©[2020] Anika Haq ALL RIGHTS RESERVED
Transcript of ©[2020] Anika Haq ALL RIGHTS RESERVED
©[2020]
Anika Haq
ALL RIGHTS RESERVED
APPLICATION OF SOLUBILITY-PHYSICOCHEMICAL-THERMODYNAMIC (SPT)
THEORY FOR DESIGNING A TOPICALLY APPLIED THYMOQUINONE POLYMER
FILM TO TREAT INFECTED WOUNDS
By
ANIKA HAQ
A dissertation submitted to the
School of Graduate Studies
Rutgers, The State University of New Jersey
In partial fulfillment of the requirements
For the degree of
Doctor of Philosophy
Graduate Program in Pharmaceutical Sciences
Written under the direction of
Bozena Michniak-Kohn
And approved by
New Brunswick, New Jersey
May, 2020
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ABSTRACT OF THE DISSERTATION
APPLICATION OF SOLUBILITY-PHYSICOCHEMICAL-THERMODYNAMIC
(SPT) THEORY FOR DESIGNING A TOPICALLY APPLIED
THYMOQUINONE POLYMER FILM TO TREAT INFECTED WOUNDS
By ANIKA HAQ
Dissertation Director:
Professor Bozena Michniak-Kohn
Skin has significant barrier properties that inhibit the passive transport of many
active molecules. Different strategies are developed to overcome this skin barrier such as,
chemical enhancement techniques using penetration enhancers and targeted drug delivery
using topical and/or transdermal formulations. Usually these approaches are tested using
human or animal skin. Human skin is not easily accessible and animal skin has significant
biological and barrier differences when compared with human skin. Due to these issues the
possibility of having a synthetic skin membrane is an attractive option. In this thesis, firstly,
we investigated different formulations containing various enhancers from the aspect of
their ability to enhance or reduce the delivery of nicotine through human cadaver skin and
correlated that to Strat-M® synthetic membrane to examine the usefulness of this
membrane as a convenient screening tool to investigate topically applied formulations and
TDDS (Transdermal Delivery System). Formulations containing nicotine and a chemical
penetration enhancer (CPE) were used for evaluating drug penetration to understand how
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each enhancer impacts the permeability of nicotine as a model compound. The permeability
measurements of human cadaver skin and Strat-M® membrane were performed with Franz
diffusion cell methods accompanied by HPLC analysis. The study demonstrated the good
correlation (R2=0.99) of the permeability data obtained through human cadaver skin and
Strat-M® membrane. Our data suggests that although Strat-M® lacks the highly organized
stratum corneum (SC) intercellular structure and provided higher nicotine flux compared
to human cadaver skin where the highly structured SC significantly reduced nicotine
permeability, both membranes still provided similar enhancement factors for a given
enhancer. These studies suggest that the Strat-M® synthetic membrane lipid composition
probably closely mimics that of human cadaver skin based on the data obtained. The time
point correlation between Strat-M® and human cadaver skin were in the range 0.90-0.99.
This work suggests that some of the main transport mechanisms for drug diffusion and
permeation of Strat-M® membrane could be similar to an ex vivo human skin model.
Secondly, we report on that the overall mechanism of action of skin penetration
enhancers is best explained by the Solubility-Physicochemical-Thermodynamic (SPT)
theory. The SPT theory puts forward the concept that the mode of action of enhancers is
related to solubility parameters, physicochemical interactions and thermodynamic activity.
We have discussed these concepts by using experimentally derived permeation data,
various physicochemical and solubility parameters (ingredient active gap (IAG), ingredient
skin gap (ISG), solubility of active in the formulation (SolV) and the formulation solubility
in the skin (SolS)) generated by using FFE (Formulating for EfficacyTM - ACT Solutions
Corp) software. Our data suggests that there is an inverse relationship between measured
flux and IAG values given that there is an optimum ingredient skin gap, SolV and SolS
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ratio. The study demonstrated that the flux is actually proportional to a gradient of
thermodynamic activity rather than the concentration and maximum skin penetration and
deposition can be achieved when the drug is at its highest thermodynamic activity. This
work will connect the solubility and physicochemical properties of the active and
enhancers/ingredients with the thermodynamic activity of the model drug used in order to
explain the mode of action of enhancers in a given formulation with that specific drug.
Thirdly, we studied the effect of an ethanol and propylene glycol donor solvent
system along with various compositions of receptor solvents to investigate the feasibility
of transdermal delivery of thymoquinone (TQ). The effects of penetration enhancers on the
in vitro skin permeation and TQ skin absorption were studied using human cadaver skin in
Franz diffusion cells. The permeation of saturated solutions of TQ was investigated with
5% v/v of each of the following enhancers: Azone (laurocapram), Transcutol® P (Tc), oleic
acid, ethanol, Polysorbate 80 (Tween 80), and N-methyl-pyrrolidone (NMP). Our data
suggests that Azone, oleic Acid and Tc were able to provide adequate TQ flux and may be
the agents of choice for use in a novel transdermal formulation of TQ. These penetration
enhancers were also able to generate TQ reservoirs in the skin that may be useful to provide
sustained release of TQ from the stratum corneum over longer periods of time. The study
also demonstrated pull or drag effect of permeation enhancers and vehicle on TQ skin
deposition. These studies suggest that ethanol was able to pull more drug into the skin and
all the enhancers used in this study showed low “pulling” effect. Rather these enhancers
(Azone, oleic acid and Tc) showed enhanced permeation as the enhancers has permeation
enhancing effect. Finally, we synthesized and characterized a biocompatible novel topical
polymeric film system that has the potential to deliver antibacterial/anti-inflammatory
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agent thymoquinone (TQ) directly to the skin target site and that may be useful for the
treatment and management of wound infections. The polyvinyl pyrrolidone (PVP) matrix-
type films containing TQ were prepared by the solvent casting method using dibutyl
phthalate as a plasticizer and Azone (laurocapram) as a penetration enhancer. The
developed films were evaluated for thickness, drug content uniformity, weight variation,
flatness, folding endurance, percentage of moisture content and uptake which were found
to 1.17 ± 0.04 mm, 100 ± 6.4 %, 82.04 ± 1.9 mg, 100%, 68 ± 2.38, 14.12 ± 0.42 %, and
2.26 ± 0.47 % respectively. FESEM photograph of the film showed polymer networks
inside the film and a homogeneous dispersion of drug inside the polymer networks. In vitro
skin permeation studies on human cadaver skin produced a mean flux of 2.3 µg/cm2/h. In
vitro scratch assay results revealed that 100 ng of TQ had significant wound closure activity
in human dermal fibroblast cells compared to both control (p = 0.0014) and positive control
(p = 0.0004). Using human keratinocyte cell line, 100 ng TQ group showed 85% wound
closure activity at day six which was significantly higher (p = 0.0001) than the control
group. In a zone-of-inhibition (ZOI) assay, the presence of TQ-containing films completely
wiped out Staphylococcus aureus in a 10 cm in diameter TSA (Tryptone soya agar) plates
while 500 ug/mL gentamicin containing filters gave 10 mm of ZOI. In an ex vivo model,
the presence of TQ-film eradicated the bacterial colonization on human cadaver skin.
Furthermore, in the BALB/c mice wound model, TQ-films showed significant activity in
controlling Staphylococcus aureus infection and promoting wound closure compared to
control film. These results indicate, TQ/PVP films developed in this study have potential
for the treatment and management of wound infection.
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Acknowledgments
I am grateful to all of those with whom I have had the pleasure to work during my Ph.D.
dissertation studies and other related projects.
I would like to express my sincere gratitude to my advisor Dr. Bozena Michniak-Kohn,
Professor of Pharmaceutics, Ernest Mario School of Pharmacy for the continuous support
and motivation. Thank you for teaching me how to be an independent researcher and for
providing the scientific platform that helped me to build my knowledge on skin research
through attending numerous seminars, building networks and collaborations that further
developed into research projects. Thank you for trusting me and for encouraging me to
pursue my passion in science.
I am also very grateful to Dr. Suneel Kumar at Department of Biomedical Engineering,
Rutgers University for assisting me with in vitro and in vivo wound healing study and for
teaching me all the required techniques for the animal study. A sincere and heartfelt
gratitude to Dr Yong Mao, Research Associate Professor, New Jersey Center for
Biomaterials for sharing your valuable input in the bacterial study design. Both of your
invaluable knowledge, experience and expertise in your respective fields taught me various
necessary research skills. I am truly inspired by both of your pleasant personality and
scientific mind.
I would like to thank my thesis committee members, Dr. Tamara Minko, Distinguished
Professor and Chair, Ernest Mario School of Pharmacy and Dr. Leonid Kagan, Associate
Professor, Ernest Mario School of Pharmacy for their insightful comments,
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encouragement, valuable time and guidance throughout my thesis defense process. A
special thanks to Dr. Francois Berthiaume, Professor, Department of Biomedical
Engineering to allow me to collaborate with his research team in the wound infection
project and for providing me with all the necessary tools to conduct the animal study.
I would especially like to thank Mark Chandler (President, ACT Solutions Corp.) my
outside thesis committee member. As my mentor, he has shown me, by his example, what
a good scientist and person should be. Thank you for inspiring me with your positive
attitude and energy.
I would like to express my gratefulness to Dr. Tony Kong, Director, Graduate Program in
Pharmaceutical Science and Ms. Hui Pung, Senior Program Coordinator of Pharmaceutical
Sciences Graduate Program for their help and support throughout my graduate studies.
My sincere thank also goes to Dr. Firouz Asgarzadeh, Dr. Simone Carvalho, Mitul Patel,
Kenneth Banks and Joe Abrantes, who provided me an opportunity to join their team as
intern at Evonik Corporation, and who gave access to the laboratory and research facilities.
Without their precious support and knowledge sharing it would not be possible to conduct
my internship project.
I want to take this opportunity to thank the past and current members of Center for Dermal
Research (CDR): Ben Goodyear, Dina Ameen, Jemima Shultz, Anna Froelich, Rose
Soskind, Sonia Trehan, Parinbhai Shah, Vinam Puri and Julia Zhang; whom I have had the
joy of working with. Thank you all for being the awesome lab mates. You all made my
Ph.D. journey memorable and fun.
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I would also like to thank the Center for Dermal Research (CDR) for providing funding for
this research and the Berthiaume laboratory for funds for the animal studies. In addition, I
acknowledge the Division of Life Sciences, School of Arts and Sciences and Ernest Mario
School of Pharmacy at Rutgers University for providing me with a Teaching Assistantship
from 2016 to 2020.
Finally, I would like to express my love and respect for my parents for raising me as a
strong woman and as an empathetic human being. Thank you for believing in me and for
guiding me through my life. You both are my role models. You both encourage me to be
the best version of myself and to live my life up to my full potential while caring and
serving for others. You taught me to believe in the power of being happy as a whole with
everyone in my life. Most importantly, I wish to thank my loving and supportive husband,
Atik, my two beautiful sisters, Lima and Tanny, and my two wonderful nephews, Tahsin
and Tahan. You all are the blessings of my life and a great source of comfort. You all
provide unending inspiration.
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Table of Contents
Abstract of the Dissertation…………………………………………………………… ii
Acknowledgments………………………………………………………………………. vi
List of Tables …………………………………………………………………………. xvii
List of Figures ………………………………………………………………………… xix
Chapter 1: Background and Specific aims …………………………………………… 1
1.1. Skin physiology …………………………………………………………………….. 1
1.2. Artificial membrane ………………………………………………………………… 8
1.3. Skin penetration enhancement …………………………………………………….. 11
1.4. Topical and transdermal drug delivery ……………………………………………. 18
1.5. Thymoquinone …………………………………………………………………….. 21
1.6. Specific aims ……………………………………………………………………… 22
Chapter 2: Evaluating Strat-M® Synthetic Membrane as a Screening Tool for
Topical/Transdermal Formulation ………………………………………………….. 27
2.1. Introduction ………………………………………………………………………... 27
2.2. Materials and Methods ……………………………………………………………. 30
2.2.1. Materials ………………………………………………………………………. 30
2.2.2. Preparation of formulations …………………………………………………… 31
2.2.3. In vitro skin permeation test (IVPT) studies ………………………………….. 31
x
2.2.4. High performance liquid chromatography (HPLC) …………………………… 32
2.2.5. Data analysis ………………………………………………………………….. 33
2.2.6. Statistical analysis …………………………………………………………….. 33
2.3. Results and discussion ……………………………………………………………. 33
2.3.1. Effect of Azone ……………………………………………………………….. 34
2.3.2. Effect of Propylene glycol ……………………………………………………. 36
2.3.3. Effect of Eucalyptol and Tween 80 …………………………………………… 37
2.3.4. Effect of N-methyl pyrrolidone ………………………………………………. 38
2.3.5. Enhancement factor ………………………………………………………….. 38
2.3.6. Correlation of Strat-M membrane to human cadaver skin ……………………. 39
2.4. Conclusions ………………………………………………………………………. 40
Chapter 3: Solubility-Physicochemical-Thermodynamic Theory of Penetration
Enhancers Mechanism of Action …………………………………………………… 44
3.1. Introduction ………………………………………………………………………. 44
3.1.1. Theoretical background ………………………………………………………. 47
3.2. Materials and Methods ……………………………………………………………. 51
3.2.1. Materials ………………………………………………………………………. 51
3.2.2. Preparation of formulation and solubility determination ……………………… 52
3.2.3. Permeation procedure for enhancer studies …………………………………… 52
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3.2.4. Skin deposition study ………………………………………………………… 53
3.2.5. High performance liquid chromatography (HPLC) ………………………….. 53
3.2.5.1. Nicotine ………………………………………………………………….. 54
3.2.5.2. Thymoquinone …………………………………………………………… 54
3.2.6. Calculated solubility and physicochemical parameters and permeation data … 54
3.2.7. Data and statistical analysis …………………………………………………… 55
3.3. Results and discussion …………………………………………………………….. 55
3.3.1. Nicotine ………………………………………………………………………. 55
3.3.2. Thymoquinone ………………………………………………………………... 65
3.3.3. Concentration dependency of Oleic acid ……………………………………... 74
3.4. Conclusions ……………………………………………………………………….. 77
Chapter 4: Effects of Solvents and Penetration Enhancers on Transdermal Delivery
of Thymoquinone: Permeability and Skin Deposition Study ……………………… 78
4.1. Introduction ……………………………………………………………………….. 78
4.2. Materials and Methods ……………………………………………………………. 80
4.2.1. Materials ……………………………………………………………………… 80
4.2.2. Solubility determination ……………………………………………………… 81
4.2.3. In vitro skin permeation test (IVPT) studies ………………………………….. 81
4.2.4. High-performance liquid chromatography (HPLC) method development and
validation for TQ ……………………………………………………………… 82
4.2.4.1. Method characteristics ……………………………………………………. 82
4.2.4.2. Standard solutions and calibration curve …………………………………. 82
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4.2.4.3. Method validation ……………………………………………………… 83
4.2.5. Determination of TQ concentration in the skin ……………………………… 83
4.2.6. Data and statistical analysis …………………………………………………… 84
4.3. Results and discussion ……………………………………………………………. 85
4.3.1. HPLC method validation ……………………………………………………... 85
4.3.2. Thymoquinone solubility study ……………………………………………… 88
4.3.3. Effect of propylene glycol and ethanol donor solvent ……………………….. 89
4.3.4. Effect of receiver solvent composition ………………………………………. 94
4.3.5. Effect (pull or drag) of permeation enhancers and vehicle on TQ skin deposition
……………………………………………………………………………….. 96
4.4. Conclusions ………………………………………………………………………. 99
Chapter 5: Thymoquinone Loaded Polymeric Films and Hydrogels for the
Treatment of Wound Healing and Bacterial Skin Infections…………………….. 100
5.1. Introduction ……………………………………………………………………… 100
5.2. Materials and Methods …………………………………………………………… 104
5.2.1. Materials …………………………………………………………………….. 104
5.2.2. Fourier Transform Infrared (FTIR) analysis ……………………………....... 105
5.2.3. Fabrication of films …………………………………………………………. 105
5.2.4. Preparation of TQ hydrogel formulations ………………………………........ 106
5.2.5. Field Emission Scanning Electron Microscopic (FESEM) studies …………. 106
5.2.6. Physicochemical characterization of films ………………………………..... 107
5.2.6.1. Film thickness …………………………………………………………… 107
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5.2.6.2. Drug content uniformity ……………………………………………… 107
5.2.6.3. Weight variation ……………………………………………………… 108
5.2.6.4. Flatness ……………………………………………………………….. 108
5.2.6.5. Folding endurance ……………………………………………………. 108
5.2.6.6. Percentage of moisture content ………………………………………. 108
5.2.6.7. Percentage of moisture uptake ……………………………………….. 109
5.2.7. Physicochemical characterization of the prepared hydrogels ……………... 109
5.2.7.1. Visual inspection ……………………………………………………… 109
5.2.7.2. pH determination ……………………………………………………... 109
5.2.7.3. Spreadability test …………………………………………………….. 109
5.2.7.4. Drug content uniformity ……………………………………………… 110
5.2.8. Rheological characterization of hydrogel formulation …………………… 110
5.2.8.1. Oscillation stress sweep ……………………………………………... 110
5.2.8.2. Frequency sweep …………………………………………………….. 110
5.2.9. In Vitro skin permeation studies …………………………………………. 111
5.2.10. High-performance liquid chromatography (HPLC) …………………….. 111
5.2.11. Skin deposition study …………………………………………………… 112
5.2.12. Stability study …………………………………………………………... 112
5.2.13. In vitro antibacterial activity of TQ films and hydrogels ………………. 113
5.2.14. Ex vivo antibacterial activity of TQ films and hydrogels using human
cadaver skin explants ………………………………………………….. 113
5.2.15. Cyto-compatibility study ……………………………………………… 114
5.2.16. Scratch assay for wound closure activity ……………………………… 114
5.2.17. In vivo bacterial skin infection study …………………………………. 115
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5.2.18. Behavioral response of mice ……………………………………………… 116
5.2.19. Histopathological examination …………………………………………… 117
5.2.20. Data and statistical analysis ……………………………………………… 117
5.3.Results and discussion …………………………………………………………… 118
5.3.1. Fourier Transform Infrared (FTIR) Spectroscopic studies ……………....... 118
5.3.2. Physicochemical characterization of films ………………………………… 118
5.3.3. Characterization of the TQ hydrogels ……………………………………... 121
5.3.4. In Vitro skin permeation and deposition studies …………………………... 125
5.3.5. Stability study ……………………………………………………………… 125
5.3.6. Cyto-compatibility study …………………………………………………... 127
5.3.7. In vitro and Ex vivo bacterial inhibition study ……………………………. 128
5.3.8. Scratch assay for wound closure activity ……………………………......... 130
5.3.9. Wound healing and anti-bacterial activity of TQ film in vivo ……………. 132
5.3.10. Histological examination ………………………………………………… 136
5.4. Conclusions ……………………………………………………………………... 137
Appendix A: Development of Lidocaine Loaded EUDRAGIT® RLPO Transdermal
Patch Application …………………………………………………………………... 139
A.1. Introduction ……………………………………………………………………. 139
A.2. Materials and Methods ………………………………………………………… 141
A.2.1. Materials …………………………………………………………………….. 141
A.2.2. Preparation of lidocaine loaded transdermal patches ……………………… 142
A.2.3. Patch characterization ……………………………………………………….. 144
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A.2.3.1. Thickness …………………………………………………………… 144
A.2.3.2. Weight variation ……………………………………………………. 144
A.2.3.3. Content uniformity …………………………………………………. 144
A.2.4. High performance liquid chromatography (HPLC) ………………........... 144
A.2.5. Mechanical properties …………………………………………………… 145
A.2.6. Loop tack test or adhesive strength study ……………………………….. 145
A.2.7. Rheology ………………………………………………………………… 146
A.2.7.1. Oscillation stress sweep …………………………………………….. 146
A.2.7.2. Frequency sweep ……………………………………………………. 146
A.2.8. In vitro release study …………………………………………………….. 147
A.2.9. Water vapor transmission of transdermal patch system ………………… 147
A.2.10. Shower resistance study ……………………………………………….. 148
A.2.11. SEM-EDS (Scanning Electron Microscopy Energy
Dispersive Spectroscopy) ……………………………………………… 148
A.2.12. Differential scanning calorimetry studies ……………………………… 149
A.2.13. Data and statistical analysis ……………………………………………. 149
A.3. Results and discussion …………………………………………………………. 149
A.3.1. Appearance and patch thickness ………………………………………… 150
A.3.2. Content uniformity ………………………………………………………. 150
A.3.3. The effect of Eudragit® RLPO in adhesive and cohesive strength ……… 151
A.3.4. The effect of drug loading and Eudragit® RLPO on the
mechanical properties of transdermal patches …………………………. 153
A.3.5. The effect of drug loading on rheological behavior
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of the formulations ……………………………………………………. 155
A.3.6. In vitro release study …………………………………………………….. 157
A.3.7. Scanning electron microscopy …………………………………………... 159
A.3.8. Differential scanning calorimetry ……………………………………….. 161
A.3.9. WVP evaluation …………………………………………………………. 164
A.3.10. Evaluation of shower effect on the patches …………………………… 165
A.4. Conclusions …………………………………………………………………. 166
References ………………………………………………………………………… 168
Thesis summary and future perspectives ………………………………………. 177
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List of Tables
Table 1.1. Regional variations in drug permeability of stratum corneum. 4
Table 1.2. A list of various skin models and membranes used for skin research. 8
Table 1.3. Examples of chemical penetration enhancers on the bases of their 13
structure.
Table 2.1. Penetration parameters of nicotine through Strat-M® membrane after 35
8 hours.
Table 2.2 Penetration parameters of nicotine through human cadaver skin after 35
8 hours.
Table 3.1. Hansen solubility parameters and molar volume for nicotine and different 57
solvents/enhancers.
Table 3.2. Physicochemical parameters of nicotine and different 58
enhancers.
Table 3.3. Penetration parameters of nicotine through human cadaver skin after 65
8 hours.
Table 3.4. Hansen solubility parameters and molar volume of thymoquinone and 67
different solvents/enhancers.
Table 3.5. Physicochemical parameters of thymoquinone and different enhancers. 67
Table 3.6. Penetration parameters of thymoquinone through human cadaver skin 70
(N=5) after 24 hours.
Table 3.7. Summary of the solubility study results showing the effect of 5% 71
penetration enhancers on the solubility of TQ using propylene glycol. The values
represent the concentration of TQ ± SD (N=3) in mg/mL at 48 hours.
Table 4.1. Intra-day variability of TQ standard solutions of three separate runs 87
in one day.
Table 4.2. Inter-day variability of TQ standard solutions of two separate runs 87
in two days.
Table 4.3. Summary of the Solubility Study Results. The values represent the 88
concentration of TQ ± SD (N=3) in mg/mL at 48 hours.
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Table 4.4. Penetration parameters of thymoquinone through human cadaver skin 91
(N=5) after 24 hours using propylene glycol vehicle.
Table 4.5. Penetration parameters of thymoquinone through human cadaver skin 92
(N=5) after 24 hours using ethanol vehicle.
Table 4.6. Penetration parameters of thymoquinone through human cadaver skin 95
(N=5) after 24 hours using ethanol vehicle and ethanol : PBS pH 7.4 (60:40)
receptor solvents.
