minimum effective concentration (therapeutic level) of the drug in the

blood followed by prolonged released of the drug from the 60% coated granules. The

146


improved results of Type-I Capsules and Type-II Capsules in terms of sustained

release profile can be envisioned in the mentioned Figure 4.46.

ii. f1 and f2 Values for Prepared Capsules of Famotidine

The prepared Type-I Capsules (100% uncoated granules) showed f1 72 and f2 19 in

comparison to marketed product (Kamcid® 20 mg, Bloom Pharmaceuticals, (PVT)

LTD, Pakistan). While the Type-II Capsules (40% uncoated & 60% coated granules)

showed f1 76 and f2 18. The data analysis clearly showed that the prepared Type-I

Capsules and Type-II Capsules have the desired remarked difference as compared to

marketed product.

0

9.12

16.86

24.23

32.1239.19

45.9852.85

07.16

13.8419.26

27.13

35.1340.91

46.63

0

23.23

33.58

47.49

58.51

71.82

87.2

100

0

20

40

60

80

100

120

0 40 80 120 160 200

Cum

ulat

ive

Dru

g R

elea

sed

(%)

Sampling Time (Minutes)

Type-I Capsule of FamotidineType-II Capsule of FamotidineMarketed Drug

Figure 4.47: Drug release profile of FFH-4 nanoformulation and marketed drug

Among the mentioned three different formulations, the prepared Type-II

Capsules (40% uncoated & 60% coated granules) showed much better results in terms

of sustained drug release profile. During the conducted study of 180 minutes, the

Type-II Capsules released almost 46% of the entraped drug. The advantageous nature

of the Type-II Capsules can be exposed by saying that it released the entraped drug of

147


40% uncoated granules earlier then the 60% coated granules. The earlier release of the

drug from the 40% uncoated granules maintained the therapeutic level of the drug in

the blood followed by sustained release of the drug from the 60 % coated granules for

prolonged period of time. The Type-I Capsules released almost 52% of the entraped

drug, while marketed product released almost 100% of the drug in 200 min. The

improved results of the optimized FFH-4 nanoformulation (Type-I Capsules and

Type-II Capsules) in terms of sustained release profile can be envisioned in the

mentioned Figure 4.47.

148

CONCLUSION

CONCLUSIONThe main goal of the current investigation was to develop formulation design by

encapsulating Famotidine and Roxithromycin in Solid Lipid Nanoparticles

(SLNs). SLNs loaded with Famotidine and Roxithromycin were prepared by three

different methods i.e. Solvent Injection, Solvent Evaporation and Hot Melt

Encapsulation method. These techniques were simple, reproducible as well as also

have the potential to easily scale-up for bulk production.

During preparation process, optimized loaded SLNs formulations showed best

particle size (<200 nm), polydispersity index (<0.5), zeta potential (>±30), entrapment

efficiency (>83%) and drug loading capacity (>2.6%).

Morphological study using SEM micrographs showed spherical shaped SLNs

with well-defined periphery. The particles size was also in concordance to the particle

size analysis data attained from Dynamic Light Scattering (DLS). Further

characterization revealed no drugs-excipients interaction with successful reduction in

crystalline nature. During stability study, maximum stability was observed at

refrigerated temperature. In-vitro drug release study exhibited sustained release

behavior.

Moreover, tray drying as alternative to lyophilization was investigated and

found that this technique can also be employed for SLNs drying purpose especially

for bulk production. Scanning Electron Microscopy (SEM) was conducted for the

samples being prepared by tray drying technique in order to compare with the

lyophilized samples, the white patches in the micrographs of both samples were

almost similar in size and shape. After tray drying, the resultant dried powder was

converted to granules and consequently filled in capsule shells. In-vitro study of the

prepared capsules was conducted for dissimilarity (f1) and similarity (f2) factors

149

CONCLUSION

determination. Dissimilarity factor was greater then 65 (f2>65) showing a remarked

difference compared to the marketed products.

The in-vivo pharmacokinetic study indicated that loaded SLN formulations

are vastly significant demonstrating almost 2-fold increase in oral bioavailability.

Drug plasma concentrations were significantly elevated in rabbits treated with loaded

SLN formulations compared to marketed product. Overall, these results indicate that

Famotidine and Roxithromycin absorption is significantly boosted by SLNs

nanoformulations compared to marketed product (conventional dosage form).