Table 5.1. Composition of TQ topical hydrogels (% w/w). 107
Table 5.2. Physicochemical properties of TQ films (data shows 121
mean of five determinations with ± standard deviation).
Table 5.3. Physicochemical properties of TQ topical hydrogel formulations 122
(F1- F10).
Table 5.4. Penetration parameters of thymoquinone through human cadaver 126
skin (N=5) after 8 hours.
Table A.1. Lidocaine-loaded patch composition % (w/w) at different drug 143
loading ranging from 4% to 20% (Formulation A-D).
Table A.2. Physical and mechanical properties of transdermal 151
patches containing lidocaine. Data represents N=3, mean ± SD.
xix
List of Figures
Figure 1.1. Structure of the skin. 2
Figure 1.2. Anatomy and physiology of the skin. 3
Figure 1.3. Schematic representation of penetration pathways. 5
Figure 1.4. Scanning electron microscopic image of a cross-section of Strat-M™. 9
The first (i), second (ii), and third layer (iii) of Strat-M™.
Figure 1.5. Transmission scanning electron microscopic observation of a 10
cross-section of Strat-M™. The first (i), second (ii), and third layer (iii) of
Strat-M™. Lipids in the layer were stained with a black color. Arrows show the
lipid region.
Figure 1.6. Lipophilic and hydrophilic pathways of drug penetration and mode of 12
action of penetration enhancers.
Figure 1.7. Franz diffusion cell. 14
Figure 1.8. A typical plot of permeation study. 17
Figure 1.9. Schematic representation of the process involved in drug transport from 19
topical or transdermal formulation.
Figure 1.10. Nigella sativa, black cumin seed and chemical structure of 21
thymoquinone.
Figure 2.1. Multilayered structure of Strat-M® membrane. 29
Figure 2.2. Cumulative amounts of nicotine per cm2 of membrane/skin permeated 36
after 8 hours through Strat-M® membrane (n=6) and human cadaver skin (n=6)
samples.
Figure 2.3. Schematic representation of Azone disrupting the stratum corneum 37
intercellular lipids.
Figure 2.4. Human cadaver skin and Strat-M® membrane enhancement factors for 39
different formulation enhancers.
Figure 2.5. Nicotine penetration profiles of transdermal formulations (A) human 41
cadaver skin, (B) Strat-M® membrane, and (C) time point correlations between the
amounts of drug penetrated through human cadaver skin and Strat-M® membrane.
Means plus minus S.D. and correlation coefficients.
xx
Figure 2.6. Correlation of flux between Strat-M® synthetic membrane and human 42
cadaver skin.
Figure 3.1. Nicotine permeation profiles of transdermal formulations (A) with the 59
Franz diffusion cell method using human cadaver skin, (B) the correlations between
the calculated and measured permeation of nicotine.
Figure 3.2. Position of the active nicotine and penetration enhancers/ingredients in 60
3D Hansen Space.
Figure 3.3. The influence of physicochemical interactions (IAG, SolV) 64
between penetration enhancer and active (Nicotine) on the driving force for diffusion
and, the influence of various physicochemical and solubility parameters (ISG, SolS)
of the formulation on the skin affinity of the penetrant is illustrated. (A) The
possible mechanism of action of skin penetration enhancers; (B) a representation
of the active-enhancer and stratum corneum interactions promoting partitioning
into the stratum corneum.
Figure 3.4. Thymoquinone permeation profiles of transdermal formulations 68
(A) with the Franz diffusion cell method using human cadaver skin, (B) the
correlations between the calculated and measured permeation of Thymoquinone.
Figure 3.5. Position of the active Thymoquinone and penetration enhancers/ 69
ingredients in 3D Hansen Space.
Figure 3.6. The influence of physicochemical interactions (IAG, SolV) 73
between penetration enhancer and active (Thymoquinone) on the driving
force for diffusion and, the influence of various physicochemical and solubility
parameters (ISG, SolS) of the formulation on the skin affinity of the penetrant
is illustrated. (A) The possible mechanism of action of skin penetration enhancers;
(B) a representation of the active-enhancer and stratum corneum interactions
promoting partitioning into the stratum corneum.
Figure 3.7. Amount of Thymoquinone detected at 24 hours in human cadaver skin. 75
Figure 3.8. Thymoquinone permeation profile in propylene glycol vehicle containing 76
different concentration of Oleic Acid. Time points were measured at 3, 4, 6, 8, 10, 12
and 24 hours. Each point represents the mean ± S.D. of five experiments. ***p<0.02.
Figure 4.1. Thymoquinone chromatogram peak at retention time of 4.2 min. 85
Figure 4.2. Thymoquinone standard curve for HPLC assay. 86
Figure 4.3. Thymoquinone permeation profile in propylene glycol vehicle. 90
Time points were measured at 3,4,6,8,10,12 and 24 hours. Each point represents the
xxi
mean ± S.D. of five experiments.
Figure 4.4. Thymoquinone permeation profile in ethanol vehicle. Time points were 91
measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five
experiments.
Figure 4.5. Thymoquinone permeation profile in ethanol vehicle and ethanol:PBS 93
(pH 7.4) receptor. Time points were measured at 3,4,6,8,10,12 and 24 hours. Each
point represents the mean ± S.D. of five experiments.
Figure 4.6. Amount of TQ (PG vehicle) detected after 24 hours in human cadaver 96
skin (N=5, mean ± SD).
Figure 4.7. Amount of TQ (ethanol vehicle) detected after 24 hours in human 98
cadaver skin (N=5, mean ± SD).
Figure 4.8. Amount of TQ (ethanol : PBS receptor) detected after 24 hours 99
in human cadaver skin (N=5, mean ± SD).
Figure 5.1. Physicochemical characterization of TQ films. (A) FTIR spectrum of 119
TQ pure drug, PVP, physical mixture of drug and polymer, freshly prepared
films containing drug and polymer, stored films containing drug and polymer; (B)
control films (i); Field emission scanning electron microscopic (FESEM) images
showing surface morphology of control film (ii-iii) at different magnifications; and
(C) TQ films (i); FESEM images showing surface morphology of TQ films (ii-iii)
at different magnifications.
Figure 5.2. Rheological characterization of TQ hydrogel formulations (F1-F10). 124
(A) Oscillation frequency sweep data. The elastic modulus (i); The viscous modulus
(ii) were plotted against angular frequency. TQ permeation and skin deposition from
film and gel formulations (B). TQ permeation profile for different hydrogel formulations
(i). Time points were measured at 1, 2, 3, 4, 5, 6 and 8 hours. Each point represents the
mean ± S.D. of five experiments; TQ permeation from film formulation across human
cadaver skin (mean ± S.D., n=5) (ii); Amount of TQ detected after 8 hours in human
cadaver skin (N=5, mean ± SD) using different TQ hydrogel formulations (iii).
Figure 5.3. Cytocompatibility study of TQ film. Cell viability of TQ film with HDF 128
and HaCat cells using alamarBlue® assay.
Figure 5.4. Bacterial inhibition study. (A) Inhibition of bacterial growth on agar plate 129
by Control negative (i); Gentamicin positive control 50 µg/mL (ii) right upper and
500 µg/mL (ii) right lower; Control film (iii); TQ hydrogel (iv) and TQ film (v)
against Staphylococcus aureus; (B) Ex vivo antibacterial activity by Control (i);
Control film (ii); Gentamicin sulfate USP, 0.1% marketed cream (iii); TQ hydrogel
(iv); TQ film (v) and Log of bacterial reduction with different treatment groups (vi).
Data represent mean ± SD of four replicates. ***p = < 0.001 and ^^^p = < 0.05.
xxii
Figure 5.5. Effect of different treatment groups on the wound healing of 131
keratinocytes and fibroblasts. (A) Representative micrographs from control, 1 ng/mL
and 100 ng/mL of TQ, showing the original wound and the wound after 6 days;
(B) Quantitative analysis of wound closure as a function of time. The wound area
was determined as the wound area at a given time relative to the original wound area.
Data are presented as the means ± SD (n=5-6). ***p<0.001 (control vs 100 ng/mL)
and ^p<0.05 (Control vs 1 ng/mL). (C) Representative micrographs from control, 1
ng/mL and 100 ng/mL of TQ, showing the original wound and the wound after 24 hour;
(D) Quantitative analysis of wound closure as a function of time. The wound area
was determined as the wound area at a given time relative to the original wound area.
Data are presented as the means ± SD (n=6). **p<0.01 and ***p<0.001 (control vs
100 ng) and ^p<0.05 (Control vs 1 ng); (E) Quantitative measurement of cells
number migrating in the corresponding scratched wound areas at different
treatment groups. The values plotted were means of 6 determinations (𝑛 = 6).
***p<0.001 (100 ng/mL vs control/1 ng/mL/10 ng/ml).
Figure 5.6. Macroscopic observations, wound closure and bacterial reduction. 134
(A) Photographs of wounds in BALB/c mice in which the wounds received TQ
loaded film and Gentamicin. The animal with bacterial wounds and wound with no
bacterial served as control group and animal with control film served as a vehicle
control. Representative photographs of the wounds were taken at 0, 3, 7, 10, 14,
and 21 days post-wounding; (B) Log of bacterial reduction at each time point (Day 1,
2, 3 and 7) using different experimental groups. Data are presented as the means ± SD
(n=2-4). ***p<0.001 (Bacterial wound vs TQ Film) and ^^^p<0.001 (Bacterial wound
vs Gentamicin); (C) Percentage of wound closure in all experimental groups at 0, 3, 7,
10, and 14 days post-wounding.
Figure 5.7. Masson’s trichrome staining of the different samples at day 21 post- 137
wounding (control wound (i); Gentamicin sulfate USP, 0.1% marketed cream (ii);
bacterial wound (iii); control film (iv); TQ film (v); TQ film + dose (vi)) indicates
epidermis and indicates dermis.
Figure A.1. Schematic representation of solvent evaporation method. 143
Figure A.2. Peak adhesive force of different formulations containing Eudragit® 152
RLPO and different concentration of Lidocaine. Data represents mean ± SD (n=3).
Figure A.3. Cohesive properties (A) formulation with EUDRAGIT® RLPO, 153
(B) formulation without EUDRAGIT® RLPO.
Figure A.4. The effect of different formulations on tensile stress. Data represents 154
mean ± SD (n=3), ***p < 0.005.
Figure A.5. The effect of different formulations on % Elongation. Data represents 154
mean ± SD (n=3), ***p < 0.005.
xxiii
Figure A.6. Rheological behavior of different drug loaded patch formulations in 156
terms of oscillation frequency sweep data (A) the elastic or storage modulus and
(B) the viscous or loss modulus were plotted against angular frequency.
Figure A.7. In vitro release profile of lidocaine from experimental and marketed 157
patch formulations in phosphate buffer at pH 7.4 (N=3).
Figure A.8. Higuchi release kinetics, in phosphate buffer at pH 7.4 after 25 h 159
(N=3).
Figure A.9. SEM-EDS photographs of (A) lidocaine 4% transdermal patch (B) 160
lidocaine 10% transdermal patch after 30 days and (C) marketed 5% lidocaine patch.
Figure A.10. Differential scanning calorimetry profiles of different components 162
in transdermal patches: (A) Pure lidocaine; (B) 1:1:1 physical mixture of
EUDRAGIT® RLPO, HPMC and chitosan; (C) 1:1:1:1:1 physical mixture
of EUDRAGIT® RLPO, HPMC, chitosan, TEC and lidocaine; (D) Ex lidocaine
patch 10% and [E] Ex lidocaine patch 20%.
Figure A.11. Effect of shower or 40 psi water pressure on marketed (A-C) 166
and experimental patches (D-F).
1
Chapter 1. Background and Specific aims
1.1.Skin physiology
The skin is a complex arrangement of structures and has a multifunctional role-
provides a physical barrier to the environment by acting as a protective barrier against the
ingress of foreign material, maintains homeostasis and thermoregulation by limiting the
loss of water, electrolytes, and heat and prevents microbial colonization [1]. It is the largest
organ in the body and occupies about 16% of the total body weight of an adult and has a
surface area of about 2 m2 [2]. It weighs approximately 3-5 kg, twice as much as the brain.
Even though it is structurally continuous throughout the body, skin varies in thickness
according to the function, age of the individual and area of the body (on the eyelids, the
skin is only 0.5 mm thick, whereas on the soles of the feet it can has the thickness of 3-4
mm). In general, skin is 1-2 mm thick. In about one square centimeter of skin there are 10
hair follicles, 100 sweat glands, 15 sebaceous glands, 12 nerves, 360 cm of nerves and 3
blood vessels [3]. Hair, nails, sweat glands and sebaceous glands are considered to be skin
appendages or derivatives. The skin is a multilayered organ (Figure 1.1 and 1.2) and can
be considered to have four distinct mutually dependent tissue layers [4]:
1. Non-viable epidermis (stratum corneum)
2. Viable epidermis
3. Viable dermis
4. Subcutaneous connective tissue (hypodermis)
Its multilayered structure reflects the barrier properties of the skin (Figure 1.2) and each
layer is known to represent different levels of cellular or epidermal differentiation.
2
Figure 1.1. Structure of the skin [5].
Non-viable epidermis (stratum corneum)
The stratum corneum (SC) is the uppermost layer and consists of 10-15 layers
of corneocytes and varies in thickness from approximately 10-15 µm in the dry state to
40 µm when hydrated. These corneocytes are denucleated, nonliving, flattened cells-
34-44 µm long, 25-36 µm wide, 0.5- 0.20 µm thick with a surface area of 750 to 1200
µm2.
3
Figure 1.2. Anatomy and physiology of the skin [6].
So, the SC is comprised of multi-layered “brick and mortar” like structure of keratin-
rich corneocytes (bricks) embedded in a complex matrix of organized lipid bilayers
(mortar) composed primarily of free fatty acids (10-15%), long chain ceramides (40-
50%), cholesterol (25%) and 5% of other lipids (triglycerides, cholesterol sulfate and
sterol/wax esters) [7-11]. The barrier property of the SC is governed by the presence of
79-90% of protein and 5-15% of lipids and was first established in the 1940s [12]. The
protein part of the SC primarily contains approximately 70% of α-keratin, 10% of β-
keratin and 5% of the cell envelope. The SC is lipophilic and contains 13% of water
and the skin’s hydrophilic properties increase from the surface as its depth increases.
4
The SC is crucially important in controlling the percutaneous absorption of most drugs
and other chemicals. Table 1.1 represents the regional variations in drug permeability
through SC.
Table 1.1. Regional variations in drug permeability of stratum corneum [13].
It is generally thought that drug can be penetrated by three pathways in the SC (Figure 1.3)-
1. Transcellular Route: most direct route and require transport through densely packed
keratin-filled corneocytes followed by multiple transfers between the corneocytes
and the lipid filled intercellular areas.
2. Paracellular Route: the most common penetration pathway of drug molecules. In
this pathway, drug remains in the lipid moiety and stay around keratin and follows
a tortuous diffusion pathway.
3. Transappendgeal Route: the route via skin appendages (hair follicles, sweat glands)
which form shunt pathways through the intact epidermis.
5
Figure 1.3. Schematic representation of penetration pathways [6].
Viable epidermis
Below the SC, the remainder of the epidermis is viable tissue called viable
epidermis. It has a thickness of 50-100 µm and contains nucleated cells called
keratinocytes. It is a region for drug binding, metabolism, active transport, and
surveillance. As the water content of epidermis is about 90%, the density of this region is
not much different than water. The epidermis is avascular (without blood vessels) and it
6
depends on blood vessels of the dermis for oxygenation, metabolic provision and removal
of metabolic waste products. The epidermis is made up of a number of layers-
➢ Stratum basale- is the nearest layer to the dermis and is made up of a single row of
columnar keratinocytes. It is the only layer within the epidermis that consists of
cells capable of division. Keratinocytes in the stratum basale undergo mitosis and
produce two daughter cells. One remains in this layer while the other migrates up
through the other layers to the surface of the epidermis. As the daughter cells move
away from the stratum basale, they receive less nutrition and the cells die. This
whole process takes approximately 28 days. In healthy skin there is a balance
between the formation of new keratinocytes in the stratum basale and the shedding
of dead keratinocytes from the stratum corneum. The stratum basale also contains
melanocytes, which produce skin pigment called melanin that protects the skin
from the harmful effects of ultraviolet (UV) light. Merkel cells are also found in
the stratum basale. They make contact with the flattened process of a sensory
neuron called a Merkel disc. Merkel cells and their discs together detect the
sensation of touch.
➢ Stratum spinosum- as the daughter cells move to the stratum spinosum they lose
their ability to divide. This layer is 5 to 12 cells thick. Langerhans cells are found
in this layer. They are produced in the red bone marrow and then migrate to the
stratum spinosum where they become involved in immune responses against
microorganisms. They are also important for antigen presentation and for the
activation of T lymphocytes to destroy the appropriate cells.
7
➢ Stratum granulosum- is composed of 3 to 5 layers of flattened keratinocytes. In this
layer the cells go through a process of programmed cells death known as apoptosis.
➢ Stratum lucidum- is only found in areas where the skin is thick, such as the palms
of the hands and soles of the feet. It contains 3 to 5 layers of flattened, dead
keratinocytes and provides some degree of waterproofing to the skin.
Viable dermis
A dermal-epidermal junction separates the viable epidermis from the dermis. The
dermal-epidermal junction is not flat and distinct papillae or rete pegs can be observed and
it plays a role in the permeation of large molecular weight proteins and peptides. The
dermis is 3 to 5 mm thick and is composed of connective tissue containing collagen and
elastic fibers. This layer plays an important role in the regulation of body temperature, it
delivers nutrients and oxygen to the skin while removing waste products and toxins.
Subcutaneous connective tissue (hypodermis)
It is the deepest layer of the skin and is formed from loose connective tissue and fat
(50% of the body fat). The dermis and subcutaneous layers contain blood vessels,
lymphatics, nerve cells and skin appendages. It may also contain sensory pressure organ.
It provides thermal insulation, mechanical protection, and an energy reserve.
8
1.2. Artificial membrane
Although human skin is the most relevant membrane to evaluate permeation of a
molecule [14], there are different factors which can also affect the penetration through the
skin, such as- skin age and site, skin temperature, state of the skin (normal, diseased or
abraded), degree of hydration of the skin, pretreatment of the skin etc [6]. As the use of
human skin in research encounters ethical, health, and supply problems there is a need for
the development of skin parallel artificial membrane [15]. Table 2 shows the advantages
and disadvantages of various skin model.
Table 1.2. A list of various skin models and membranes used for skin research [5].
Artificial skin models are convenient and reproducible alternatives to in vivo and
ex vivo tests with human and animal skins. The artificial skin models range from simple
homogeneous polymer materials to lipid-based parallel artificial membrane-permeability
assay (PAMPA) [16].The synthetic membranes can be classified into two groups: group 1-
consisting of polysulfone, acrylic polymer, glass fiber, silicone, and mixed cellulose ester,
9
showed higher drug permeation compared to group 2, which included
polytetrafluoroethylene–polyethylene, mixed cellulose ester (of greater thickness), and
polypropylene [5]. Although they are not capable of mimicking the complexity of
biological skin properties, they are relatively reproducible due to their simple standardized
construction. As they eliminate the complexity of human skin, their simple structure is
particularly advantageous for understanding the basic mechanisms controlling passive
transport through a membrane [17].
Strat-M™ (Merck Millipore, USA) is a commercially available skin-mimic
artificial membrane without lot-to-lot variability, safety and storage limitations. This
membrane is composed of multiple layers of polyester sulfone (two layers of
polyethersulfone and one layer of polyolefin) with a very tight top layer creating
morphology similar to human skin [18] (Figure 1.4). These layers are increasingly porous
and larger in thickness. The polyolefin and polyethersulfone present in this membrane
represent the epidermis and dermis layer of the skin. It is generally known that SC is the
Figure 1.4. Scanning electron microscopic image of a cross-section of Strat-M™. The
first (i), second (ii), and third layer (iii) of Strat-M™ [19].
10
rate limiting barrier in the passive transport of most molecules across the skin due to the
presence of protein rich corneocytes and intercellular lipids [20]. Therefore, an artificial
membrane should have a blend of lipids in order to mimic skin barrier properties. To
provide the Strat-M™ membrane with its lipophilic quality the polymeric layers are
impregnated with a proprietary blend of synthetic lipids (Figure 1.5) that finally imparts a
skin-like properties to the synthetic membrane. Artificial membrane can be potentially used
as a screening tool to narrow the selection of formulations to be evaluated with a more
biologically relevant model. Additionally, they can be easily manufactured, easy to handle,
doesn’t require membrane preparation time and more reproducible.
Figure 1.5. Transmission scanning electron microscopic observation of a cross-section of
Strat-M™. The first (i), second (ii), and third layer (iii) of Strat-M™. Lipids in the layer
were stained with a black color. Arrows show the lipid region [19].
11
1.3. Skin penetration enhancement
The use of chemical penetration enhancers is the most widely used approach to skin
penetration enhancement across the SC barrier. There are a variety of mechanisms for
penetration enhancement by these chemical enhancers [21]:
• Lipid-fluidization of the SC leads to decreased barrier function;
• Influence the thermodynamic activity of the drug in the vehicle and the skin;
• Affect the partition coefficient of the drug in order to increase its release from the
formulation into the upper layers of the skin;
• Increase drug diffusivity in the skin;
• Create drug reservoir within the skin.
Penetration enhancers may act by one or more of these mechanisms. Figure 6 shows
the influence of enhancers on the lipophilic and hydrophilic pathway of drug penetration
[22]. Based on their mechanism of permeation enhancement more than 300 penetration
enhancers can be classified into three groups [23]:
• Group 1- enhancers that weaken the barrier by extracting skin lipids (e.g., ethanol)
• Group 2- enhancers increase drug solubility within the skin (e.g., propylene glycol)
• Group 3- enhancers disorder intercellular lipids (e.g., Azone (laurocapram))
12
Figure 1.6. Lipophilic and hydrophilic pathways of drug penetration and mode of action
of penetration enhancers [22].
Table 1.3 shows the principal classes of penetration enhancers. For the better
selection of enhancers its physicochemical properties should be compared with the drug
(e.g, the solubility parameter of the enhancer should be similar to the skin solubility, that
is 10 (cal/cm3)1/2) [21]. Penetration enhancers should contain the following properties [24]:
• It should be non-toxic, non-irritant and non-allergen
• It should be pharmacologically inert
• It should be unidirectional- should allow therapeutic agents into the body whilst
preventing the loss of endogenous material from the body
• It should be compatible with both excipients and drugs
• It should work rapidly, and the activity and duration of effect should be both
predictable and reproducible
13
• Its action should be reversible, barrier properties should return both rapidly and
fully.
Table 1.3. Examples of chemical penetration enhancers on the bases of their structure [25].
The simplest way to model the process of skin penetration is to consider the skin as
a passive membrane through which the drug has to pass. With in-vitro skin permeation
studies, a membrane is clamped between two compartments (Figure 1.7). The donor
compartment contains a drug formulation and the receiver compartment holding a receptor
solution provides a sink condition (essentially zero concentration).
14
Figure 1.7. Franz diffusion cell.
The diffusion of the compound through the skin is described by Fick’s First Law:
𝐽 = −𝐴𝐷 (𝑑𝑐
𝑑𝑥) (1)
This equation describes the rate of transfer or flux (J) of the diffusing substance through
unit area A of the membrane as being proportional to the velocity of molecular movement
through the diffusional medium or diffusion coefficient D and to the differential
concentration change 𝑑𝑐 over the differential distance 𝑑𝑥 (concentration gradient
measured across the membrane (𝑑𝑐
𝑑𝑥)). The negative sign indicates the diffusion process
occurs in the opposite direction to increased concentration or the flow is in the direction of
decreasing thermodynamic activity. Fick’s First Law is combined with the differential
15
mass balance existing in a membrane and resulted in Fick’s Second Law. This second law
of diffusion is also based on some assumptions like, the compound is not metabolized, it
does not bind with the membrane and its diffusion coefficient does not vary with position
or composition.
𝜕𝐶
𝜕𝑡= 𝐷
𝜕2𝐶
𝜕𝑥2 (2)
Equation 2 may be written as Equation 3, given that the drug is applied to the membrane
at a maximum fixed concentration in the donor compartment and maintaining sink
conditions in the receptor compartment.
𝐽 = 𝐴𝐷𝐶𝑚
ℎ (3)
Where Cm is the concentration of the compound at the donor-membrane interface and h is
the effective diffusional pathlength of the membrane. The vehicle-membrane partition
coefficient (k) can be defined as the ratio between the concentrations of the permeant in
the membrane at the donor-membrane interface and the vehicle in which it is applied (CV).
By replacing Cm in Equation 3 a modified form of Fick’s first law of diffusion can be
obtained-
𝐽𝑠𝑠 =𝐴𝐷𝐾𝐶𝑣
ℎ (4)
Equation 4 for passive drug permeation enhancement indicates that increased drug flux
should be achieved by a change in D, K and C and it can be rewritten in terms of
thermodynamic activities:
16
𝐽 =𝛼𝐷
𝛾ℎ (5)
𝛼 = 𝑡ℎ𝑒𝑟𝑚𝑜𝑑𝑦𝑛𝑎𝑚𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑤𝑖𝑡ℎ𝑖𝑛 𝑓𝑜𝑟𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛
𝛾 = 𝑡ℎ𝑒𝑟𝑚𝑜𝑑𝑦𝑛𝑎𝑚𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑤𝑖𝑡ℎ𝑖𝑛 𝑚𝑒𝑚𝑏𝑟𝑎𝑛𝑒
Equation 2 can also be simplified as-
𝑑𝑚
𝑑𝑡=
𝐷𝐶𝑜
ℎ (6)
Where m is the cumulative mass of diffusant that passes per unit area through the
membrane in time t, Co is the concentration of diffusant in the first layer of the membrane
at the skin surface contacting the source of the penetrant, and h is the membrane thickness.
In most diffusion experiments it is difficult to measure Co, but Cʹo (the concentration of
diffusant in the donor phase bathing the membrane) is usually known. Both Co and Cʹo are
related by:
𝐶𝑜 = 𝑃𝐶ʹ𝑜 (7)
Where P is the partition coefficient of the diffusant between the membrane and the bathing
solution. Substituting Equation 7 into Equation 6 gives:
𝑑𝑚
𝑑𝑡=
𝐷𝐶ʹ𝑜𝑃
ℎ (8)
The cumulative amount of drug crossing a unit area of skin against time gives a
permeation profile of the drug through the membrane (Figure 1.8). Extrapolation of the
17
pseudo steady-state portion of the graph to the intercept on the time axis gives the lag time
(L). This is the period during which the rate of diffusion across the membrane is increasing
and the steady-state conditions prevail after approximately 2.7 times the lag time [23]. The
lag time L is related to the diffusion coefficient D and D may be obtained by measuring L:
𝐿 =ℎ2
6𝐷 (9)
Figure 1.8. A typical plot of permeation study [25].
From the above equation the ideal characteristics of a molecule that would penetrate SC
well can be deduced. These are [26]:
18
• Adequate solubility in oil and water to ensure high membrane concentration
gradient
• Low molecular mass, less than 600 Da is preferred when diffusion coefficient is
high
• Optimal partition coefficient (1-3)
• Low melting point (<200°F), correlating with good solubility as predicted by ideal
solubility theory
• It should not metabolize in skin.
1.4.Topical and transdermal drug delivery
Skin formulation can be divided into three depending on different functions that
can be achieved when applying drugs to the human skin. 1) Epidermal formulation- when
the active is desirable to remain on the surface of the skin, e.g. insect repellent, sunscreen.
2) Dermal formulation- delivery of the active into the skin to target the pathological sites
within the skin, e.g. skin cancer, psoriasis, eczema and microbial infections while ensuring
minimal systemic absorption. 3) Transdermal formulation- by using skin as the application
site the active diffuses through the various layers of the skin and into the systemic
circulation to exert a therapeutic effect.
Once applied to the skin initially, the drug must be released from the
topical/transdermal formulation followed by partitioning into the SC. As a result of
concentration gradient, drug molecules will subsequently diffuse through the SC before a
further partitioning process into the viable epidermis, and further diffusion through the
viable epidermis towards the dermis. Finally, the vasculature and lymphatic vessels in the
19
dermis will clear the drug from the skin and will make it available into the systemic
circulation. The process involved in drug transport from topical or transdermal formulation
are illustrated in Figure 1.9.
Figure 1.9. Schematic representation of the process involved in drug transport from topical
or transdermal formulation [27].
20
Topical and transdermal route gives an alternative to oral and intravenous delivery.
There are several advantages of cutaneous delivery [1, 13]:
1. The avoidance of first pass metabolism and stomach environment where the drug
can be degraded,
2. Sustained and controlled delivery over a prolonged period of time,
3. Improves bioavailability,
4. Reduction in side effects associated with systemic toxicity,
5. Improved patient compliance,
6. Ease of dose termination,
7. Convenient and painless administration,
8. Suitable route for unconscious or vomiting patient,
9. Best route for pediatric patients.
There are also several limitations associated with cutaneous delivery [13]-
1. A molecular weight less than 500 Da is essential to ensure ease of diffusion across
the SC,
2. Required enough aqueous and lipid solubility,
3. Variability associated with the different skin types,
4. Pre-systemic metabolism due to the presence of skin enzymes that might
metabolize the drug into a therapeutically inactive form,
5. Skin irritation and sensitization.
21
1.5.Thymoquinone
Thymoquinone (TQ) (2- isopropyl- 5- methyl- 1,4- benzoquinone) is the main
constituent of Nigella sativa (Black cumin) seeds [28]. This herb has been popularly called
as the “seed of blessing” by the Arab population [29]. Black seed contains up to 30-48%
of TQ along with 15 amino acids, proteins, carbohydrates, fixed oils, volatile oils,
alkaloids, saponins, crude fiber, minerals such as, calcium, iron, sodium and potassium
[30]. TQ is a yellow crystalline molecule and has a basic quinone structure consisting of a
para substituted dione conjugated to a benzene ring to which a methyl and an isopropyl
side chain groups are added in positions 2 and 5, respectively (Figure 1.10). TQ has many
pharmacological properties such as anticancer, anti-inflammatory, antioxidant,
antiasthmatic and immunomodulatory effect [31].
22
Figure 1.10. Nigella sativa, black cumin seed and chemical structure of thymoquinone
[32].
The ability to formulate TQ is hindered by various factors. It is a lipophilic
molecule (log P = 2.54) and poorly soluble in aqueous medium that causes bioavailability
issues. Hence, it shows poor formulation characteristics into a conventional dosage forms
such as tablets or capsules. Additionally, its thermo-labile nature limits the application of
nano-formulation techniques to enhance its bioavailability. On the other hand, it’s low
molecular weight (164.2 gmol-1), low melting point (44-45°C), and it’s lipophilic
characteristic can be useful to formulate it in a topical or transdermal delivery system.
1.6.Specific aims
Skin has significant barrier properties that inhibit the passive transport of many
active molecules. Different strategies are developed to overcome this skin barrier such as,
chemical enhancement techniques using penetration enhancers and targeted drug delivery
using various topical or transdermal delivery system. Usually these approaches are tested
using human or animal skin. Human skin is not easily accessible and animal skin has
significant biological and barrier differences when compared with human skin. Due to
these issues the possibility of having a synthetic skin membrane is an attractive option. The
goal of this research was to further investigate the effectiveness of a synthetic membrane
compared with human skin samples for drug permeability testing, to propose for the first
time a Solubility-Physicochemical-Thermodynamic theory to define the action of
penetration enhancers and to investigate the feasibility of transdermal delivery of
23
thymoquinone (TQ) using different topical/transdermal formulation approaches and to
show its effectiveness in the treatment of wound healing and bacterial skin infections. To
achieve this goal following specific aims were identified for this research:
Specific Aim 1: Evaluating Strat-M® synthetic membrane as a screening tool for
topical/transdermal formulation.
To provide data on applicability of a synthetic membrane for in-vitro diffusion
studies in transdermal arena in place of human or animal skin as a model. To compare
different formulations containing various enhancers regarding their ability to enhance or
reduce the delivery of nicotine as a model drug through human cadaver skin and to correlate
that to a novel synthetic membrane (Strat-M® EMD Millipore, MA) to examine the
usefulness of this membrane as a convenient screening tool to investigate topically applied
formulations and TDDS (Transdermal Delivery System). To obtain the aforementioned
goal we aimed to investigate the correlation of permeation behavior of transdermal
formulations through Strat-M® membrane and human cadaver skin. Strat-M® membranes
were designed with the intent to share similar structural and chemical characteristics found
in the human skin however, omitting any biological behavior due to the absence of viable
cells. Both human skin and the membrane display a layered structure with a very tight top
layer. Additionally, the Strat-M® membrane contains a combination of lipids in a specific
ratio similar to what is found in the human stratum corneum (SC). Formulations containing
nicotine and a chemical penetration enhancer (CPE) were used for evaluating drug
penetration to understand how each enhancer impacts the permeability of nicotine as a
model compound.
24
Specific Aim 2: Solubility-physicochemical-thermodynamic theory of penetration
enhancer mechanism of action.
To date, there is limited research that demonstrate the underlying mechanism of
action of penetration enhancer. Thus, it is very important to clearly define their actions in
the formulation with the drug. The objective of this study was to propose for the first time
a Solubility-Physicochemical-Thermodynamic (SPT) theory to define the action of
penetration enhancers. The hypothesis for this investigation was that the overall
mechanism of action of skin penetration enhancers is best explained by the SPT theory.
The SPT theory puts forward the concept that the mode of action of enhancers is related to
solubility parameters, physicochemical interactions and thermodynamic activity. This
study discusses these concepts by using experimentally derived permeation data, various
physicochemical and solubility parameters (ingredient active gap (IAG), ingredient skin
gap (ISG), solubility of active in the formulation (SolV) and the formulation solubility in
the skin (SolS)) generated by using FFE (Formulating for EfficacyTM - ACT Solutions
Corp) software. This work will connect the solubility and physicochemical properties of
the active and enhancers/ingredients with the thermodynamic activity of the model drug
used in order to explain the mode of action of enhancers in a given formulation with that
specific drug.
Specific Aim 3: Effects of solvents and penetration enhancers on transdermal
delivery of thymoquinone: permeability and skin deposition study.
Thymoquinone (TQ) is a quinone-based phytochemical and was first identified in
1963 in Nigella sativa (black cumin seed) by El-Dakhakhany. Based on the ideal
characteristics of transdermal delivery, TQ can be an attractive candidate for TDDS
25
(transdermal drug delivery system). The aim of this study was to investigate for the first
time the feasibility of transdermal delivery of thymoquinone (TQ) and to assess the effect
of ethanol and propylene glycol (PG) as solvents together with the effects of selected
chemical penetration enhancers on the in vitro human skin deposition and permeation of
TQ. To investigate both transdermal flux and skin deposition of thymoquinone and the
various conditions that may influence these including various vehicle/solvents, receiver
composition and permeation enhancers. The effects of penetration enhancers on the in vitro
skin permeation and TQ skin absorption were studied using human cadaver skin and Franz
diffusion cell method. The permeation of saturated solutions of TQ was investigated with
5% concentrations of each of the following enhancers: Azone (laurocapram), Transcutol®
P (Tc), oleic acid, ethanol, Polysorbate 80 (Tween 80), and N-methyl-pyrrolidone (NMP).
Specific Aim 4: Thymoquinone loaded polymeric films and hydrogels for wound
healing and the treatment of bacterial skin infections.
The purpose of this study was to synthesize and characterize a biocompatible novel
topical polymeric film and hydrogel system that has the potential to deliver
antibacterial/anti-inflammatory agent thymoquinone (TQ) directly to the skin target site
and that may be useful for the treatment and management of skin wound infections. TQ
loaded polyvinyl pyrrolidone (PVP) matrix-type films and hydrogels with different
polymers were prepared. The developed films were evaluated for thickness, drug content
uniformity, weight variation, flatness, folding endurance, percentage of moisture content
and uptake. The surface morphology of the film was recorded with a Zeiss field emission
scanning electron microscopy (FESEM) and in vitro skin permeation studies were
performed on human cadaver skin by using Franz diffusion cells (FDC). Human dermal
26
fibroblasts (HDFs) and Human keratinocytes (HaCaT) cell lines were used for wound
healing scratch assay. The proliferative effect of different concentration of TQ (1-1000 ng)
were also investigated on HDF and HaCat cell lines. Antibacterial activity of TQ loaded
films and hydrogels against Staphylococcus aureus were assessed using the disc diffusion
method (in vitro) and an ex vivo human cadaver skin explant. To evaluate the TQ film’s
preclinical and in vivo efficacy wound infection animal model was used. This animal
model was made by inoculating Staphylococcus aureus at a concentration of 108 CFU/mL
to create an in vivo bacterial wound infection at their dorsal side. Bacterial samples were
taken from the animal site at pre-determined time points and were analyzed for the bacterial
numbers. At the end of the study, all the animals were sacrificed, and the histology of the
skin samples were examined. Efficacy was determined by the % of wound closure and log
of bacterial reduction of the wound site.
27
Chapter 2. Evaluating strat-M® synthetic membrane as a screening tool for
topical/transdermal formulation
2.1. Introduction
Synthetic membranes for in vitro permeation studies were originally developed to
be used as an alternative to using a human skin models [33]. Determination of drug
permeation of formulations using ex vivo human skin methods possesses several
drawbacks which hinder reproducibility data of drug candidate screening including:
variations of skin thickness from skin donors, diseased skin states, skin storage conditions,
membrane preparation complexity, density of hair follicles, age of donor, and high
laboratory costs [34, 35]. Some advantages of using a synthetic membrane are: controlled
membrane thickness, faster membrane preparation time, low storage space, and relatively
low cost. The human stratum corneum (SC) is commonly the rate limiting step for
successful API (active pharmaceutical ingredient) delivery [36, 37]. It consists of 10-15
parallel layers of corneocytes embedded in an intercellular lipid matrix of mainly
ceramides (50%), cholesterol (25%) and free fatty acids (15%), in a bricks and mortar
arrangement [22]. There are several techniques such as chemical enhancement, physical
enhancement, and drug modification that have been employed to change the barrier
properties of stratum corneum [26]. Among these using chemical penetration enhancers is
the most widely used technique since these compounds can reversibly alter the stratum
corneum’s barrier function [38]. Usually chemical enhancers act by lipid disruption and at
acceptable concentrations they interact and affect the stratum corneum intercellular lipid
domain or organization and make the stratum corenum more permeable [39].
28
Understanding the physiochemical relationship of API/vehicle interactions through a
membrane barrier is critical for selection of optimal formulation penetration enhancement
efficacy [40]. In this regard, we report here on the development of nicotine solutions
containing penetration enhancers to evaluate the permeability correlations of Strat-M®
(EMD Millipore, MA) synthetic membrane with human cadaver skin. Nicotine is
commonly used for nicotine replacement therapy (NRT) to encourage successful smoking
cessation from tobacco products [41]. Particular chemical properties such as: low
molecular weight (162.23 g/mol), logP (1.2) and its wide-spread use make this compound
an ideal candidate for testing. It is commercially available as a TDDS (Transdermal
Delivery System).
Most commonly used synthetic membranes models lack the biological composition
of a highly structured stratum corneum, metabolic processes, and interactions of proteins
found in the human epidermis. Strat-M® membranes were designed with the intent to share
similar structural and chemical characteristics found in the human epidermis. The multiple
layers of stratum corneum undergo a process called keratinization. In this process as the
cells formed and migrated upwards to the skin surface from the basal layer stem cells the
concentration of oxygen and nutrients decrease, and the cells become flatter and
accumulation of keratin and lipids occurs. Strat-M® membrane was engineered to mimic
the layered structure and lipid chemistry of human skin (Figure 2.1). The thickness of each
Strat-M® membrane is approximately 300 µm; comprising a top layer supported by two
layers of porous polyether sulfone (PES) on top of one single layer of polyolefin non-
woven fabric support. Membrane layers are increasingly more porous and open and also
increasingly larger in thickness to mimic different layers of human skin (epidermis, dermis
29
and subcutaneous tissue). These multiple layers of the membrane create a morphology
similar to that of human skin. Both human skin and the membrane display a layered
structure with a very tight top layer. The porous membrane was treated with a proprietary
blend of synthetic lipids. Skin contains various lipids, such as phospholipids and ceramides,
which impart hydrophobic character to skin. This synthetic membrane contains a
combination of lipids (ceramides, cholesterol, free fatty acids, and other components) in a
specific ratio similar to what is found in the human SC.
Figure 2.1. Multilayered structure of Strat-M® membrane.
Strat-M® serves a purpose to be a cost-effective membrane for testing and
optimizing pharmaceutical formulations with good reproducibility to increase confidence
during early stage drug/formulation development. This synthetic membrane can be used
30
for high throughput formulation screening during the early stages of formulation
development to test for API’s, personal care products, pesticides, cosmetic actives, and
chemical warfare protective formulations. Furthermore, a need for high quality methods
that help determine safety and bioequivalence for formulations including those containing
CPE’s are sought by regulatory agencies to speed up the long approval times needed in
order for generic drug clearance and approvals [42]. Bioequivalence studies are typically
conducted using human cadaver skin or animal models. Unfortunately these models
experience a number of drawbacks that make them not very suitable for development
including: complex sample preparation, strict sample storage requirements, biohazard
issues and expensive study costs [43].
The objective of the present study was to compare different formulations containing
various enhancers regarding their ability to enhance or reduce the delivery of nicotine
through human cadaver skin and to correlate that to Strat-M® synthetic membrane to
examine the usefulness of this membrane as a convenient screening tool to investigate
topically applied formulations and TDDS.
2.2.Materials and Methods
2.2.1. Materials
Polysorbate 80 (Tween 80), eucalyptol, N-methyl-2-pyrrolidone (NMP), propylene
glycol, sodium phosphate monobasic were purchased from Sigma-Aldrich Co. (St. Louis,
MO, USA). Nicotine was purchased from Alfa Aesar (Haverhill, MA, USA) and
Laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA). Phosphate-
buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals, LLC (Solon,
31
OH, USA) and o-Phosphoric Acid 85% was purchased from Fisher Scientific (Hampton,
NH, USA). Dermatomed human cadaver skin from the posterior torso of three different
donors were obtained from New York Firefighter Skin Bank (NY, USA). Strat-M®, high-
performance liquid chromatography (HPLC) grade water and acetonitrile was a gift from
EMD Millipore (Danvers, MA, USA).
2.2.2. Preparation of formulations
Five formulations were prepared (10 mL) containing 1% nicotine, with or without
5% of enhancer (Azone, Tween 80, eucalyptol, or N-methyl-2-pyrrolidone) in propylene
glycol.
2.2.3. In vitro skin permeation test (IVPT) studies
Each of the five formulations (as described above) were applied to dermatomed
human cadaver skin with the dermal side in contact with filtered PBS (pH 7.4) and Strat-
M® membrane with the shiny side in contact with the donor compartment, both mounted
on Franz diffusion cells with a donor area of 0.64 cm2 and a receptor volume of 5.0 mL
(Permegear Inc., Hellertown, PA). Dermatomed human cadaver skin samples (~500 μm)
from the posterior torso of three different donors (2 white males at the age of 68, 45 and
one white female at the age of 34) obtained from New York Firefighters Skin Bank (New
York, NY) were used for skin permeation study. Prior to using the skin, the samples were
slowly thawed, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for 15
minutes. Strat-M® membrane (EMD Millipore, MA, USA) does not require any
32
pretreatment, thus was used immediately after removing from the packaging. The skin and
the Strat-M® membrane was not occluded in the Franz cells and the receptor compartment
of each cell was filled with filtered PBS (pH 7.4) and maintained at 37oC under
synchronous continuous stirring using magnetic stirrers at 600 rpm. The diffusion cells
were allowed to equilibrate at 37oC for 15 minutes. Then at time zero 200 µL of formulation
was added to the donor compartment of each Franz diffusion cell using a positive
displacement pipette set and the dose was spread through the surface with the tip of pipette.
At each time point (1, 2, 3, 4, 5, 6, 7 and 8 hours) 300 µL of receptor were withdrawn from
the sampling port. At the end of 8 hours, all receptor samples were analyzed using a valid
HPLC method described below.
2.2.4. High-performance liquid chromatography (HPLC)
Nicotine was quantified using a validated HPLC method and an Agilent 1100 series
HPLC (Agilent Technologies, CA, USA) coupled with UV (259 nm) and a diode array
detector (DAD). A mobile phase of 65% sodium phosphate buffer (adjusted to pH 3.2 with
85% orthophosphoric acid) and 35% acetonitrile was pumped at a flow rate of 1.0 mL/min
through a Phenomenex Luna® 5 µm C18(2) 100 Å Column 250 X 4.6 mm (ambient
temperature). The retention time for nicotine was 2.5 minutes. The method was linear at a
concentration range 4 -500 µg/mL with R2 of 1. The limit of quantification is 1.2 µg/mL
and the limit of detection is 0.35 µg/mL.
33
2.2.5. Data analysis
Penetration parameters were obtained from the cumulative amount of nicotine
permeated per unit skin surface area (µg/cm2) versus time (hours) plot. The effectiveness
of the penetration enhancers (EF= enhancement factor) was determined using Equation 1.
EF = Flux with the enhancer/Flux without the enhancer (1)
2.2.6. Statistical analysis
Results are reported as mean ± SD (n=6). The statistical analysis of the data was
performed by using one-way Anova and Student’s t-test, and p-values < 0.05 were
considered significant.
2.3. Results and discussion
Penetration enhancers are employed across various areas of drug delivery and are
classified into separate classes depending on their chemical composition [44]. In this
experiment 5% of different enhancers were selected to represent enhancer groups (amide,
surfactant, pyrrolidone, glycol, terpene) due to the fact that each class of enhancers
modifies skin lipids in different ways and the mechanism of action is linked to their ability
to interact with skin lipids [27]. The results showed that the rank order for nicotine flux for
each enhancer for both Strat-M® and human cadaver skin are: Azone > eucalyptol + Tween
80> control> N-methyl pyrrolidone. Effect of each enhancer is discussed below.