Future Work

Solid Lipid Nanoparticles (SLNs) showed significant in-vitro and in-vivo results i.e.

enhanced oral bioavailability and sustained drug release profile. Still additional

assessments of the presented nanoformulations should be conducted to ensure their

safety and toxicological parameters.

The in-vitro cytotoxicity assay (MTT-assay) and also calculation of their

lethal doses should be conducted. In-vitro cell permeability and cellular up-take

studies have to be conducted for further confirmation of its effectiveness. Despite

of available literature which supports the biocompatible nature of stearic acid,

self safety evaluation study for stearic acid should be conducted. The long term

in-vivo study is required to evaluate the continuous exposure of the

nanoformulations to the animals model to clarify any doubt concerning toxicity

and safety of the formulation excipients and drugs in chronic administration.

All these mentioned studies have to be conducted to bring forward this

reported work for conducting clinical-trials in human volunteers to evaluate

different pharmacokinetic parameters of the presented optimized nanoformulations

of Roxithromycin and Famotidine.

150

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183

PUBLICATIONS

List of Publications

1. PUBLICATION FROM THESIS

Muhammad Shafique, Mir Azam Khan, Waheed S. Khan, Maqsood-ur-Rehman,

Waqar Ahmad, and Shahzeb Khan (2017) Fabrication, Characterization, and In-

vivo Evaluation of Famotidine Loaded Solid Lipid Nanoparticles for Boosting

Oral Bioavailability. Journal of Nanomaterials.

https://doi.org/10.1155/2017/7357150 (Impact Factor 1.871)

Muhammad Shafique, Mir Azam Khan, Waheed S. Khan, Maqsood-ur-Rehman,

Shahzeb Khan, Waqar Ahmad, (2017) Famotidine loaded solid lipid

nanoparticles: Physico-chemical characterization and in-vivo evaluation of

boosted oral bioavailability Acta Pharmaceutica (Accepted) (Impact Factor

1.26)

Muhammad Shafique, Mir Azam Khan, Maqsood-ur-Rehman, Waqar Ahmad,

Shahzeb Khan “Enhancing oral bioavailability of roxithromycin by nano-

emulsifying drug delivery system”. (Under Review)

Muhammad Shafique, Mir Azam Khan, Waqar Ahmad, Maqsood-ur-Rehman,

Shahzeb Khan “Fabrication and characterization of roxithromycin loaded solid

lipid nanoparticles: comparative in-vivo evaluation confirming enhanced oral

bioavailability” (Submitted)

2. PUBLICATIONS FROM SAME PROJECT

Maqsood ur Rehman, Mir Azam Khan, Waheed S. Khan, Muhammad Shafique,

Munasib Khan (2017). Fabrication of Niclosamide loaded solid lipid

nanoparticles: In-vitro Characterizationand Comparative in-vivo Evaluation.

Artificial cells, nanomedicine, and biotechnology 1-9.

DOI:10.1080/21691401.2017.1396996 (Impact Factor 5.605)

184

https://doi.org/10.1080/21691401.2017.1396996

https://doi.org/10.1155/2017/7357150

PUBLICATIONS

Mir Azam Khan, Maqsood-ur-Rehman, Waheed S. Khan, Shahzeb Khan, Waqar

Ahmad, Muhammad Shafique (2017) Fabrication of sulfasalazine loaded solid

lipid nanoparticles, in-vitro Characterization and Comparative in-vivo Evaluation

to enhance oral bioavailability Evaluation. Acta Pharmaceutica (Accepted)

(Impact Factor 1.26)

Maqsood ur Rehman, Waheed S. Khan, Mir Azam Khan, Muhammad Shafique,

Ayesha Ihsan. Explore solid lipid nanoparticles to augment oral bioavailability

of Niclosamide: pharmaceutical and stability study. (Under Review)

Mir Azam Khan, Maqsood ur Rehman, Waheed S. Khan, Muhammad Shafique,

Waqar Ahmad. Solid lipid nanoparticles for sulfasalazine: fabrication,

characterization, in-vitro and in-vivo assessment for enhanced oral bioavailability.

(Submitted).

185

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