34
2.3.1. Effect of Azone
Azone (1-dodecylazacycloheptan-2-one) was the first compound specifically
designed as a chemical penetration enhancer [45]. It is a colorless and odorless liquid and
has a melting point of -7°C. Structurally, Azone comprises a polar headgroup attached to
a C12 chain and is a highly lipophilic material with a logPoctanol/water value around 6.2. Due
to its non-polar nature Azone dramatically affected the lipid structure and showed no
protein interaction. It further suggests that at a low concentration Azone does not enter the
cells in significant amount. Azone is known to significantly enhance penetration of API’s
in transdermal formulations by disrupting the normal lipid bilayer packing structure
arrangement in the SC [26, 46]. It is highly effective when used in conjunction with
propylene glycol. Our study also showed its effectiveness with propylene glycol. Tables
2.1 and 2.2 and Figure 2.2 show the amount of nicotine permeated per square cm from
formulation 4 (Nicotine 1% in propylene glycol + 5% Azone) in both Strat-M® and human
cadaver skin after 8 hours (Q8) was significantly greater when compared to control and all
other formulations (p<0.05). This behavior can be explained by the fact that Azone
enhances intercellular drug diffusion only and cannot affect intracellular protein contents.
On the other hand, propylene glycol enhances intracellular transport. So, the combination
of propylene glycol and Azone is more effective. The mechanism of perturbation is
suggested to be caused by Azone attaching and separating the junctions between polar
heads and lipids tails of the SC causing fluidization of the intercellular lipids which result
in a more fluid-like structure (Figure 2.3) [47]. The above mechanism of action was
supported by a ‘soup spoon’ model for Azone’s conformation within stratum corneum
lipids [48].
35
Table 2.1 Penetration parameters of nicotine through Strat-M® membrane after 8 hours.
Formulation Q at 8 hours (µg/cm2) J (µg/cm²/hr) EF
through Strat-M
Control 1220 ± 64 163 -
NMP 1181 ± 73 157 0.96
Tween 80 1802 ± 135 250 1.53
Azone 2606 ± 302 338 2.07
Eucalyptol 1845 ± 278 264 1.61
Q, cumulative amount of nicotine penetrated per cm2 at 8 hours (mean ± SD, n=6); J is
flux determined from slope of the cumulative amounts of nicotine permeated versus time
profiles; EF, flux of nicotine from the formulation containing an enhancer divided by the
flux of nicotine from the control formulation without an enhancer.
Table 2.2 Penetration parameters of nicotine through human cadaver skin after 8 hours.
Formulation Q at 8 hours (µg/cm2) J (µg/cm²/hr) EF
through Skin
Control 87 ± 8 7 -
NMP 81 ± 17 6 0.85
Tween 80 167 ± 43 14 2
Azone 484 ± 86 21 3
Eucalyptol 168 ± 41 16 2.28
Q, cumulative amount of nicotine penetrated per cm2 at 8 hours (mean ± SD, n=6); J
is flux determined from slope of the cumulative amounts of nicotine permeated versus
time profiles; EF, flux of nicotine from the formulation containing an enhancer
divided by the flux of nicotine from the control formulation without an enhancer.
36
Figure 2.2. Cumulative amounts of nicotine per cm2 of membrane/skin permeated after 8
hours through Strat-M® membrane (n=6) and human cadaver skin (n=6) samples.
2.3.2. Effect of Propylene Glycol
Propylene glycol is considered to be part of the polyols class which has superior
solubilizing capabilities in both water and oil. Since 1932 propylene glycol has been used
either as a co-solvent and/or to enhance drug permeation through skin. This co solvent was
selected due formulation compatibility to be used with CPE’s for increasing the effects of
solubility [49-51]. Bouwstra et al. (1989) suggested that propylene glycol decreased the
hydration of skin [52]. It might be incorporated in the head group regions of the lipids and
0
500
1000
1500
2000
2500
3000
Control N-Methyl
Pyrrolidone
Tween 80 Azone Eucalyptol
Cu
mu
lati
ve
am
ou
nt
of
nic
oti
ne
per
mea
ted
(ug
/cm
2)
Strat-M
Skin
37
probably acts by solvating alpha-keratin and occupying hydrogen-bonding sites. Evidence
suggests a very mild enhancement effect of propylene glycol for estradiol and 5-
fluorouracil [24].
Figure 2.3. Schematic representation of Azone disrupting the stratum corneum
intercellular lipids.
2.3.3. Effect of Eucalyptol and Tween 80
In this study, eucalyptol and Tween 80 shared similar trends by providing lower
nicotine flux in comparison to the formulation containing 5% Azone and higher flux of
nicotine when compared to control and N-methyl pyrollidone (5%). Tween 80 is a non-
ionic surfactant widely used to make emulsion formulations. The mechanism of enhancing
drug delivery is different than that of Azone and this surfactant works by allowing polar
molecules to partition across the barrier more easily by the incorporation of micelles [53].
38
Micelles may use some SC barrier lipids to favor permeation of these vehicles containing
hydrophilic API’s. Eucalyptol was selected from the class of terpenes and can be rapidly
metabolized by the skin making them a useful penetration enhancer for transdermal drug
delivery systems [54]. The mechanisms of enhancement include the modification of the
structure of the SC lipids to increase diffusivity of hydrophilic compounds [55].
2.3.4. Effect of N-methyl pyrrolidone
The formulation with 5% N-methyl pyrrolidone did not show any enhancement
effect on nicotine permeation through Strat-M® membrane and human cadaver skin. The
mechanism of enhancement is different than others mentioned in which reservoirs or
clusters containing drug in vehicle are formed between the lipids of the SC and are more
soluble providing a prolonged sustained release of drug from vehicle [56, 57].
2.3.5. Enhancement factor
Tables 2.1 and 2.2 and Figure 2.4 shows the enhancement factor for a given
enhancer of nicotine permeation for Strat-M® and human cadaver skin. Although, Strat-M®
lacks the highly organized SC intercellular structure and provided higher nicotine flux
compared to human cadaver skin where the highly structured SC significantly reduced
nicotine permeability, both membranes still provided similar enhancement factors for a
given enhancer. These studies suggest that the Strat-M® synthetic membrane lipid
composition probably closely mimics that of human cadaver skin based on the data
obtained.
39
Figure 2.4. Human cadaver skin and Strat-M® membrane enhancement factors for different
formulation enhancers.
2.3.6. Correlation of Strat-M® membrane to human cadaver skin
Several studies have investigated the correlation of different synthetic membrane
to human cadaver skin. Shah et al., prepared hydrocortisone creams and tested using pure
cellulose acetate, cellulose and polysulfone synthetic membranes [58]. These membranes
had good reproducibility and were eventually used for quality control assurance for batch
uniformity, but studies concluded that no correlations existed between the synthetic
membranes and ex vivo human skin models. Another study performed by Ng et al., used
an ibuprofen formulation to test drug penetration using various polymeric membranes [59].
This work concluded that no correlations with human skin existed with membrane pore
0
2
4
N-Methyl Pyrrolidone Tween 80 Azone Eucalyptol
En
ha
nce
men
t F
act
or
Strat-M
Skin
40
size, thickness, and polymer molecular weight and that only the polymer matrices
themselves were limiting drug vehicle interactions. Additionally, no correlations were
drawn comparing the flux of drug with the membrane and human skin. In a recent study
conducted by Simon et al., synthetic membranes were used to compare with an ex vivo pig
ear skin model for transdermal drug permeation of rivastigmine [60]. Results concluded
that most polymeric membranes tested had low correlation factors (R2 = < 0.90). In this
study, Strat-M® synthetic membrane demonstrated a relatively high correlation to human
skin for nicotine flux with a R2 value of 0.99 (Figure 2.6). Moreover, the time point
correlation between Strat-M® and human cadaver skin were in the range 0.90-0.99 (Figure
2.5C). This work suggests that some of the main transport mechanisms for drug diffusion
and permeation could be similar to an ex vivo human skin model. But more studies needed
to be performed with different hydrophilic and lipophilic compounds to further confirm
these suggestions.
2.4. Conclusions
In summary, this study has demonstrated that a novel Strat-M® synthetic membrane
has potential to be used as an early screening model to select the best performing CPE for
transdermal formulations containing nicotine. Our findings suggest that Strat-M® can
provide useful predictions based on the major role partitioning plays in passive drug
diffusion. The synthetic membrane also offers an additional value by providing rank
ordering of penetration enhancers from enhancement effectiveness standpoint. Although
41
Fig. 6.
(C)
0
25
50
75
100
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Control
0
1000
2000
3000
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Tween 80
0
1000
2000
3000
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Eucalyptol
0
500
1000
1500
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
N-Methyl Pyrrolidone
0
100
200
300
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Tween 80
0
25
50
75
100
0 500 1000 1500
Q (
Skin
, ug/c
m-2
)
Q (Strat-M, ug/cm-2)
Control
0
25
50
75
100
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
N-Methyl Pyrrolidone
0
25
50
75
100
0 500 1000 1500
Q (
Skin
, ug/c
m-2
)
Q (Strat-M, ug/cm-2)
N-Methyl Pyrrolidone
0
50
100
150
200
0 1000 2000
Q (
Skin
, ug/c
m-2
)
Q (Strat-M, ug/cm-2)
Tween-80
0
50
100
150
200
0 1000 2000Q (
Skin
, ug/c
m-2
)
Q (Strat-M, ug/cm-2)
Eucalyptol
0
100
200
300
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Eucalyptol
0
200
400
600
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Azone
0
1000
2000
3000
4000
0 2 4 6 8
Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Azone
0
1
2
3
0 1000 2000 3000
Q (
Skin
, ug/c
m-2
)
Q (Strat-M, ug/cm-2)
Azone
0
500
1000
1500
0 2 4 6 8Per
mea
tio
n (
ug/c
m-2
)
Time (h)
Control
(A) (B)
R²=0.98
R²=0.99
R²=0.99
R²=0.98
R²=0.90
42
Figure 2.5. Nicotine penetration profiles of transdermal formulations (A) human cadaver
skin, (B) Strat-M® membrane, and (C) time point correlations between the amounts of drug
penetrated through human cadaver skin and Strat-M® membrane. Means plus minus S.D.
and correlation coefficients.
Figure 2.6. Correlation of flux between Strat-M® synthetic membrane and human cadaver
skin.
Strat-M® will only provide information on trends and correlations and not match the
absolute permeability values for human skin, still ranking the order of penetration
enhancement efficiency is helpful for understanding which modes are the most effective
for developing an optimized formulation. This however, is information that can be used in
preliminary screening efforts in the pharmaceutical, cosmetic and personal care industries
to select formulations or penetration enhancers or vehicles for drugs/actives. Moreover, the
synthetic membranes are a convenient tool to investigate and to determine the most
effective chemical penetration to be used in vivo with human skin. The Strat-M® synthetic
R² = 0.99
0
5
10
15
20
25
0 50 100 150 200 250 300 350 400
Hu
ma
n C
ad
av
er S
kin
Flu
x (
µg
/cm
²)
Strat-M Synthetic Membrane Flux (µg/cm²)
43
membrane serves as a rapid, cost-effective, easy and ready to use method with no solvent
activation requirements. Thus, synthetic membranes can be successfully used as a
screening tool in order to choose the best performing chemical enhancers and to evaluate
the transdermal formulations through the human cadaver skin.
The Strat-M® membranes share similar drug permeability behavior of nicotine
permeation to an ex vivo human skin model; which is mainly driven by drug partitioning.
Major structure organizational differences between both models; limiting the applicability
to be used as a direct correlation prediction. Enhancement of physiochemical behavioral
properties in formulations show similar trends between both models. Future work in this
area is still needed to understand the impact of CPE’s from other classes on other drug
candidates including ones with lipophilic properties.
44
Chapter 3. Solubility-physicochemical-thermodynamic theory of penetration
enhancers mechanism of action
3.1.Introduction
The passage of the drugs through skin mainly follows laws of passive diffusion and
can be described by Fick’s first law [27]. According to this law a permeant will move down
a concentration gradient [60] from a region of high concentration to a region of lower
concentration. Mathematical models derived by Higuchi explained this passive diffusion
process in terms of percutaneous absorption [61]. Moreover, Higuchi used
physicochemical principles in describing the importance of the thermodynamic activity of
the permeating molecule [62]. The thermodynamic driving force for the partitioning of
drug depends on the difference between the chemical potential of drug in a particular
vehicle and skin [63]. From the thermodynamic point of view, the steady state flux (J) can
be expressed by [61] -
J = αD/ϒ L (1)
where α is the thermodynamic activity of the drug in its vehicle, ϒ is the activity
coefficient of the drug in the skin, D and L are diffusion coefficient of the drug in the skin
and skin thickness respectively.
The extent of the percutaneous drug absorption from a topically applied formulation
is greatly influenced by the physicochemical properties of the drug. Due to the dependency
on physicochemical properties there is only a very small group of drugs that can be
45
delivered through the transdermal route utilizing passive diffusion alone. There are several
techniques among which is the use of chemical enhancers – a most convenient and popular
technique to modify the permeation through the skin. By using different penetration
enhancers, we can modify the thermodynamic activity of the drug in the formulation and
as a result can manipulate permeant flux. In this way we can also increase the range of
drug candidates that can be effectively delivered through transdermal route.
To date, there is limited research that demonstrate the underlying mechanism of
action of these enhancers [64]. Thus, it is very important to clearly define their actions in
the formulation with the drug. Lipid-Protein-Partitioning theory [65] is based on the fact
that enhancers usually work by one or more of the following three mechanisms-
1) The disruption of the lipid domains of the stratum corneum;
2) Interaction with intracellular protein; and
3) Increasing partitioning of a drug, co-enhancer or co-solvent into the tissue.
These are all broadly described mechanisms that do not provide details why a given
enhancer will not increase the permeation of all types of drugs and why the permeation
ability of enhancers depends on their concentration and why some of them are most
effective at lower rather than higher concentrations. In this manuscript we provide evidence
that the mechanism of action of enhancers is related to solubility parameters,
physicochemical interactions as well as thermodynamic activity. The objective of the
present investigation was to propose for the first time a Solubility-Physicochemical-
Thermodynamic theory to define the action of penetration enhancers. While developing
this approach, the following important factors had to be considered-
46
1) The potency of penetration enhancers appears to be drug specific due to the
importance of physicochemical interactions between drug and enhancers and thus
should be compared. Here we describe this interaction as the “ingredient active
gap” (IAG).
2) The effectiveness of most penetration enhancers is concentration dependent and it
must be remembered that penetration enhancers also permeate through the skin with
the vehicle and active molecule.
3) The affinity of the enhancers to the skin. There is a link between the mechanism of
action of enhancers and their affinity to skin. Here we describe this affinity as the
“ingredient skin gap” (ISG).
4) The thermodynamic activity effect provided by the addition of an enhancer.
Enhancers can increase or optimize the thermodynamic activity of the drug in the
formulation as well as the effects in the skin.
5) The solvent properties of the skin that affect the permeants. Thus, solubility of
active in the formulation (SolV) and the formulation solubility in the skin (SolS)
should also be considered as both influence drug permeation.
This work is the missing piece of the puzzle that will connect the physicochemical
properties of the active and enhancers/ingredients with the thermodynamic activity of the
drug in order to explain the mode of action of enhancers in a given formulation with a
specific drug. There is definitely a need for theoretical bases that will allow prediction of
the effect of enhancers on skin flux of drugs. Here we propose the basis of this theory that
47
allows the prediction of outcomes and to correlate these with in vitro experimental
diffusion cell data.
3.1.1.Theoretical background
The cohesive energy density which is equal to the energy of vaporization divided
by the molar volume reflects the degree of attractive forces holding the molecules together
[66]. In 1950 Hildebrand introduced solubility parameter δt, the square root of the cohesive
energy density (ced) [67, 68].
δt = (ced)1/2 = (𝛥𝐸𝑣
𝑉)1/2 (2)
where 𝛥𝐸𝑣 is the latent energy of vaporization, V is the molar volume. The latent energy
of vaporization is usually calculated by-
𝛥𝐸𝑣 = 𝛥𝐻𝑣 -RT (3)
where 𝛥𝐻𝑣 is the latent heat of vaporization, 𝑅 is the universal gas constant, and 𝑇 is the
absolute temperature.
It was realized by Charles M. Hansen in the mid-1960s that in the evaporation
process all of the cohesive bonds holding the liquid together were broken and led to an
expansion of the Hildebrand solubility parameters δt into three dimensions [69] : dispersive
interactions (δD) – the dispersion or van der Waals properties of a molecule, or the amount
of polarizable electrons. It has a strong correlation with refractive index which is, at a
deeper level, a correlation with the polarizability of a molecule. Those molecules with more
electrons able to move freely at the surface have a higher polarizability, a higher refractive
48
index, stronger van der Waals interactions and a higher δD. polar interactions (δP) – the
polar contribution – related to (and calculable from) dipole moment. This is generally
thought of as classic polarity. Something with a large dipole moment will have a large δP.
and hydrogen bonding (δH) – the hydrogen bonding contribution is usually found by
subtracting the polar and dispersion parameters from the total parameter, or in terms of
energies, by subtracting the dispersion and polar energies from the cohesive energy. This
is important for a number of the solvents and solutes in the study and should not be ignored.
(δt)2 = (δD)2 + (δP)2 + (δH)2 (4)
These three solubility parameters can be visualized using FFE (Formulating for
EfficacyTM - ACT Solutions Corp) Software with a three-dimensional coordinate system,
the so-called Hansen space with axes δD, δP and δH. The Hansen Solubility Parameter (HSP)
methodology is based on the Hansen Solubility Parameters in Practice (HSPiP) program
from Steven Abbott, Charles Hansen and Hiroshi Yamamoto. It incorporates the Y-MB
(Yamamoto Molecular Breaking) methodology for calculating HSP from SMILES
developed by Hiroshi Yamamoto. The Formulating for Efficacy (FFE) software program
was written for Dr. Johann Wiechers, a noted expert in active delivery by Professor Steven
Abbott and based on the HSPiP software with additions and adaptations for utility in
pharmaceutical and cosmetic applications. Among the additions is the Diffusion Modeler
which mimics the dynamics of a Franz diffusion cell, using accepted principles of diffusion
and HSP of the stratum corneum.
The closer the HSP (Hansen solubility parameters) values of two substances or
smaller the distance between the coordinates of two substances in the 3D HSP space the
49
higher is their similarity and the stronger is their affinity [70]. The distance between the
materials (Ra) on such plots is calculated by the following equation [69]:
Ra2 = 4(δD1 - δD2)2 + (δP1 – δP2)
2 + (δH1 – δH2)2 (5)
In this equation subscript 1 and 2 refer to material 1 and 2. For high affinity between
two materials Ra must be less than the R0 (radius of a Hansen solubility parameter sphere)
of the test material. R0 describes how large or small the interaction range is. Relative
Energy Difference (RED) value is used to quantify relative distances between Ra and R0
and RED < 1 represents high affinity whereas RED > 1 represents low affinity [71]
RED = Ra/ R0 (6)
The FFE software plots the δD, δP and δH of each compound along the three axes of
a 3D graph after which the program calculates a sphere whose center coordinates give the
δD, δP and δH of the skin. This software was chosen as it has an integrated suite of programs
that allow calculation of various physicochemical and solubility properties including
ingredient active gap (IAG), ingredient skin gap (ISG), solubility of active in the
formulation (SolV) and the formulation solubility in the skin (SolS) [72]. IAG; this is the
difference in polarity between an Ingredient and an Active, expressed in this work through
comparison of the bonding energies of each – Dispersion, Dipolar, and Hydrogen Bonding
forces of the molecules. In another sentence, the IAG is the sum of the square root of the
difference of the squares between the HSP’s of the Ingredient and the Active. An Active
with a high IAG between it and a specific Ingredient indicates that there are vast differences
in the polarity between the molecules and likely not a good match for solvency or delivery,
but possibly could be used to induce a driving force in a mixture with an Ingredient with a
50
low IAG. The smaller the IAG, the more alike are the Active and the Ingredient and as
“like dissolves like” the solubility will be higher. ISG; analogous to the Ingredient Active
Gap. This is the difference in polarity between an Ingredient and the skin; expressed in
this work through comparison of the bonding energies of each – Dispersion, Dipolar, and
Hydrogen Bonding forces of the molecules; an Ingredient with a low ISG may be more apt
to penetrate the skin and carry an Active, but other such factors as SolS, IAG, MVol (molar
volume), and SolV should be taken into account. The smaller it is, the more compatible the
ingredient is with the stratum corneum. This means that it will also ‘mix’ better with the
skin. Skin penetration can be seen as a way of ‘mixing’ the skin and the ingredient. So, the
smaller the ISG, the more the ingredient will penetrate the skin. This ISG was formerly
called the PPG, the Penetrant Polarity Gap. SolV; predicted solubility of an Active in a
specific Ingredient, expressed as a percentage; uses IAG and comparisons of MVol, and
Melting Point (MPt. ⁰C) to predict the percentage of Active that will remain soluble in an
Ingredient at room temperature; this Hansen model has been used for more than 50 years,
mainly in industrial applications, to predict solubility with excellent reliability. SolS;
solubility in the skin; whether an Ingredient has low or high solubility in the barrier is a
factor to consider when evaluating an Ingredient for use in delivery enhancement;
expressed as a percentage.
The aim of the present study was to investigate whether various physicochemical
properties derived from Hansen solubility parameters of actives, excipients, and the skin
and FFE software can be used to describe the ability of drugs and enhancers/excipients to
cross the biological membrane. The purpose of these studies was to develop a model to
explain the enhancers mode of actions in drug permeation. The overall concept may be
51
referred as the Solubility-Physicochemical-Thermodynamic (SPT) theory so that this
designation may facilitate our consideration of enhancer action based on solubility
parameters and physicochemical interactions governing the thermodynamic activity of
drug.
3.2.Materials and Methods
3.2.1. Materials
Polysorbate 80 (Tween 80), eucalyptol, N-methyl-2-pyrrolidone, propylene glycol,
sodium phosphate monobasic, thymoquinone (TQ), High-performance liquid
chromatography (HPLC) grade water and acetonitrile were purchased from Sigma-Aldrich
Co. (St. Louis, MO, USA). Nicotine was purchased from Alfa Aesar (Haverhill, MA, USA)
and laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA).
Phosphate-buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals,
LLC (Solon, OH, USA), ethanol was purchased from Decon Labs, Inc. (King of Prussia,
PA, USA) and O-phosphoric Acid 85% was purchased from Fisher Scientific (Hampton,
NH, USA). Dermatomed human cadaver skin from the posterior torso (2 Females at the
age of 34, 69 and 2 males at the age of 68, 45) were obtained from New York Firefighter
Skin Bank (NY, USA). Oleic acid and Transcutol® P was a gift from Croda (Edison, NJ,
USA) and Gattefosse (Paramus, NJ, USA) respectively.
52
3.2.2. Preparation of formulation and solubility determination
10 mL of five different formulations containing 1% nicotine, with or without 5%
of one type of enhancer (Azone, oleic acid, Tween 80, eucalyptol, and N-Methyl-2-
pyrrolidone) in propylene glycol (as vehicle) were prepared. Saturated solutions of TQ
were prepared by adding an excess amount of TQ and 250 µL of the enhancer to 5 mL of
propylene glycol in a well-closed container. Using Julabo SW22 shaker (Julabo USA Inc.,
Allentown, PA) all the amber containers with TQ in their respective solvents were agitated
at 37°C for 48 hours. After 48 hours all the samples were filtered through a 0.2 µm PES
syringe filter media with polypropylene housing. All experiments were performed in
triplicate and the drug content was measured by HPLC after appropriate dilution.
3.2.3. Permeation procedure for enhancer studies
Franz diffusion cells (FDC) with a donor area of 0.64 cm2 and a receptor volume
of 5.0 mL (Permegear Inc., Hellertown, PA) were used throughout the study. The samples
of dermatomed human cadaver skin were slowly thawed, cut into appropriate pieces to fit
the Franz cells and then soaked in filtered PBS (pH 7.4) for 15 minutes. After that they
were mounted on FDC with the epidermal side in contact with the formulation or donor
compartment. The receptor compartment of each cell was filled with filtered PBS (pH 7.4)
and was maintained at 37oC under synchronous continuous stirring using a magnetic stirrer
at 600 rpm. The diffusional membranes were left to equilibrate at 37oC for 15 minutes.
Once reached equilibrium, at time zero 200 µL of nicotine formulation and 500 µL of
thymoquinone formulation was added to the donor compartment of each Franz diffusion
53
cell. At each time point (1, 2, 3, 4, 5, 6, 7 and 8 hours for nicotine and 3, 4, 6, 8, 10, 12 and
24 hours for thymoquinone) 300 µL of receptor samples were withdrawn from the sampling
port. At the end of experimental hours, receptor aliquots of 300 µL were then analyzed
using a valid HPLC method described below.
3.2.4. Skin deposition study
At the end of permeation study, the skin was removed from the diffusion cell and
was cut around the diffusional area, air dried, and accurately weighed. The samples were
then placed into bead bug tubes and using a scissor they were cut into very small pieces. 1
mL ethanol was added to each tube and they were homogenized for 9 minutes (3 min of 3
cycles) by using BeadBugTM Microtube homogenizer, D1030 (Benchmark Scientific,
Sayreville, NJ). All the skin samples were then placed in a Julabo SW22 shaker (Julabo
USA Inc., Allentown, PA) and were agitated at 37 °C for 24 hours. After that all the skin
samples were centrifuged at 1200 rpm for 5 minutes and were filtered through a 0.45 μm
polypropylene filter media with polypropylene housing. TQ concentrations were expressed
as ng of TQ per skin weight in mg.
3.2.5. High-performance liquid chromatography (HPLC)
A validated HPLC method was used for this study. The HPLC instrument used
was Agilent 1100 series instrumentation (Agilent Technologies, CA, USA) coupled with
54
UV detection (DAD) and HP Chemstation software V. 32. The HPLC method of analysis
for each compound is provided below:
3.2.5.1. Nicotine
For the analysis of nicotine, a mobile phase of 65% sodium phosphate buffer
(adjusted to pH 3.2 with 85% orthophosphoric acid) and 35% acetonitrile was pumped
through a Phenomenex Luna® 5 µm C18(2) 100 Å Column 250 X 4.6 mm. Injection
volumes of 10 uL with a flow rate of 1.0 mL/minute set to 25°C with UV detection of 259
nm were used with retention time of 2.5 minutes. The method was linear at a concentration
range 0.9 -500 µg/mL with R2 of 0.999.
3.2.5.2. Thymoquinone
For the analysis of TQ, a mobile phase of 80% acetonitrile and 20% water was
pumped through an Agilent Eclipse XDB-C18 5 µm, 250 X 4.6 mm column. Injection
volumes of 20 uL with a flow rate of 1.0 mL/minute was set to 23°C with UV detection of
250 nm were used with retention time of 4.2 minutes. The method was linear at a
concentration of 0.39-100 µg/mL with R2 value of 0.998.
3.2.6. Calculated solubility and physicochemical parameters and permeation data
The FFE Software can be used to derive the Hansen Solubility Parameters of actives
and excipients with the input of a linear chemical structure expression called SMILES;
from there, solubility profiles, permeation and different physicochemical properties of drug
55
actives and excipient ingredients are calculated automatically. Measured drug permeation
data were compared with the calculated permeation data and solubility parameters of the
drugs. Eight-hour diffusion studies for nicotine and twenty-four-hour diffusion studies for
TQ were modeled for each formulation were run using the software.
3.2.7. Data and statistical analysis
The cumulative amounts of nicotine and TQ permeated per unit area were plotted
against time. The flux was calculated by determination of the slope of the linear portion of
the permeation profile. Results are reported as mean ± SD (n=6). The statistical analysis of
the data was performed by using one-way Anova and Student’s-t test, and p-values < 0.05
were considered significant.
3.3.Results and discussion
3.3.1. Nicotine
If a penetration enhancer is added into the formulation, then its affinity to the skin
must be considered. For example, if a penetration enhancer has lower ISG indicating it has
higher affinity to the skin that will further increase the formulation solubility in the skin
(SolS) (Figure 3.3A and B). The addition of certain penetration enhancers can also increase
the solubility of active in the formulation (SolV). The more extreme the difference in
solubility between the formulation (SolV) and the skin (SolS) the greater this driving force
for partitioning into the stratum corneum. In another scenario, if a penetration enhancer has
56
lower skin affinity (increased ISG) that will lead to the reduction of formulation solubility
in the skin and can further increase the permeability of the active across the skin given that
there is an increased active solubility in the formulation and an increased physicochemical
interaction between the enhancer and active (Figure 3.3A and B). If there is an increase in
the thermodynamic activity of the drug in the formulation, then an increase in rate of
delivery/flux of the drug can be achieved which will improve the clinical effect as well.
The desired modification of the thermodynamic activity of the drug can be achieved by
thoughtfully selecting the penetration enhancers for the formulation or by changing the
concentration or relative composition of the enhancers. Such modifications can benefit
both topical and transdermal delivery of either pharmaceutical or cosmetic active
ingredients.
In the present investigation the three-dimensional Hansen solubility parameters
were used to correlate the experimental permeation data of drugs. It was demonstrated that
the permeation of drug can be predicted by analyzing different physicochemical (Table
3.2) and solubility parameters (Table 3.1) of drug and ingredients that were assessed using
FFE Software. The three solubility parameters (δD, δP and δH) from Table 3.1 can be further
visualized using FFE ((Formulating for EfficacyTM - ACT Solutions Corp) Software with
a three-dimensional coordinate system, the so-called Hansen space with axes δD, δP and δH
(Figure 3.2). The closer the HSP (Hansen solubility parameters) values of two substances
in the 3D HSP space the higher is their similarity and the stronger is their affinity. Figure
3.1A represents the measured permeation data from the Franz diffusion cell method using
human cadaver skin and Figure 3.1B represents the correlations between the calculated and
measured permeation of nicotine, where X axis represent calculated permeation data from
57
the software model (µg/cm2) and Y axis represent measured permeation data from the
Franz diffusion cell study (µg/cm2). Through this graph we have also found out the
theoretical value of the correlation of permeation that exist between the measured
permeation data and the calculated permeation data. It is represented by R2 value, the
coefficient of multiple determination for multiple regression. Figure 3.1B shows a linear
correlation between measured and calculated permeation of nicotine from various
formulation using different ingredients. Among other ingredients Azone showed lower
regression coefficient value of R2 = 0.62. This result supports our previous study of
comparing the permeability of different formulations using various enhancers to human
cadaver skin and Strat-M® synthetic membrane [73].
Table 3.1. Hansen solubility parameters and molar volume for nicotine and different
solvents/enhancers.
1
2
Solvent Diffusion Coefficient δD δP δH Mvol 3 (m2/s) 4 5 Nicotine 5.49E-10 18.6 5.1 5 159.9 6 Propylene Glycol 1.30E-09 16.8 10.4 21.3 73.7 7 Tween 80 8.72E-15 16.2 6.6 9.6 1265 8 Eucalyptol 5.09E-10 16.6 2.7 2.5 167.5 9 N-Methyl Pyrrolidone 1.02E-09 18.1 10.3 6.6 98.1 10 Azone 1.20E-10 17 1.6 3.2 311.7 11 Oleic Acid 1.14E-10 16.5 3.2 5.7 317.5 12 13
14
58
Table 3.2. Physicochemical parameters of nicotine and different enhancers.
This study has also shown lower R2 value while comparing the permeation of drug
using Azone with human skin and synthetic membrane. This is probably due to the fact
that both synthetic membrane and software-based model are unable to account for the more
biological interaction of penetration enhancer and skin. In case of Azone the biological
interaction with the skin is more prominent as it causes the fluidization of the intercellular
lipids by creating a ‘soup spoon’ model within stratum corneum lipids [47, 48]. This unique
characteristic of Azone must have played a significant role while driving formulation
through skin. On the other hand, this biological influence was absent while running
diffusion using FFE software and this same effect was also noticeable while performing
permeation study using synthetic membrane. Moreover, Azone is associated with removal
of skin lipids and provide additional effects on the membrane components by acting as
1 2
Penetration Enhancers ASG IAG ISG SolV SolS 3
Nicotine 5.26 - - 100 100 4 Propylene Glycol - 17.51 9.96 0.9 100 5 Tween 80 - 6.82 33.66 100 0.6 6 Eucalyptol - 5.29 12.86 100 3.4 7 N-Methyl Pyrrolidone - 5.53 3.41 100 100 8 Azone - 5.07 24.94 100 0.2 9 Oleic Acid - 4.66 17.19 100 0.2 10 11 ASG – Active skin gap; IAG – Ingredient active gap; ISG – Ingredient skin gap; SolV – 12
Solubility of active in the formulation; SolS – The formulation solubility in the skin 13
59
A B
Figure 3.1. Nicotine permeation profiles of transdermal formulations (A) with the Franz
diffusion cell method using human cadaver skin, (B) the correlations between the
calculated and measured permeation of nicotine.
60
Figure 3.2. Position of the active nicotine and penetration enhancers/ingredients in 3D
Hansen Space.
surfactants as well as solvents [74]. The amount of nicotine permeated per square cm from
formulation 4 (nicotine 1% in propylene glycol + 5% Azone) and from formulation 6
(nicotine 1% in propylene glycol + 5% oleic acid) after 8 hours (Q8) were significantly
greater when compared to control and all other formulations (p<0.05) (Table 3.3). There is
synergy between Azone and propylene glycol since propylene glycol assists Azone
penetration into the stratum corneum and Azone also promotes the flux of propylene
glycol. Barry, B.W. has demonstrated the importance of the correct choice of vehicle or
co-solvent for Azone and oleic acid. For such enhancers to reach the polar surface of the
lipid bilayer in sufficient amounts, they may require a co-solvent such as propylene glycol
or ethanol [65]. The study conducted by Wotton, P.K. et al. has demonstrated that
61
metronidazole penetration is enhanced both by 1% Azone and by propylene glycol alone
and together they produced a synergistic effect by giving highest permeation of the
compound [75]. In another study Squillante, E., et al. have identified positive synergistic
interactions among the following formulation components; propylene glycol, cis-oleic
acid, and dimethyl isosorbide—which strongly affected nifedipine permeation [76]. The
synergistic action of propylene glycol with oleic acid may be due to the co-solvent’s ability
to reduce the polarity of the aqueous regions of the stratum corneum, so increasing the
ability of the stratum corneum to solubilize oleic acid [77]. Our previous study showed
highest permeation of active using 5% of Azone and oleic acid from propylene glycol and
ethanol vehicle compared to other enhancers [78]. It may be deduced from this study that
there must be other factors such as the change in the barrier properties by these solvents,
that have played an additional role besides the synergy present between the solvent and
enhancer to increase the flux of same active from two different solvents. The formulation
with 5% N-Methyl Pyrrolidone did not show any enhancement effect on nicotine
permeation through human cadaver skin. Eucalyptol and Tween 80 shared similar trends
by providing lower nicotine flux in comparison to the formulation containing 5% oleic
acid, 5% Azone, and higher flux of nicotine when compared to N-Methyl Pyrrolidone
(5%). The rank order of each enhancer/ingredient for the enhancement of nicotine skin
permeation was as follows: Oleic acid>Azone>Eucalyptol + Tween 80>N-Methyl
Pyrrolidone. This permeation ranking can be further understood by analyzing the ratio of
SolV and SolS, IAG and ISG values showed on Table 3.2 and illustrated in Figure 3.3A
and B. By using FFE Software it was found that the SolV and SolS ratio of NMP is 100:100,
which is the same as nicotine. For this reason, in spite of having lowest ISG (3.41) value
62
among all other ingredients, most likely the N-Methyl Pyrrolidone did not achieve high
enough thermodynamic activity to drive the active from the formulation into the skin
efficiently (Figure 3.3B). On the other hand, oleic acid, with SolV and SolS ratio (100:0.2)
and low IAG (4.66) value compared to all other ingredients used in this experiment, was
found to be best in providing maximum nicotine flux. Additionally, Azone shares the same
SolV and SolS values with oleic acid but showed lower nicotine flux than oleic acid due
likely to higher IAG and ISG values (Figure 3.3A and B).
There appears to be an inverse relationship between measured flux and IAG values
given that there is an optimum ingredient skin gap, SolV and SolS ratio. The sensitivity of
the Hansen solubility and physicochemical parameters can be further understood by
analyzing the behavior of eucalyptol and Tween 80. From the experimental study, Q at 8
hours (µg/cm2) for nicotine permeation using eucalyptol was 168 ± 41 and Q at 8 hours
(µg/cm2) for nicotine permeation using Tween 80 was 167 ± 43. The experimental data
showed that in spite of having different functional group they influenced the
thermodynamic activity of the nicotine in the formulation at a same rate. Interestingly, we
can explain this behavior using SPT theory. According to this theory, after analyzing their
physicochemical characteristics it was found that eucalyptol has higher affinity to the skin
with a SolS value 3.4 compare to Tween 80 (SolS value 0.6). Additionally, IAG value of
eucalyptol is 5.29 and for Tween 80 is 6.82. It can be assumed that in spite of having higher
skin solubility (SolS) this small fraction of reduction of IAG values of eucalyptol may
influenced the thermodynamic activity of nicotine in a better way by giving slightly
increased flux compared to Tween 80 (Figure 3.3A). It is to be remembered that what is
being evaluated here, especially regarding Tween 80 systems, are SPT effects and not
63
[A]
Penetration enhancers Action 1
Oleic acid Increased physicochemical interaction with nicotine due to small IAG value (4.66) 2
and optimum SolV (100) and SolS (0.2) values. Increased thermodynamic activity 3
drove nicotine from the formulation efficiently for the drug to enter and pass through 4
the skin. Higher skin permeability and flux was observed. 5
Azone Physicochemical interaction between Azone and nicotine was reduced compared to 6
Oleic acid due to slightly increased IAG value (5.07). Azone showed optimum SolV 7
(100) and SolS (0.2) values. Due to lower physicochemical interaction Azone 8
provided lower skin permeability of nicotine compared to oleic acid. 9
Eucalyptol Increased IAG value (5.29) reduced physicochemical interaction of eucalyptol and 10
nicotine. Eucalyptol has higher affinity to the skin due to lower ISG value (12.86) 11
that produced improved/increased formulation solubility in the skin (SolS value 3.4). 12
Provided slightly increased flux of nicotine compared to Tween 80 due to small 13
reduction in IAG value. 14
Tween 80 Physicochemical interaction was reduced with increasing IAG value (6.82). Tween 15
80 has lower affinity to the skin compared to eucalyptol due to increased ISG 16
(33.66) that further reduced the formulation solubility in the skin as indicated by 17
SolS value of 0.6. 18
NMP Reduction in physicochemical interaction due to increased IAG (5.53). Produced 19
highest SolV and SolS ratio (100:100). Higher affinity to the skin compared to 20
other ingredients as indicated by lower ISG (3.41). Due to the same SolV:SolS 21
ratio like nicotine NMP has provided lower skin permeability of nicotine thus 22
lower flux. 23
24
64
[B]
Figure 3.3. The influence of physicochemical interactions (IAG, SolV) between
penetration enhancer and active (Nicotine) on the driving force for diffusion and, the
influence of various physicochemical and solubility parameters (ISG, SolS) of the
formulation on the skin affinity of the penetrant is illustrated. (A) The possible mechanism
of action of skin penetration enhancers; (B) a representation of the active-enhancer and
stratum corneum interactions promoting partitioning into the stratum corneum.
Vehicle Stratum Corneum Lipid
Physicochemical interaction
between nicotine and Oleic
acid
IAG 4.66
Increased thermodynamic
activity drove nicotine from
the formulation efficiently to
enter and pass through the
skin. Higher skin permeability
and flux was observed.
ISG 17.19
SolS 0.2
Physicochemical interaction
between nicotine and NMP
IAG 5.53
Due to the same SolV:SolS
(100:100) ratio like nicotine
NMP has provided lower
skin permeability of
nicotine thus lower flux.
Increasing affinity for stratum
corneum ISG 3.41
65
colloidal effects. Further enhancement based on such colloidal structures as
microemulsions or lamellar gel networks can be overlain on the SPT approach.
Table 3.3. Penetration parameters of nicotine through human cadaver skin after 8 hours.
3.3.2.Thymoquinone
After analyzing the applicability of Solubility-Physicochemical-Thermodynamic
(SPT) theory using 1% nicotine and 5% of each enhancer from five different groups
(Amides-Azone; fatty acids- oleic acid; surfactants- Tween 80; pyrrolidones- N-Methyl
Pyrrolidone, terpenes- eucalyptol) now we want to use another drug of a solid form
(thymoquinone) to measure their experimental solubility in different enhancers to see
whether we can correlate their solubility, permeability and skin deposition experimental
66
data with the measured permeability, solubility and physicochemical parameters to
understand the influence of enhancers on thermodynamic activity of thymoquinone.
Tables 3.4 and 3.5 represent the Hansen solubility and physicochemical parameters
of thymoquinone and different solvents/enhancers. Penetration parameters of
thymoquinone through human cadaver skin (N=5) after 24 hours have been provided in
Table 3.6. The rank order of each enhancer/ingredient for the enhancement of
thymoquinone skin permeation was as follows: Azone + Oleic Acid>Transcutol®
P>Control + Tween 80>Ethanol>NMP. Figure 3.4A shows the measured permeation data
from the Franz diffusion cell method using human cadaver skin and Figure 3.4B shows the
correlations between the calculated and measured permeation of TQ, where X axis
represent calculated permeation data from the software model (µg/cm2) and Y axis
represent measured permeation data from the Franz diffusion cell study (µg/cm2). Figure
3.4B shows a linear correlation between measured and calculated flux of TQ from various
formulation using different enhancers and Figure 3.5 shows the position of thymoquinone
and penetration enhancers in 3D Hansen Space.
67
Table 3.4. Hansen solubility parameters and molar volume of thymoquinone and different
solvents/enhancers.
Table 3.5. Physicochemical parameters of thymoquinone and different enhancers.
1
2
Solvent Diffusion Coefficient δD δP δH Mvol 3 (m2/s) 4 5 Thymoquinone 5.72E-10 18.3 9.2 5.1 159.9 6 Propylene Glycol 1.30E-09 16.8 10.4 21.3 73.7 7 Tween 80 8.72E-15 16.2 6.6 9.6 1265 8 N-Methyl Pyrrolidone 1.02E-09 18.1 10.3 6.6 98.1 9 Azone 1.20E-10 17 1.6 3.2 311.7 10 Oleic Acid 1.14E-10 16.5 3.2 5.7 317.5 11 Ethanol 1.51E-09 15.4 9.2 19.6 58.7 12 Transcutol® P 7.03E-10 16.3 7.1 11.9 135.2 13
14
1 2
Penetration Enhancers ASG IAG ISG SolV SolS 3
Thymoquinone 4.08 - - 73.4 100 4 Propylene Glycol - 16.52 9.96 1.3 100 5 Tween 80 - 6.68 33.66 21.7 0.6 6 N-Methyl Pyrrolidone - 1.9 3.41 81.6 100 7 Azone - 8.25 24.94 22.4 0.2 8 Oleic Acid - 7.02 17.19 35.3 0.2 9 Ethanol - 15.62 7.1 4.9 100 10 Transcutol® P - 8.16 5.73 67 100 11
ASG – Active skin gap; IAG – Ingredient active gap; ISG – Ingredient skin gap; SolV – 12
Solubility of active in the formulation; SolS – The formulation solubility in the skin 13
68
A B
Figure 3.4. Thymoquinone permeation profiles of transdermal formulations (A) with the
Franz diffusion cell method using human cadaver skin, (B) the correlations between the
calculated and measured permeation of Thymoquinone.
69
Figure 3.5. Position of the active Thymoquinone and penetration enhancers/ingredients in
3D Hansen Space.
In spite of having lowest IAG (1.9) value compared to all other enhancers NMP
was not able to provide better TQ permeation due to its high solubility in the skin and in
the formulation (Figure 3.6A). From the solubility data (Table 3.7) it was found that
thymoquinone has highest solubility in ethanol. On the other hand, the permeation data
showed that thymoquinone flux was lower than the control formulation with 5% of ethanol
(Table 3.6). It shows that the flux is actually proportional to a gradient of thermodynamic
activity rather than the concentration. The thermodynamic activity of thymoquinone was
70
reduced as ethanol has highest IAG value and it is also very soluble in the skin (Figure
3.6A).
Table 3.6. Penetration parameters of thymoquinone through human cadaver skin (N=5)
after 24 hours [78].
TQ Flux TQ Q 24 ER
Formulation (µg/cm²/h) (µg/cm²)
Control 11.02±1.2 208±23
Tween 80 11.09±1.5 208±16 1
NMP 9±1.5b 167±38 0.81
Azone 49.3±5.6a 854±93 4.47
Ethanol 10.59±1 180±55 0.96
Oleic Acid 46.3±4.5a 865±113 4.2
Transcutol® P 14.23±1.4a 247±26 1.29
ER= Enhancement Ratio
a, significant increase in TQ flux (p<0.05)
b, significant reduction in TQ flux (p<0.05)
71
Table 3.7. Summary of the solubility study results showing the effect of 5% penetration
enhancers on the solubility of TQ using propylene glycol. The values represent the
concentration of TQ ± SD (N=3) in mg/mL at 48 hours [78].
Enhancers Solubility (mg/mL) ± SD
Propylene glycol 8.6 ± 0.3
Tween 80 9.4 ± 1.1
N-Methyl Pyrrolidone 8.5 ± 0.2
Azone 15.0 ± 1.4
Ethanol 15.7 ± 0.5
Oleic Acid 13.6 ± 2.1
Transcutol® P 11.1 ± 0.7
Table 3.4 and 3.5 and Figure 3.6A and B demonstrated that although Tween 80 has lower
IAG value compared to Azone and oleic acid it showed lower permeability due to its
highest ISG (33.66) and Mvol (1265). Transcutol® P has lower IAG value but does not
possess an optimum SolV : SolS ratio, thus did not provide better skin flux of TQ (Figure
3.6B). Oleic acid has a smaller IAG value than Azone (Table 3.5). Additionally, both
compounds have similar SolS values, but oleic acid has a higher SolV value than Azone.
It can be stated that, the more extreme the difference in solubility between the formulation
and the skin the greater the driving force for partitioning of the active into the stratum
corneum. With thymoquinone Azone and oleic acid showed similar trends in terms of
permeability. Whereas there was significant increase (p<0.05) of nicotine flux with Azone
compared to oleic acid. It is possible that this reversed behavior of Azone and oleic acid
72
[A]
Penetration enhancers Action 1
NMP Increased physicochemical interaction with TQ due to small IAG value (1.9). 2
Due to increased solubility of active in the formulation (SolV 81.6) and formulation 3
solubility in the skin (SolS 100) the driving force for partitioning into the stratum 4
corneum was reduced. Thus, lower skin permeability and flux was observed. 5
Ethanol Physicochemical interaction between TQ and ethanol was reduced with increasing 6
IAG value (15.62). It has higher affinity to the skin as indicated by the lower ISG 7
value (7.1). Most probably due to the higher IAG and lower ISG value there was 8
lower SolV (4.9) and higher SolS (100) value. This further reduced the flux of TQ. 9
Tween 80 Increased IAG value (6.68 ) reduced the physicochemical interaction of Tween 80 10
and TQ. Has lower affinity to the skin due to higher ISG value (33.66) that further 11
reduced the formulation solubility in the skin (SolS value 0.6). Provided lower skin 12
permeability and flux. 13
Transcutol Physicochemical interaction was reduced with increasing IAG value (8.16). It has 14
higher affinity to the skin (ISG 5.73). No optimum SolV, SolS ratio 15
(67:100) resulting in lower flux of TQ. 16
Oleic acid Reduction in physicochemical interaction due to increased IAG (7.02). Higher 17
affinity to the skin compared to Azone due to lower ISG (17.19). SolV, SolS ratio 18
was found to be (35.3:0.2) and provided higher flux of TQ. 19
Azone Increased IAG value (8.25) reduced the physicochemical interaction between 20
Azone and TQ. Lower affinity to the skin compared to oleic acid due to increased 21
ISG (24.94). Optimum SolV:SolS ratio (22.4:0.2) resulted in higher flux of TQ. 22
23
73
[B]
Figure 3.6. The influence of physicochemical interactions (IAG, SolV) between
penetration enhancer and active (Thymoquinone) on the driving force for diffusion and,
the influence of various physicochemical and solubility parameters (ISG, SolS) of the
formulation on the skin affinity of the penetrant is illustrated. (A) The possible mechanism
of action of skin penetration enhancers; (B) a representation of the active-enhancer and
stratum corneum interactions promoting partitioning into the stratum corneum.
Vehicle Stratum Corneum Lipid
Physicochemical interaction
between TQ and Azone
IAG 8.25
The more extreme difference in
solubility between the formulation
(SolV) and the skin (SolS) provided the
greater driving force for partitioning of
the active into the stratum corneum and
increased flux.
ISG 24.94
SolS 0.2
Physicochemical interaction
between TQ and Transcutol
IAG 8.16
No optimum SolV:SolS
ratio (67:100) resulting in
lower skin permeability and
lower flux of TQ.
Increasing affinity for stratum
corneum ISG 5.73
74
with the two different drugs is due to the complex concentration dependency of oleic acid
since oleic acid is more effective at lower concentration. We should also take in account
that penetration enhancers also permeate through the skin together with the vehicle and
active molecule.
When experiments were carried out for 8 hours with nicotine 5% oleic acid showed
highest permeability of the nicotine. On the other hand, use of 5% oleic acid did not result
in better permeability of thymoquinone compared to Azone when the experiment was
continued for 24 hours. It can be assumed that oleic acid was permeating together with the
active and at some point, there was not enough oleic acid to drive the thermodynamic
activity of the drug to influence its permeation. To understand the concentration
dependency of oleic acid we performed another experiment with 3% and 10% of oleic acid.
Figure 7 shows the amount of thymoquinone detected at 24 hours in human cadaver skin.
Since Azone and oleic acid showed the highest skin deposition of TQ it can be stated that
the maximum skin penetration and deposition was achieved when the drug is at its highest
thermodynamic activity.
3.3.3.Concentration dependency of oleic acid
Thymoquinone permeation profiles at different oleic acid concentrations (Figure
3.8) clearly show that thymoquinone permeated optimally with 3% oleic acid. In addition,
as the enhancer concentration increased the TQ permeation decreased. At the beginning,
for first 6 hours of experiment TQ permeated at a similar rate from each of the formulation
containing different concentrations (3%, 5% and 10%) of oleic acid (Figure 3.8). Then
gradually with increasing time the permeation become influenced by the varied
75
concentration of oleic acid. This may be due to the fact that with increasing concentration
and with increasing time higher amount of oleic acid was also leaving the formulation with
active that in terms reduced the thermodynamic activity of TQ. In this case, the
thermodynamic activity of the drug which is affected by the vehicle composition
determines the drug permeation [79].
Figure 3.7. Amount of Thymoquinone detected at 24 hours in human cadaver skin.
76
Figure 3.8. Thymoquinone permeation profile in propylene glycol vehicle containing
different concentration of oleic acid. Time points were measured at 3, 4, 6, 8, 10, 12 and
24 hours. Each point represents the mean ± S.D. of five experiments. ***p<0.02.
The concurrent use of FFE Software and FDC (Franz diffusion cell) methodologies
has demonstrated some interesting aspects of how physicochemical interaction of drug and
enhancers regulate the thermodynamic activity of the system. It was shown that the model
developed in the present study to describe the permeation of drugs and enhancers action
enables prediction of permeation behavior of active compounds through the skin.
Although, differential scanning calorimetry (DSC), X-ray diffraction, infrared (IR) and
confocal Raman spectroscopy offer some possibilities to shed light on the mechanism of
action of enhancers in terms of the interactions of the stratum corneum lipid systems with
***
77
the enhancers, none of these methods provide important information about the
thermodynamic activity regulated by the physicochemical properties of both drug and
enhancers that further governs the permeation of drug.
3.4. Conclusions
Better understanding of the physicochemical properties and solubility parameters
of the active and enhancers, interaction of enhancers with the drug and skin will aid in to
simplify our concept of enhancers behavior. By considering thermodynamic activity
previously described simple generalized enhancers mechanism of action can be improved
significantly to the point where one can explain the mode of action of enhancers in terms
of the specific experimental set-up (drug, vehicle, enhancers and its composition and
concentration differences, experimental duration). The knowledge of physicochemical
interactions and thermodynamic influence of enhancers on the permeant flux can not only
enable us to modify permeation but can also help in designing new and exciting chemical
enhancers. The solubility and physicochemical parameter is a fundamental thermodynamic
property. So, understanding Solubility-Physicochemical-Thermodynamic (SPT) theory
can open scope to design new chemical enhancers and can answer regulatory related
inquiry in the process of approval of the upcoming new chemical enhancers. Moreover, the
adoption of this SPT theory can save countless hours of trial and error in drug/active
formulation and delivery and can benefit pharmaceutical, cosmetic and chemical industry
and the related entity.
78
Chapter 4. Effects of solvents and penetration enhancers on transdermal delivery of
thymoquinone: permeability and skin deposition study
4.1. Introduction
The alternative route of oral drug administration is achieving growing interest. The
experts predict that the transdermal and intradermal drug delivery system market will
exceed $25 billion by 2018. It was found that approximately 74% of orally administered
drugs failed to exert desired pharmacological effectiveness [80]. In such case transdermal
delivery should be considered as an attractive alternative route to oral delivery. There are
many important advantages associated with transdermal drug delivery system (TDDS).
This delivery system can be very beneficial in avoiding hepatic first pass effect [81] and
stomach environment; a potential site of drug degradation [82]. It can also provide steady
state plasma level, improved bioavailability, decreased side effects and ultimately can
improve patient compliance [80, 83]. In 1979, FDA first approved scopolamine for
transdermal delivery system [83]. Until the present only nicotine, lidocaine, estradiol,
testosterone, fentanyl, nitroglycerine etc. represent this novel group of TDDS [39]. It
indicates that there are very small group of drugs that are able to cross the skin barrier in
an amount that will be sufficient to give desired therapeutic concentrations and
effectiveness. One should consider several physicochemical properties of active molecules
while choosing them for transdermal delivery. An ideal candidate for transdermal delivery
should a) have low molecular weight (<500 gmol-1) b) Optimum lipophilicity (log P = 1-
3) and c) low melting point (<200°F).
79
There is also an increasing interest in the development of bioactive compounds
isolated from natural products as a potential drug [84]. Poor oral bioavailability has
diminished the potential of using drugs of natural origin [85], thus there is a scope for
alternative route of drug administration for natural drugs. Thymoquinone (TQ) is a
quinone-based phytochemical and was first identified in 1963 in Nigella sativa (black
cumin seed) by El-Dakhakhany [86]. Black seed has been listed by US-FDA on its GRAS
(Generally recognized as safe) list [87]. This is one of the most widely studied plants and
for more than 2000 years the seed has been used to treat various maladies [88]. The seeds
contain volatile oils, proteins, alkaloids, saponins, crude fiber, minerals and vitamins
(calcium, potassium, iron, sodium, zinc, vitamin A, vitamin B, vitamin B2, vitamin C etc.),
linoleic acid, oleic acid etc.. [88, 89]. TQ is a yellow crystalline molecule and is the main
constituents of (30-48%) Nigella sativa extract [87]. It exhibits anti-oxidant, anti-
inflammatory and anti-neoplastic properties and has been studied for the treatment of
cancer and various neurodegenerative diseases e.g., Alzheimer’s and Parkinson’s disease
[90]. Based on the ideal characteristics of transdermal delivery TQ can be an attractive
candidate for TDDS. It is lipophilic (log P = 2.54), has low molecular weight (164.2 gmol-
1) and low melting point (44-45°C). On the other hand, due to its lipophilic character
thymoquinone is not an ideal candidate for tablet or capsule formulations. So, one approach
that may be successful is to develop a transdermal formulation of thymoquinone.
Overcoming skin barrier properties that is mainly regulated by stratum corneum, is
primarily composed of multiple layers of keratin-rich corneocytes (the bricks) surrounded
with lipid lamellae (the mortar) in a bilayer form is the major challenge while delivering a
drug through the skin [62, 91]. There are several strategies to overcome skin barrier to
80
successfully deliver active molecules. They include- chemical permeation enhancers,
iontophoresis, electroporation, microneedle, ultrasound, magnetophoresis,
photomechanical waves etc. [92]. Using chemical enhancers is the commonly used
technique that reversibly decrease skin barrier function by disrupting intercellular stratum
corneum lipids [38, 93, 94]. Many chemicals have been investigated as penetration
enhancers, e.g., surfactants, n-methyl-2-pyrrolidone, terpenes, transcutol, azone, oleic acid
etc. [64].
The objective of this study was to investigate the feasibility of transdermal delivery
of thymoquinone. To the best of our knowledge, this is the first study to investigate both
transdermal flux and skin deposition of thymoquinone and the various conditions that may
influence these including various vehicle/solvents, receiver composition and permeation
enhancers. This study would be further useful for the development of novel thymoquinone
transdermal formulation.
4.2. Materials and Methods
4.2.1. Materials
Polysorbate 80 (Tween 80), N-Methyl-Pyrrolidone (NMP), propylene glycol (PG),
thymoquinone (TQ), High-performance liquid chromatography (HPLC) grade water and
acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ethanol was
purchased from Decon Labs, Inc. (King of Prussia, PA, USA) and Azone (laurocapram)
was purchased from BOC Sciences (Shirley, NY, USA). Phosphate-buffered saline tablets
(PBS, pH 7.4) was purchased from MP Biomedicals, LLC (Solon, OH, USA). Dermatomed
81
human cadaver skin from the posterior torso was obtained from New York Firefighter Skin
Bank (NY, USA). Oleic Acid and Transcutol® P was a gift from Croda (Edison, NJ, USA)
and Gattefosse (Paramus, NJ, USA) respectively.
4.2.2. Solubility determination
Saturated solutions of TQ were prepared by adding an excess amount of TQ in 5
mL of series of different solvents in well closed amber containers. Using Julabo SW22
shaker all the amber containers with TQ in their respective solvents were agitated at 37°C
for 48 hours. After 48 hours all the samples were filtered through a 0.2 µm PES syringe
filter media with polypropylene housing. All experiments were done in triplicate and the
drug content was measured by HPLC after appropriate dilution.
4.2.3. In vitro skin permeation test (IVPT) studies
The skin permeability of TQ was studied in vitro by using Franz diffusion cells
(Permegear Inc., Hellertown, PA). Dermatomed human cadaver skin with the dermal side
in contact with receptor compartment was mounted on Franz diffusion cells. The
donor/diffusion area was 0.64 cm2 and the receptor compartment was filled with PBS (pH
7.4). The precise volume of PBS that was needed to fill the receptor compartment was
measured for each cell and was included into the calculations. Before using skin, they were
slowly thawed, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for 15
minutes. The diffusion cells were allowed to equilibrate at 37oC for 15 minutes. Once
reached equilibrium, at time zero the donor compartment was filled with 0.5 mL of
saturated solutions of TQ in PG/ethanol vehicle with or without 5% of each enhancer used
82
in this study. The skin was occluded with parafilm and the receptor compartment of each
cell was maintained at 37oC under synchronous continuous stirring using a magnetic stirrer
at 600rpm. At each time point (3,4,6,8,10,12 and 24 hours) 300 µL of receptor samples
were withdrawn from the sampling port and were immediately replaced with an equal
volume of PBS (pH 7.4). At the end of 24 hours, receptor aliquots of 300 µL were then
analyzed using a validated HPLC method described below.
4.2.4. High-performance liquid chromatography (HPLC) method development and
validation for TQ
4.2.4.1. Method characteristics
TQ was quantified using a validated HPLC method by Agilent 1100 series HPLC
instrumentation (Agilent Technologies, CA, USA) coupled with UV detection (DAD). A
mobile phase of 80% Acetonitrile and 20% water was pumped through an Agilent Eclipse
XDB-C18 5 µm, 250 X 4.6 mm column. Injection volumes of 20 uL with a flow rate of
1.0mL/minute was set to 23°C with UV detection of 250 nm were used with retention time
of 4.2 minutes. The method was linear at a concentration of 0.39-100 µg/mL with R2 value
of 0.99.
4.2.4.2. Standard solutions and calibration curve
Thymoquinone standard stock solution of 200 µg/mL was prepared by dissolving
2 mg of TQ into 10 mL of mobile phase (80 Acetonitrile : 20 Water). 1 mL of the standard
stock solution was transferred to a test tube and was mixed with 1mL of mobile phase to
83
give a concentration of 100 µg/mL. Process was repeated to produce calibration standards
of 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.39 µg/mL.
4.2.4.3. Method validation
The precision of an analytical method is the closeness of a series of individual
analyte measurements applied repeatedly to multiple aliquots of the same sample and can
be calculated as a Relative Standard Deviation (RSD).
%RSD=(SD/mean)×100
The method was validated for linearity of the calibration curve. Intra and inter-day
variability was determined by running HPLC for the standard solutions three times per day
and on two different days respectively.
4.2.5. Determination of TQ concentration in the skin
At the end of the 24 hours of permeation study the left-over donor solutions and
donor compartments were carefully transferred into a 50 mL centrifuge tube. Skin surface
was then patted dry with a cotton swab. Franz diffusion cell with still skin mounted on it
was held in slightly tilted position over the 50 mL centrifuge tube and 1mL of ethanol was
placed dropwise to wash the skin surface and again few cotton swab was used to patted dry
the skin. All the cotton swab with additional 4 mL of ethanol were placed in 50 mL
centrifuge tube to further analyze the concentration of donor. The skin was then removed
from the diffusion cell and was cut around the diffusional area, air dried and precisely
84
weighed. Then it was placed into a bead bug tube, using a scissor the skin was cut into very
small pieces. The tube was then filled with 1 mL ethanol and was homogenized for 9
minutes (3 min of 3 cycles) by using bead bug homogenizer. All the skin and donor samples
were then placed in a Julabo SW22 shaker and were agitated at 37°C for 24 hours. After
24 hours all the skin samples were centrifuged at 1200 rpm for 5 minutes and were filtered
through a 0.45 µm polypropylene filter media with polypropylene Housing. To further
calculate TQ skin concentration we have divided TQ skin concentration in ng by the
respective skin weight in mg.
4.2.6. Data and statistical analysis
The flux of TQ was determined from the slope of the linear portion of the
cumulative amount of TQ permeated per unit skin surface area (µg/cm2) versus time
(hours) plot. Individual permeation profile was generated to calculate average TQ flux with
their respective standard deviations. Permeability coefficient (Kp) and the effectiveness of
the penetration enhancers (ER= enhancement ratio) were determined using equation 1 and
2 respectively,
Kp = Flux/solubility………………………………………...Equation 1 [95]
ER = Flux with the enhancer/Flux without the enhancer……………Equation 2
The lag time was calculated from extrapolation of the linear portion to the x-axis intercept
of the permeation profile. Results are reported as mean ± SD (n=5). The statistical analysis
85
of the data was performed by using one-way Anova and Student’s t - test, and p-values <
0.05 were considered significant.
4.3. Results and discussion
4.3.1. HPLC method validation
Using the current validated HPLC method the TQ chromatogram peak appeared
around 4.2 min and showed in Figure 4.1. The peak’s shape passed the requirement for
symmetry and sharpness.
Figure 4.1. Thymoquinone chromatogram peak at retention time of 4.2 min.
86
The average standard calibration curve obtained from three separate HPLC runs for TQ
solutions ranging from 0.39-100 μg/mL was shown in Figure 4.2. The method was linear
with an R2 value of 0.99.
Figure 4.2. Thymoquinone standard curve for HPLC assay.
The results of intra-day and inter-day variability assessment are provided in Table 4.1 and
4.2 respectively. For inter-day and intra-day precision the %RSD for the slope of the best
fit line was calculated as 0.11% and 0.01% respectively. These values are lower than the
requirement %RSD value of 2%.
0
2000
4000
6000
8000
10000
12000
14000
0 20 40 60 80 100 120
Response A
rea (
mA
U*s
)
Concentration of Thymoquinone (µg/mL)
y = 130.02x + 119.31R² = 0.9985
87
Table 4.1. Intra-day variability of TQ standard solutions of three separate runs in one
day.
Conc (µg/mL) Average AUC SD %RSD
100 12910.5 44.7 0.34
50 6941.7 19.9 0.28
25 3561.3 11.5 0.32
12.5 1814.2 6.3 0.34
6.25 921.9 0.1 0.01
3.125 465 2.1 0.46
1.56 236 1 0.43
0.78 119.9 0.46 0.38
0.39 60.7 0.25 0.41
Table 4.2. Inter-day variability of TQ standard solutions of two separate runs in two
days.
Conc (µg/mL) Average AUC SD %RSD
100 12902.1 54.1 0.41
50 6937.8 26.9 0.38
25 3559.3 15.5 0.43
12.5 1811.9 6.8 0.37
6.25 921.2 1 0.11
3.125 465.1 2.8 0.62
1.56 235.4 2 0.87
0.78 119.5 0.98 0.82
0.39 60.5 0.35 0.58
88
4.3.2. Thymoquinone solubility study
There is not enough literature available on TQ solubility data in commonly used
vehicle [96]. The saturation solubility of TQ in several commonly used solvents is shown
in Table 4.3. The results show that highest TQ solubility can be achieved with ethanol
solvent (19 ± 2.6 mg/ml). Adding water to ethanol in 1:1 ratio significantly decreased
(p<0.0002) the solubility of TQ (0.42 ± 0.06 mg/ml). Although TQ has good solubility in
PBS pH 7.4 (0.66 ± 0.01 mg/ml), interestingly it can be further increased to 0.79 ± 0.03
mg/ml by adding ethanol in 1:1 ratio. Thus, we can depict that ethanol synergistically
increase TQ solubility when used with another vehicle. On the other hand, water reduced
TQ solubility when used with another solvent like, ethanol and methanol.
Table 4.3. Summary of the Solubility Study Results. The values represent the
concentration of TQ ± SD (N=3) in mg/mL at 48 hours.
Solvents Solubility (mg/mL) ± SD
Methanol 0.4 ± 0.02
Ethanol 19 ± 2.6
Ethanol : Water (1:1) 0.42 ± 0.06
Methanol : Water (1:1) 0.34 ± 0.03
Propylene Glycol 9.7 ± 0.16
PBS pH 7.4 0.66 ± 0.01
Ethanol : PBS pH 7.4 (1:1) 0.79 ± 0.03
Polyethylene Glycol 2.9 ± 0.2
89
4.3.3. Effect of propylene glycol and ethanol donor solvent
Ethanol and propylene glycol are commonly used solvents for lipophilic drug [79].
Both of them are important solvents and/or co-solvents for topical and transdermal
formulation. Penetration parameters of thymoquinone using PG vehicle are summarized in
Table 4.4 and Penetration parameters of thymoquinone using ethanol vehicle are
summarized in Table 4.5. For both of the solvents the formulations were applied on the
skin as saturated suspensions with 5% of each enhancer and the study was continued for
24 hours. Figure 4.3 and 4.4 shows that 24 hours study was sufficient to reach steady state
plasma concentrations. It was found that there are significant differences in the penetration
parameters of thymoquinone in presence of various chemical enhancers and vehicles
(ethanol and PG). The rank order for the TQ flux of each enhancers from the PG vehicle
was as follows: Azone + oleic acid>Tc>control + Tween 80>ethanol>NMP. On the other
hand, the rank order for the TQ flux of each enhancers from the ethanol vehicle was as
follows: Tc>oleic acid>Azone>control>Tween 80 + NMP. Cumulative amount of TQ
penetrated per cm2 from both Azone and oleic acid formulation after 24 hours through
human cadaver skin was 1.8 folds reduced in ethanol vehicle compare to PG vehicle. In
this study, TQ solubility in PG vehicle was found to be 9.7 ± 0.16 mg/ml (Table 4.3), which
is significantly lower (p<0.0035) than the solubility of TQ in ethanol vehicle (19 ± 2.6
mg/ml). The solubility and penetration parameters data showed that although ethanol was
able to increase the solubility of TQ it was not able to increase the flux of permeant from
the saturated solutions of TQ. From the above findings, it can be postulated that
90
Figure 4.3. Thymoquinone permeation profile in propylene glycol vehicle. Time points
were measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five
experiments.
thermodynamic activity of TQ in the formulation was modified most probably by the rapid
permeation and evaporation of ethanol vehicle. Due to the varying results between PG and
ethanol vehicle we cannot suggest that only the permeation and evaporative effect of
ethanol either reduced or increased the thermodynamic activity of TQ. Moreover, different
penetration enhancers and vehicles act differently due to their varying mechanism of
action. It is believed that ethanol may disrupts cutaneous barrier function by removing
intercellular material [97]. Ethanol is also involved in lipid fluidization and extraction [98,
99]. On the contrary, PG might increase drug permeability by solvating the α-keratin
structures of the cells [79].
91
Figure 4.4. Thymoquinone permeation profile in ethanol vehicle. Time points were
measured at 3,4,6,8,10,12 and 24 hours. Each point represents the mean ± S.D. of five
experiments.
Table 4.4. Penetration parameters of thymoquinone through human cadaver skin (N=5)
after 24 hours using propylene glycol vehicle.
TQ Flux TQ Q 24 Lag time Px10-3 ER
Formulation (µg/cm²/hr) (µg/cm²) (Hr) (cm/h)±SD
Control 11.02±1.2 208±23 3.17±0.07 1.25±0.13
Tween 80 11.09±1.5 208±16 3.25±0.4 1.03±0.14 1
NMP 9±1.5b 167±38 3.9±0.3 1.07±0.19 0.81
Azone 49.3±5.6a 854±93 4.2±0.3 3.59±0.36 4.47
Ethanol 10.59±1 180±55 2.34±0.2 0.69±0.06 0.96
Oleic Acid 46.3±4.5a 865±113 3.2±0.3 3.03±0.26 4.2
Transcutol P 14.23±1.4a 247±26 3.7±0.2 1.37±0.12 1.29
ER= Enhancement Ratio
a, significant increase in TQ flux (p<0.05)
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b, significant reduction in TQ flux (p<0.05)
Table 4.5. Penetration parameters of thymoquinone through human cadaver skin (N=5)
after 24 hours using ethanol vehicle.
TQ Flux TQ Q 24 Lag time Px10-3 ER
Formulation (µg/cm²/hr) (µg/cm²) (Hr) (cm/h)±SD
Control 21.28±1.8 347±18 4.21±0.8 0.90±0.07
Tween 80 20.68±2.8 325±43 4.8±0.2 0.85±0.11 0.97
NMP 20.68±2.8 382±62 4.30±0.2 0.90±0.12 0.97
Azone 27.97±5.8a 468±98 4.38±0.3 0.97±0.20 1.31
Oleic Acid 28.25±10a 466±146 4.31±0.6 0.97±0.35 1.32
Transcutol P 29.53±3a 483±48 4.93±0.2 0.96±0.09 1.38
ER= Enhancement Ratio
a, significant increase in TQ flux (p<0.05)
Comparing the penetration parameters of two vehicle it was found that there was significant
increase (p<0.05) in TQ flux of control, Tween 80 and NMP formulation from ethanol
vehicle. On the other hand, there was significant increase (p<0.05) in TQ flux of Azone
and oleic acid formulation from PG vehicle compare to ethanol vehicle. Such behavior of
Azone and oleic acid in PG vehicle can be explained by the fact that there might be a
synergistic effect between Azone and oleic acid with PG vehicle [64]. Azone is a highly
lipophilic compound (log P = 6.2) and is the first molecule to be specifically designed as a
skin penetration enhancers [45]. Although efficacy of Azone is influenced by the vehicle
choice, it mostly shows its ability to permeate an active molecule by interacting with the
stratum corneum lipid domains [64]. Oleic acid was also found to exerts its effectiveness
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by either modifying or by interacting with the stratum corneum lipid domains [26].
Interestingly, 5% Tc performed better in ethanol vehicle by showing two folds increase in
TQ flux compare to PG vehicle. It is reported that Transcutol increase the solubility of
drugs in the skin, its exact mechanism of action is still unexplored [100]. By analyzing
penetration parameters of TQ in PG and ethanol vehicle it was found that 5% Azone in PG
vehicle showed highest permeation of TQ. Additionally, 5% NMP in PG vehicle showed
lowest permeation of TQ. Although there was significant improvement of TQ permeation
with 5% NMP in ethanol vehicle (p<0.05) compare to PG vehicle, the permeation was
lower when comparing with ethanol control and was equal with Tween 80 formulation in
ethanol vehicle. This result is not surprising as NMP acts well with hydrophilic molecule
Figure 4.5. Thymoquinone permeation profile in ethanol vehicle and ethanol:PBS (pH 7.4)
receptor. Time points were measured at 3,4,6,8,10,12 and 24 hours. Each point represents
the mean ± S.D. of five experiments.
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by providing higher flux and TQ is a highly lipophilic molecule [64]. Additionally, Tween-
80 as a non-ionic surfactant has a minor penetration enhancement effect in human skin and
can lower thermodynamic activity of permeants [27]. Moreover, there was reduction of lag
time when 5% ethanol was used in PG vehicle (Table 4.4), although this formulation was
not able to increase TQ flux compare to all other formulations except NMP in PG vehicle.
This result support the study conducted by Rao (2015) and also confirmed that ethanol can
reduce the time needed for a drug to reach the steady state [101].
4.3.4. Effect of receiver solvent composition
As from solubility study we have found adding ethanol with PBS pH 7.4 has
increased the amount of thymoquinone that can be detected, we choose to change the
receiver solvent composition to 60:40 (Ethanol : PBS pH 7.4) to further run the permeation
study. Table 4.6 summarizes the penetration parameters of thymoquinone using ethanol
vehicle and ethanol : PBS pH 7.4 (60:40) receptor solvents. The rank order for the TQ flux
of each enhancers from the ethanol vehicle and ethanol : PBS receptor was as follows:
Azone>Tc >oleic acid>Tween 80 >control> NMP. In all three different permeation studies
NMP showed lowest permeation ability of TQ. On the other hand, Azone came out as a
better penetration enhancer by providing highest flux in PG vehicle and in ethanol : PBS
receptor solvent study. Additionally, Tc was a better enhancer in ethanol vehicle and PBS
receptor. It was interesting to find out that Tween-80 acted as a control in PG vehicle and
it acted as a NMP in ethanol vehicle. By changing the receptor composition, we were able
to increase the permeability capacity of Tween-80 compare to control and NMP. May be
this is due to the phenomenon that receptor composition improved the thermodynamic
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activity of TQ in Tween-80 formulation as the flux is proportional to a gradient of
thermodynamic activity and also due to the improvement of TQ skin reservoir (Figure 4.8).
Table 4.6. Penetration parameters of thymoquinone through human cadaver skin (N=5)
after 24 hours using ethanol vehicle and ethanol : PBS pH 7.4 (60:40) receptor solvents.
TQ Flux TQ Q 24 Lag time Px10-3 ER
Formulation (µg/cm²/hr) (µg/cm²) (Hr) (cm/h)±SD
Control 160±5 3140±270 3.6±0.5 5.54±0.2
Tween 80 168±11 2966±188 4.3±0.1 5.74±0.38 1.05
NMP 155±15 2918±250 3.8±0.5 5.30±0.53 0.96
Azone 206±18a 3885±202 3.8±0.4 7.26±0.64 1.28
Oleic Acid 171±8a 3839±316 2.7±0.8 6.04±0.30 1.06
Transcutol P 177±19a 3504±353 3.4±0.2 6.22±0.66 1.11
ER= Enhancement Ratio
a, significant increase in TQ flux (p<0.05)
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Figure 4.6. Amount of TQ (PG vehicle) detected after 24 hours in human cadaver skin
(N=5, mean ± SD).
4.3.5. Effect (pull or drag) of permeation enhancers and vehicle on TQ skin
deposition
Both donor and receptor solvent composition influence the permeation enhancers
and vehicle capability to either increase or decrease TQ skin deposition. Figure 4.6 shows
the amount of TQ in ng/mg (PG vehicle) detected after 24 hours in human cadaver skin.
The rank order for TQ skin deposition of PG vehicle using different enhancers are as
follows: Azone>oleic acid> ethanol>control>Tc>NMP>Tween 80. This result shows both
Azone and oleic acid was able to provide reservoir for TQ skin deposition. Moreover, with
PG vehicle Azone showed almost two folds increase in TQ skin deposition compare to
oleic acid. Figure 5 shows the amount of TQ in ng/mg (ethanol vehicle) detected after 24
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hours in human cadaver skin and the rank order are as follows: oleic
acid>Azone>control>Tc>Tween 80> NMP. Although oleic acid, Azone and Tc in ethanol
vehicle were not as efficient as oleic acid, Azone and Tc in PG vehicle in terms of TQ flux,
but they were able to provide highest TQ skin deposition. So, with the ethanol vehicle all
the enhancers were able to create reservoirs within the skin membrane and more TQ was
deposited in the skin rather than migrating to the receptor compartment of Franz diffusion
cell. In another words, ethanol increases the capacity of the stratum corneum for drug
uptake. As both vehicle and enhancer also penetrate through the skin with the active
molecule there might be some pull or drag effect associated with this varying results by
using two different vehicle. We can further understand the pull/drag effect of ethanol
vehicle on TQ skin deposition by analyzing the skin deposition study (Figure 4.8) after
changing the receptor composition to 60:40 (Ethanol : PBS). The rank order are as follows:
control>Tc>NMP>Tween 80>Azone>oleic acid. This skin distribution ranking profile is
somewhat reversed comparing the skin permeation ranking profile. This time ethanol
control was able to provide highest skin reservoir compare to other formulation with
different enhancers and Azone and oleic acid showed lowest TQ skin deposition. From the
above result, it can be depicted that ethanol control was able to pull more drug in the skin
and as the control formulation didn’t contain any enhancer the ethanol vehicle was not
further able to enhance the drug permeability. On the other hand, all the enhancers used in
this study showed low pulling effect as they were not as efficient like control to pull more
drug in skin membrane. They rather showed enhanced permeation as the enhancers has
permeation enhancing effect. It must be noted here, that we have analyzed the whole skin
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for TQ skin deposition/concentration study and the skin samples were analyzed after 24
hours of permeation study. However, further study of TQ skin absorption is needed.
Figure 4.7. Amount of TQ (ethanol vehicle) detected after 24 hours in human cadaver
skin (N=5, mean ± SD).
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Figure 4.8. Amount of TQ (ethanol : PBS receptor) detected after 24 hours in human
cadaver skin (N=5, mean ± SD).
4.4. Conclusions
Skin penetration of TQ was influenced not only by the physicochemical properties
of the vehicle but also by the other experimental parameters such as, receiver composition
and permeation enhancers. This study showed donor-receiver inter-relationships governing
TQ penetration and skin absorption along with effects of penetration enhancers. It can be
concluded that transdermal permeation and skin deposition of TQ can be obtained by using
penetration enhancers and different vehicles. Azone, oleic acid and Tc at a concentration
of 5% was able to provide measurable TQ flux and can be the choice of penetration
enhancer to further develop a novel transdermal formulation of TQ. These penetration
enhancers were also able to generate TQ reservoirs in the skin that might be useful to exert
sustained release of TQ from the stratum corneum over longer period of time.
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Chapter 5. Thymoquinone loaded polymeric films and hydrogels for the treatment
of wound healing and bacterial skin infections
5.1.Introduction
Skin is the largest organ in the body and occupies about 16% of the total body
weight of an adult and has a surface area of about 2 m2 [2]. It is a complex arrangement of
structures and has a multifunctional role- provides a physical barrier to the environment by
acting as a protective barrier against the ingress of foreign material, maintains homeostasis
and thermoregulation by limiting the loss of water, electrolytes, and heat and prevents
microbial colonization [1, 102]. Although skin act as a shield against bacterial invasion,
bacteria can still invade the epidermis and dermis to produce localized infection and cause
a variety of pathologic changes in the skin (impetigo, furuncles, subcutaneous abscesses)
[103]. Microbial infections of the skin and underlying tissues are among the most frequent
conditions found in ambulatory care patients [104]. Staphylococcus aureus is one of the
most important human and veterinary pathogens and is the causative agent for the majority
of primary skin infections [105]. It causes infections ranging from benign to life threatening
diseases [106]. Skin and soft tissue infections (SSTIs) encompass a wide variety of clinical
outcomes, ranging from mild cases of cellulitis, erysipelas, trauma, subcutaneous tissue
infections, wound related infections to complicated deep-seated infections with systemic
sign of sepsis [107]. SSTIs may lead to severe complications and hospital admission when
associated with co-morbidities and/or bacteraemia. The most commonly reported cause of
SSTIs is Staphylococcus aureus followed by β-haemolytic streptococci (BHS) [108, 109].
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Staphylococcus aureus can internalize by a variety of nonphagocytic host cells and can
contribute to the development of persistent or chronic infections and may lead to deeper
tissue infections or dissemination [110-112].
The skin of patients with atopic dermatitis (AD), eczema and psoriasis show a
striking susceptibility to colonization with Staphylococcus aureus. About 7% of patients
with psoriasis have bacterial skin infections [113] and 80-100% of patients with AD and
eczema are colonized with Staphylococcus aureus [114, 115]. On the other hand,
Staphylococcus aureus can be isolated from the skin of only 5-30% of normal individuals,
mainly from intertriginous areas of the body (axilla of the arm, the anogenital region
between digits etc.). There is a relationship between disease severity, extent and
Staphylococcus aureus colonization of lesional and non-lesional skin and the density of
Staphylococcus aureus has been shown to correlate with cutaneous inflammation [116].
The colonization density of Staphylococcus aureus can reach up to 107 colony-forming
units cm-2 without clinical signs of infection in patients with AD. Bacterial infection can
often lead to a chronic wound [117]. Wound management is a prevalent clinical problem
as wound healing involves a series of complex process including inflammation phase,
proliferative phase (formation of granulation tissue, reepithelialization and matrix
formation) and remodeling phase [118]. Each phase of wound healing is well defined,
although they overlap with next [119]. The process of wound healing becomes delayed
when wounds are colonized and the colonizing agent is sustained [120]. In patient with
weaken immune system bacterial contamination can prolong wound healing [121, 122] and
colonization of bacteria in wounds is a serious threat. Open wounds are also at high risk of
invasive wound infections, which can further lead to amputation and disability [123].
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SSTIs including atopic dermatitis (AD), eczema, psoriasis and wound healing all rely on
efficient antibiotic therapies. As temporary eradication of Staphylococcus aureus with
antibiotics often leads to clinical improvement of AD, data from clinical studies suggest
that antimicrobial treatments should be applied in AD patients with apparent or recurrent
skin infections [124]. Antimicrobial resistance is one of the biggest challenges in the global
health sector [125]. The high incidence of methicillin resistance in hospitals complicated
the prevention and treatment of serious infections due to staphylococci [126]. As infections
due to multi-resistant Gram-positive organisms are increasing day by day their early
recognition, treatment and proper management are greatly required. Several antimicrobial
agents in different dosage forms are available for the treatment of SSTIs. Topical
application of antibiotic agents have several benefits over oral and systemic therapy [127]-
localized and targeted delivery can provide required concentration for antibiotic activity
more efficiently at the skin target site, can avoid an unnecessary exposure of gut flora that
may exert selection for resistance, can avoid side-effect and allergic reactions associated
with systemic antibiotic treatment. Therefore, topical application may highly influence the
treatment efficiency and can increase the patient compliance.
In such scenario, development of new treatment strategy is crucial to deal with the
emerging issues of skin infections. Topical delivery of antibacterial agent of medicinal
plants can be considered as a source for new therapeutic agents aimed at the treatment and
management of skin infections. Thymoquinone (TQ) (2- isopropyl- 5- methyl- 1,4-
benzoquinone) is the main constituent of Nigella sativa (Black cumin) seeds [28]. TQ is a
yellow crystalline molecule and has a basic quinone structure consisting of a para
substituted dione conjugated to a benzene ring to which a methyl and an isopropyl side
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chain groups are added in positions 2 and 5, respectively. TQ has many pharmacological
properties such as anticancer, antimicrobial, anti-inflammatory, antioxidant, antiasthmatic
and immunomodulatory effect [31]. Thus far, several experiments have explored its
anticancer and brain targeting properties - Odeh et al. loaded TQ in a liposome system and
tested that on breast cancer cell lines (MCF-7 and T47D) to evaluate its anticancer
properties [28], Ng et al. prepared TQ loaded nanostructured lipid carrier and showed its
effectiveness towards breast cancer and cervical cancer cell lines [128], Ahmad et al.
evaluated TQ loaded mucoadhesive nanoemulsion for the treatment of cerebral ischemia
[129]. However, there has no report on its delivery from a topical delivery system and its
effectiveness for the treatment of wound healing and Staphylococcus aureus associated
bacterial skin infections.
The objective of this study was to synthesize and characterize a biocompatible
novel topical polymeric film and hydrogel system that has the potential to deliver
antibacterial TQ agent directly at the skin target site that may be useful for the treatment
and management of Staphylococcus aureus related bacterial skin infections and for the
wound management. To achieve this objective, TQ loaded polyvinyl pyrrolidone (PVP)
films were prepared using solvent casting method and TQ wound hydrogels were prepared
using different polymers. The prepared films and hydrogels were characterized for physical
parameters, permeability and stability studies. Its biocompatibility was assessed, and the
antibacterial efficacy of films and hydrogels were evaluated in vitro and ex vivo on selected
strains of Staphylococcus aureus (ATCC 49230). Further, in vitro scratch assay models
using HDF (fibroblast) and HaCat (keratinocytes) cell lines were used to demonstrate its
wound healing properties. To evaluate its preclinical and in vivo efficacy biopsy punch
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wound infection animal model was used. This work demonstrates that PVP/TQ film is
effective in facilitating wound healing. Both the film and wound hydrogel can be useful in
the treatment of bacterial skin or wound infections and TQ can be a promising candidate.
5.2. Materials and Methods
5.2.1. Materials
Thymoquinone (TQ), polyvinylpyrrolidone (PVP), dibutyl phthalate (DBP),
hydroxy propyl methyl cellulose (HPMC), potassium chloride, benzoic acid, Gentamicin
solution, formalin solution (10%), triethanolamine, propylene glycol, dipropylene glycol,
alamarBlue® (resazurin) assay kit, High-performance liquid chromatography (HPLC)
grade water and acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO,
USA). Laurocapram (Azone) was purchased from BOC Sciences (Shirley, NY, USA).
Phosphate-buffered saline tablets (PBS, pH 7.4) was purchased from MP Biomedicals,
LLC (Solon, OH, USA), ethanol was purchased from Decon Labs, Inc. (King of Prussia,
PA, USA), xanthan gum was purchased from Spectrum Chemical (New Brunswick, NJ,
USA), hydroxy propyl cellulose (HPC) was purchased from Ashland (Wilmington, DE,
USA). Carbopol 980 and ultrez 10 were purchased from Lubrizol (Cleveland, OH, USA)
and drierite was purchased from Acros organics (Morris Plains, NJ, USA). Bacto tryptic
soy broth, bacto agar, Dulbecco's Modified Eagle's Medium (DMEM) and Dulbecco's
phosphate-buffered saline (DPBS) were purchased from Fisher Scientific (Hampton, NH,
USA). Human epidermal keratinocytes (HaCat) and Human dermal fibroblasts (HDF) cell
lines and Pen/strep were purchased from Life Technologies (Carlsbad, CA, USA).
Staphylococcus aureus (ATCC 49230) was purchased from ATCC (Manassas, VA, USA).
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Isoflurane was purchased from Henry Schein (Dublin, OH, USA). Fetal bovine serum
(FBS) was purchased from Atlanta Biologicals (Minneapolis, MN, USA). CellTiter 96®
was purchased from Promega (Madison, WI, USA). Gentamicin sulfate cream USP, 0.1%
was purchased from Perrigo (Allegan, MI, USA). Dermatomed human cadaver skin from
the posterior torso were obtained from New York Firefighter Skin Bank (NY, USA). Nine
to ten week old male BALB/c mice were purchased from Charles River (Wilmington, MA,
USA).
5.2.2. Fourier Transform Infrared (FTIR) analysis
FTIR spectra of samples were taken on Thermo Scientific (model Nicolet iS10)
instrument to investigate the possible interaction between the drug and polymer. FTIR
spectra of pure drug, polymer, physical mixture of drug and polymer in ratio of 1:1, films
with TQ and films without TQ were scanned in the ranged between 4000-400 cm¯¹.
5.2.3. Fabrication of films
The matrix-type polymeric films containing TQ were prepared by solvent casting
method. Accurately weighted TQ were dissolved in ethanol and were sonicated for 30
minutes to ensure solubilization. DBP was used as a plasticizer and Azone was used as a
penetration enhancer. The weighted amount of PVP, DBP (4%, v/v) and Azone (5%, v/v)
was added in the drug solution. The mixture was stirred at 200 rpm at 25°C for 20 minutes.
The solution was poured on Teflon dish (15 cm2) and placed in an oven maintained at 60°C
± 5°C. To allow complete evaporation the system was left undisturbed for 3 hour 20
minutes. The formed films were completely removed from the Teflon dish and punched
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out into 0.64 cm2 pieces. Control films without drug containing only PVP, plasticizer and
penetration enhancer were also prepared.
5.2.4. Preparation of TQ hydrogel formulations
Drug-loaded hydrogels were prepared using gelling agents, preservatives,
penetration enhancer and vehicles. Different concentrations of various polymers (gelling
agents) with or without xanthan gum were dispersed slowly in an aqueous-based solution
containing TQ (0.2% w/w), 1:1 concentration of propylene glycol and dipropylene glycol
(20% w/w, as a vehicle), benzoic acid (0.1% w/w, as a preservative), ethanol (5% w/w, as
a penetration enhancer), using an overhead mechanical stirrer at a moderate speed.
Triethanolamine was used to adjust the pH of Carbopol and Ultrez 10. The prepared
hydrogels were packed in wide mouth jar covered with screw capped plastic lid and kept
in dark and at laboratory ambient temperature. The composition of different prepared TQ
hydrogel formulations is given in Table 5.1.
5.2.5. Field Emission Scanning Electron Microscopic (FESEM) studies
The surface morphology of the film was recorded with a Zeiss field emission
scanning electron microscopy (FESEM) (FSD PRE-AMP 4CH, Germany). The film
sample was mounted on an aluminium stub with double-sided adhesive band then gold was
sputtered on the specimen (20 nm) to ensure sufficient electrical conductivity. An
accelerating voltage of 5 kV was applied and the image was photographed by secondary
electron detector.
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Table 5.1. Composition of TQ topical hydrogels (% w/w).
5.2.6. Physicochemical characterization of films
5.2.6.1. Film thickness
Film thickness was measured using Digital Caliper (Fisher Scientific, New
Hampshire, USA) at three different places, and mean value was calculated.
5.2.6.2. Drug content uniformity
A prepared film was dissolved in 10mL ethanol and stirred continuously for 24
hour. The drug content was analyzed using HPLC method.
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5.2.6.3. Weight variation
Weight variation was studied by individually weighing 5 randomly selected films.
5.2.6.4. Flatness
Three longitudinal strips were cut out from each film (one from the center, one from
the left side, and one from the right side). The length of each strip was measured and the
variation in length because of nonuniformity in flatness was measured by determining
percent constriction, with 0% constriction equivalent to 100% flatness.
% Constriction = L1- L2/L2 x 100
where L1= initial length of each strip and L2 = final length of each strip
5.2.6.5. Folding endurance
Folding endurance was determined by repeatedly folding the film at the same place
until it broke. The number of times the film could be folded at the same place without
breaking was the folding endurance value.
5.2.6.6. Percentage of moisture content
The prepared films were marked, then weighed individually and kept in a desiccator
containing drierite at room temperature for 24h. The films were weighed again and again
individually until it showed a constant weight. The percentage of moisture content was
calculated as the difference between initial and final weight with respect to final weight.
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5.2.6.7. Percentage of moisture uptake
The films were weighed and kept in a desiccator at room temperature for 24 hour.
Films were taken out and placed in a desiccator containing 100mL of saturated solution of
potassium chloride to maintain 84% relative humidity until a constant weight for the films
were obtained. The percentage of moisture uptake was calculated as the difference between
final and initial weight with respect to initial weight.
5.2.7. Physicochemical characterization of the prepared hydrogels
5.2.7.1. Visual inspection
TQ hydrogels were examined visually for their color and homogeneity (appearance
and presence of any aggregates).
5.2.7.2. pH determination
The pH of various TQ hydrogel formulations was determined using pH meter
(VWR pH meter symphony B10P, Radnor, PA, U.S.A). 1 gm of TQ hydrogel was
dissolved in 10 gm of DI water. After 2 hours pH was determined at room temperature.
5.2.7.3. Spreadability test
Spreadability was determined in mm. A 10 mg sample was placed on top of a
microscopic slide and covered with another slide, 50 gm of standardized weight was put
on it and after 1 minute the diameter of the sample was taken in mm.
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5.2.7.4. Drug content uniformity
Three samples of a specific quantity (100 µL) of each prepared hydrogel was taken
and dissolved in 10 mL of ethanol solvent. To ensure drug solubility the 20 mL glass vial
containing the gel solution was put on a magnetic stirrer at 600 rpm at 25°C for overnight.
The drug content was then determined using HPLC. The variability of TQ content in
hydrogels was reported as % RSD,
% RSD = (standard deviation/mean drug content) × 100.
5.2.8. Rheological characterization of hydrogel formulation
Rheological characterization was performed on rheometer (Kinexus Ultra +,
Malvern, UK) equipped with a 25 mm flat stainless-steel plate. All tests were done at 32ºC
and a gap of 1 mm. Following tests were carried out-
5.2.8.1. Oscillation stress sweep
The samples were subjected to increasing stress (0.1 - 500 Pa) at a constant
frequency of 1 Hz. This test allows determination of the liner viscoelastic region (LVR) of
the sample, and therefore the consequent choice of the stress value to use in the subsequent
oscillation test.
5.2.8.2. Frequency sweep
All the prepared samples were subjected to increasing frequency of 0.1–50 rad/sec
at a constant stress (5 Pa) obtained from LVR. Effect of stress on elastic modulus (G′) and
viscous modulus (G″) was monitored.
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5.2.9. In Vitro skin permeation studies
In vitro skin permeation studies were performed by using a Franz diffusion cells
(FDC) with a donor area of 0.64 cm2 and a receptor volume of 5.0 mL (Permegear Inc.,
Hellertown, PA). The samples of dermatomed human cadaver skin were slowly thawed at
room temperature, cut into appropriate pieces and then soaked in filtered PBS (pH 7.4) for
15 minutes. After that they were mounted on FDC with the epidermal side in contact with
the formulation or donor compartment. The receptor compartment of each cell was filled
with filtered PBS (pH 7.4) and was maintained at 37oC under synchronous continuous
stirring using a magnetic stirrer at 600 rpm. The diffusional membranes were left to
equilibrate at 37oC for 15 minutes. Once reached equilibrium, at time zero formulated films
and 100 µL of hydrogels were placed over the skin to the donor compartment of each Franz
diffusion cell. At each time point 300 µL of receptor samples were withdrawn from the
sampling port. At the end of experimental hours, receptor aliquots of 300 µL were then
analyzed using a valid HPLC method described below.
5.2.10. High-performance liquid chromatography (HPLC)
A validated HPLC method was used for this study. The HPLC instrument used was
Agilent 1100 series instrumentation (Agilent Technologies, CA, USA) coupled with UV
detection (DAD) and HP Chemstation software V. 32. For the analysis of TQ, a mobile
phase of 80% acetonitrile and 20% water was pumped through an Agilent Eclipse XDB-
C18 5 µm, 250 X 4.6 mm column. Injection volumes of 20 uL with a flow rate of 1.0
mL/minute was set to 23°C with UV detection of 250 nm were used with retention time of
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4.2 minutes. The method was linear at a concentration of 0.39-100 µg/mL with R2 value of
0.99.
5.2.11. Skin deposition study
At the end of permeation study, the skin was removed from the diffusion cell and
was cut around the diffusional area, air dried, and accurately weighed. The samples were
then placed into bead bug tubes and using a scissor they were cut into very small pieces. 1
mL ethanol was added to each tube and they were homogenized for 9 minutes (3 min of 3
cycles) by using BeadBugTM Microtube homogenizer, D1030 (Benchmark Scientific,
Sayreville, NJ). All the skin samples were then placed in a Julabo SW22 shaker (Julabo
USA Inc., Allentown, PA) and were agitated at 37 °C for 24 hours. After that all the skin
samples were centrifuged at 1200 rpm for 5 minutes and were filtered through a 0.45 μm
polypropylene filter media with polypropylene housing. TQ concentrations were expressed
as ng of TQ per skin weight in mg.
5.2.12. Stability study
The films were put in a petri dish and the dish was wrapped by aluminium foil and
stored at 20 °C for 60 days. The samples were analyzed for physical changes such as color,
texture and other physical parameters. The FTIR spectra of stored films were compared
with the freshly prepared films. The films were also analyzed for drug content. On the other
hand, all the hydrogel formulations were filled in glass wares and covered with aluminum
foil and were kept at laboratory ambient temperature for 8 months. The physical stability
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of the formulation was examined visually for appearance, color and odor in every two
weeks. After 8 months an antibacterial efficacy study was performed to confirm the
formulation stability of hydrogel formulation.
5.2.13. In vitro antibacterial activity of TQ films and hydrogels
The prepared control films and TQ loaded films were tested for their antibacterial
activity against Staphylococcus aureus (ATCC 49230) using disc diffusion method.
Briefly, Muller Hinton agar (MHA) plates were used for screening, prepared by pouring
15 mL of molten media into sterile petri dishes. Then 150 µL of overnight cultured bacteria
adjusted to OD concentration of 0.602 (OD 1 = 1X109/mL of bacteria) in sterile TSB
(Tryptic soy broth) was spread on the surface of MHA agar plates with the help of sterile
spreader. The disc shaped polymer film of 0.64 cm2 and 100 µL of TQ gel were then placed
on the surface of the medium and incubated at 37 °C for 24 hours. Gentamicin 500 µg/mL
and 50 ug/mL was used as a positive control, UV irradiated filter paper was used as a
negative control and control film without TQ was used as a control. At the end of
incubation, the inhibition zones were examined around the polymer disc films. The study
was performed in triplicate.
5.2.14. Ex vivo antibacterial activity of TQ films and hydrogels using human cadaver
skin explants
Human cadaver skin was thoroughly washed three times using sterile PBS. Using
surgical gloves and sterile scissors they were cut into pieces (2 cm x 2 cm) and two skin
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samples were placed on each agar plate. 5 µL of 1 X 10⁶ CFU/mL was put onto each skin
pieces followed by the application of treatment. After overnight incubation at 37°C bacteria
were extracted for counting using sterile PBS and 10 seconds of vortex. Serial dilutions of
bacteria were prepared and were plated on TSB agar plates. Bacteria were counted after
overnight incubation at 37°C.
5.2.15. Cyto-compatibility study
alamarBlue® (resazurin) assay was used to evaluate the cyto compatibility of the
TQ film using two cell lines, HaCat (Human epidermal keratinocytes, passage 8) and HDF
(Human dermal fibroblasts, passage 5). The cells were counted and were seeded into the
6 well plates at a density of 200000 cells/cm². After reaching confluency, the cells were
treated with the samples for 24 h. 1% Triton treated cells served as positive control and
cells in media without any treatment acted as negative control. After 24 h the cells were
treated with alamarBlue® and incubated for 4 h. The optical density was measured at
excitation-emission wavelength of 560-590 nm using Spark 10M multimode microplate
reader (Tecan, Switzerland). The percentage of cell viability was calculated using the
formula given below,
% Cell viability = [ [Fluorescent intensity] test/[Fluorescent intensity] control] X 100
5.2.16. Scratch assay for wound closure activity
HDF (Passages 2-4) cells were counted and were seeded into the 24 well plates at
a density of 50000 cells/well. After reaching confluency, the culture media was replaced
with sterile base media (DMEM with 1% P/S) and scratch wounds were created in the cell
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monolayer using a 200 µL sterile pipette tip [130]. TQ in DMEM at different concentration
(1 ng- 1000 ng) was put into the culture media and the experiment was continued for 24
hours. Base media was used as a control and 100 ng FGF 2 (Fibroblast Growth Factor) was
used as a positive control. Images were taken at 0, 4, 8, 12 and 24 hours. HaCat (Passage
37-39) cells were counted and were seeded into the 24 well plates at a density of 250k
cells/well. After reaching confluency, the culture media was replaced with sterile base
media (DMEM with 1% FBS+ 1% P/S) and scratch wounds were created in the cell
monolayer using a sterile pipette tip. TQ in DMEM at different concentration (1 ng- 1000
ng) was put into the culture media and the experiment was continued for six days. Base
media was used as a control and 10% DMEM was used as a positive control. Images were
taken at 0, 24, 48, 72 and 144 hours.
5.2.17. In vivo bacterial skin infection study
Animal infection experiments were performed at the Nelson Biological
Laboratories, Rutgers University (Piscataway, NJ, USA) in accordance with a protocol
approved by the Rutgers University Institutional Animal Care and Facilities Committee
(ACFC). Mice were housed under standard conditions of light and temperature and were
fed standard diet and water ad libitum. Adult male mice (BALB/c, 10 weeks) were used
for all experiments [131, 132]. Prior to the experiment day, the mice were anesthetized
using an inhaler beginning with 5% isoflurane, and then decrease to 2-3% to maintain
sedation for the remainder of the procedures. The hair over the dorsum (head to tail) were
shaved with an electric clipper. To remove the remaining hair the depilatory cream was
applied for 3 min. Finally, the shaved area was washed with wet scrub, and the animals
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were returned to their cages. On next day, 10 mm biopsy punch was used to create the
wound on the dorsum of the animals. A bacterial infection at the wound site was initiated
by placing on the skin a 10 µL droplet containing 108 CFU/mL cells of concentrated
Staphylococcus aureus from an overnight bacterial culture in stationary phase. Mice were
divided into following groups namely, Control wound (10 mm biopsy skin wound),
Bacterial wound (skin wound infected with bacteria), Control film (wound infected with
bacteria and then treated with control film without TQ), TQ film (wound infected with
bacteria and then treated with TQ loaded film), Gentamicin (wound infected with bacteria
and then treated with gentamicin marketed cream formulation)
Film with or without TQ were applied at the wound infection site on Day 0, 1, 2
and 3. Gentamicin was applied similarly as TQ film. Wounds were covered using Tegaderm
film (3M, Saint Paul, MN, USA). At each time point (Day 1, 2, 3 and 7) bacterial samples
were taken by taking out the Tegaderm film from the wound site and collected in 2 mL
microtube containing PBS. The tubes were then vortexed for 10 sec to extract the bacteria.
Different bacterial dilutions were made by adding 10 µL of bacterial solution to the 990 µL
of TSB solution and were plated on TSB agar plates. After overnight incubation at 37°C
bacteria were counted. The experiments were continued for 21 days. Wounds were visually
monitored for local inflammatory reaction and photographed at Day 0, 3, 7, 10, 14 and 21
days. All the animals were euthanized at the end of day 21.
5.2.18. Behavioral response of mice
The mice were observed at least once each day for signs of fatigue, stress, and
aggressiveness. The mice were weighed at each time point.
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5.2.19. Histopathological examinations
Immediately after the animals were killed, wound skin samples were collected
and immediately fixed in phosphate-buffered (pH 7.4) formalin (10%) for 48-72 hours at
room temperature and then switched into 70% ethanol and stored at 4°C until process.
Skin tissues including wound scar area were sectioned (5um thickness) and stained with
Masson’s trichrome.
5.2.20. Data and statistical analysis
The cumulative amounts of TQ permeated per unit area were plotted against time.
The flux was calculated by determination of the slope of the linear portion of the
permeation profile. The % wound closure for each time interval was determined by the
following formula and were calculated using ImageJ software-
𝑤𝑜𝑢𝑛𝑑 𝑐𝑙𝑜𝑠𝑢𝑟𝑒 (%) =𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑛 𝑑𝑎𝑦 0 − 𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟𝑜𝑛 𝑜𝑛 𝑑𝑎𝑦 𝑛
𝑤𝑜𝑢𝑛𝑑 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑛 𝑑𝑎𝑦 0 𝑋 100 (1)
Results are reported as mean ± SD. The statistical analysis of the data was performed by
using one-way Anova, the Tukey post-hoc tests and Student’s-t test, and p-values < 0.05
were considered significant.
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5.3.Results and discussion
5.3.1. Fourier Transform Infrared (FTIR) Spectroscopic studies
The FTIR analysis was employed to study the compatibility of the drug with the
polymer used (Figure 5.1A). The samples were scanned in the region of 4000-400 cm -1.
The IR spectral analysis of pure TQ showed that the major peaks were observed at
wavenumbers 2967.30 (C-H stretching of aliphatic group), 1640.54 (C = C stretching),
1462.23, 1358.15 (C-H methyl rock), 1246.90, 1133.03, 1023.62 (C-H in-plane bend),
1006.33, and 933.08, confirming the purity of the drug (Figure 5.1A). A weaker band
observed at a higher wavenumber (3253.95) corresponds to the stretching observed in the
vinylic C-H in the C = C-H groups [133]. In the IR spectra of the physical mixture of TQ
and PVP (Figure 5.1A) the major peaks of TQ were observed at wavenumbers 2966.73,
1644.49, 1461.38, 1374.84, 1248.05, 1133.32, 1023.30, 1006.10 and 933.43. Infrared
spectra of physical mixture of TQ and polymer showed all the characteristic peaks
indicating the absence of any possible interaction between the drug and polymer.
Therefore, it can be stated that the drug and polymer are compatible and can be formulated
into films. The characteristic peaks of the drug can also be seen in TQ films of both freshly
prepared and stored films (Figure 5.1A).
5.3.2. Physicochemical characterization of films
The results of the physicochemical studies are summarized in Table 5.2. The drug
content in the prepared films was found to be 100% with a low standard deviation,
indicating good uniformity in drug content. The thickness and weight variation of films are
associated with the uniformity and accuracy of dosing [134]. Uniformity of thickness of
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Figure 5.1. Physicochemical characterization of TQ films. (A) FTIR spectrum of TQ pure
drug, PVP, physical mixture of drug and polymer, freshly prepared films containing drug
and polymer, stored films containing drug and polymer; (B) control films (i); Field
emission scanning electron microscopic (FESEM) images showing surface morphology of
control film (ii-iii) at different magnifications; and (C) TQ films (i); FESEM images
showing surface morphology of TQ films (ii-iii) at different magnifications.
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each film and minimal weight variation was ensured as low standard deviation values were
observed in the thickness of films and weight variation studies. The flatness study showed
that the films had the same strip length before and after cutting, indicating 100% flatness.
These data also indicate 0% constriction in the films meaning they could maintain a smooth
surface when applied onto the skin. In other words, the films provided intimate contact
with skin and hence better drug permeation. Folding endurance test results indicated that
the films would not break and would maintain their integrity when folded. The low
moisture uptake at laboratory ambient condition protects the material from microbial
contamination and avoids extra bulkiness of the films. The moderate moisture content of
the prepared films could assist the formulation stability by preventing drying and
brittleness. These results indicated that the polymeric combinations showed good film-
forming properties and the process employed to prepare films in this study could produce
films with uniform drug content and minimal film variability.
The surface morphology of the drug loaded film was assessed using field emission
scanning electron microscopy (FESEM) and shown in Figure 5.1C. FESEM images were
taken at different magnifications 100X, 500X and 1000X to investigate the surface of films.
At all magnifications the film surface appeared smooth and compact. FESEM photograph
of TQ film shows polymer networks inside the film and homogeneous dispersion of drug
inside the polymer networks (Figure 5.1C).
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Table 5.2. Physicochemical properties of TQ films (data shows mean of five
determinations with ± standard deviation).
Physicochemical TQ polymeric
parameters films
Drug content (%) 100 ± 6.4
Thickness (mm) 1.17 ± 0.04
Weight variation (mg) 82.04 ± 1.9
Flatness (%) 100
Folding endurance 68 ± 2.38
Moisture content (%) 14.12 ± 0.42
Moisture uptake (%) 2.26 ± 0.47
5.3.3. Characterization of the TQ hydrogels
Table 5.3 shows the results of physicochemical properties of prepared TQ hydrogel
formulations (F1-F10). All the prepared hydrogels were light yellow in color and either
opaque or clear. Most of the formulations showed good homogeneity with no lumps and
smooth homogeneous texture. pH values of the formulations were found in the range of
3.91-4.86. The relative standard deviation (% RSD) of prepared hydrogel formulations
ranged from 0.12 to 0.34%. Good spread ability is one of the criteria for gel as it shows the
behavior of the gel when it comes out form the tube. It is the term that is used to indicate
the extent of area to which gel readily spreads on application. It was observed that spread
ability of TQ hydrogels decreased by increasing the polymer concentration and the values
were in the range of 12-21 mm.
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Table 5.3. Physicochemical properties of TQ topical hydrogel formulations (F1- F10).
+: not good; ++: good; +++: very good
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Rheological properties of gel formulation provide important information regarding
physical form, appearance, texture and flow behavior [135]. The determination of linear
viscoelastic region (LVR) is used to understand the physical form or microstructure of the
gel formulation. In this study, oscillation stress sweep was used to obtain the LVR. The
range of stress over which the elastic modulus G′ is independent of the applied stress
amplitude is called the LVR. So, it presents a critical stress beyond which the sample may
show significant structural breakdown. The mean stress value of 5 Pa obtained from LVR
was used for other oscillation tests such as frequency sweep.
To obtain information about viscoelastic behavior of the prepared samples
oscillation frequency sweep test was performed to measure the response of a system as a
function of frequency at constant stress amplitude (within LVR). Elastic modulus (G′) and
viscous modulus (G″) were determined as a function of frequency. G″ is a measure of the
energy lost per cycle and reflects the fluid-like component whereas G′ is a measure of
energy stored per cycle and reflects the solid like component of the viscoelastic material.
The G′ will be large if a material is predominantly elastic or highly structured. In this
experiment, the addition of Carbopol and Ultrez 10 showed drastic increase in G′, whereas
addition of HPMC and HPC in formulation reduced G′. The order of increment in G′ was
F10 > F9 > F4 > F3>F1 (Figure 5.2A).
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Figure 5.2. Rheological characterization of TQ hydrogel formulations (F1-F10). (A)
Oscillation frequency sweep data. The elastic modulus (i); The viscous modulus (ii) were
plotted against angular frequency. TQ permeation and skin deposition from film and gel
formulations (B). TQ permeation profile for different hydrogel formulations (i). Time
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points were measured at 1, 2, 3, 4, 5, 6 and 8 hours. Each point represents the mean ± S.D.
of five experiments; TQ permeation from film formulation across human cadaver skin
(mean ± S.D., n=5) (ii); Amount of TQ detected after 8 hours in human cadaver skin (N=5,
mean ± SD) using different TQ hydrogel formulations (iii).
5.3.4. In Vitro skin permeation and deposition studies
Penetration parameters of thymoquinone are summarized in Table 5.4. The results
showed that the rank order for thymoquinone flux from each formulation are: F 2 > F 5 >
F 3 > F 7 > F 10 > F 9 > F 6 > F 8 > F 4 > F 1. Figure 5.2B shows the thymoquinone
permeation profile and amount of TQ detected after 8 hours in human cadaver skin. It was
observed that formulation 7 and 9 was able to retain more drugs in human cadaver skin
compared to other formulations that might be useful in the treatment and management of
wound infections.
5.3.5. Stability study
After storage, no significant changes in color and texture of the film were observed.
The drug content of the stored films was comparable and were within limits. Additionally,
FTIR spectra of stored film and freshly prepared films can be superimposed, indicating the
stability of the films (Figure 5.1A). Hence, the film can be used after storage of two months
without any loss of physical and chemical attributes. After storage for 8 months, hydrogels
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Table 5.4. Penetration parameters of thymoquinone through human cadaver skin (N=5)
after 8 hours.
Formulation Q at 8 hours (µg/cm2) TQ Flux
(µg/cm²/hr)
1 62 ± 9 5.38 ± 0.3
2 77 ± 13 9.56 ± 1.0
3 73 ± 11 7.87 ± 0.9
4 76 ± 16 5.59 ± 0.7
5 67 ± 15 9.19 ± 2.0
6 36 ± 7 6.70 ± 1.8
7 99 ± 17 7.11 ± 1.6
8 52 ± 9 6.24 ± 1.1
9 48 ± 10 6.89 ± 1.4
10 40 ± 9 7.1 ± 2.3
Q, cumulative amount of thymoquinone penetrated per cm2 at 8
hours (mean ± SD, n=5)
did not show any change in color and odor. Additionally, no phase separation occurred.
The antibacterial study with the stored hydrogel showed similar efficacy like the marketed
gentamicin cream against Staphylococcus aureus. This indicated that the drug was stable
in gels even after 8 months of storage and the gel formulations were physically and
chemically stable.
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5.3.6. Cyto-compatibility study
The viability of HDF and HaCat cells at the presence of TQ film was analyzed
using alamarBlue® assay- is a cell viability assay reagent which contains the cell
permeable, non-toxic and weakly fluorescent blue indicator dye called resazurin. Resazurin
is used as an oxidation-reduction (REDOX) indicator that undergoes colorimetric change
in response to cellular metabolic reduction. The reduced form resorufin is pink and highly
fluorescent. As the intensity of fluorescence produced is proportional to the number of
living cells respiring, the viable cells continuously convert resazurin to resorufin,
increasing the overall fluorescence and color of the media surrounding cells. Through
detecting the level of oxidation during respiration alamarBlue® acts as a direct indicator to
quantitatively measure cell viability and cytotoxicity. The results of the test are shown in
Figure 5.3. As can be seen from the graph, the cell viability is not affected by the addition
of TQ film in the media. TQ film showed 90% cell viability on HaCat and 96% cell viability
on HDF cell lines after 24 h of incubation. According to ISO 10993–5: 2009 [136]
standards, it can be confirmed that the prepared samples were nontoxic in nature, because
the cell viability is > 80%
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Figure 5.3. Cytocompatibility study of TQ film. Cell viability of TQ film with HDF and
HaCat cells using alamarBlue® assay.
5.3.7. In vitro and Ex vivo bacterial inhibition study
Prior to in vivo experiments, the in vitro antibacterial efficacy of TQ film was first
validated along with ex vivo antibacterial activity. For this, the materials were inoculated
with Staphylococcus aureus and their antibacterial efficacies against bacterial growth were
assessed after 24 h incubation. Results from in vitro study showed that the presence of
gentamicin generated a zone of growth inhibition of Staphylococcus aureus on the plate in
a dosage dependent manner (Figure 5.4A). However, there was no growth of bacteria in
the presence of TQ films and hydrogels (Figure 5.4A) and suggested that a complete
inhibition of Staphylococcus aureus. The completely absence of bacteria in the presence
of TQ films was verified by three independent experiments. Furthermore, the results from
0
20
40
60
80
100
120
Media TQ Film Control Film Stored Film Triton
Cell
via
bili
ty (
%)
HaCat
HDF
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ex vivo antibacterial study showed complete eradication of bacteria using TQ film from
human cadaver skin. Whereas, gentamicin cream and TQ hydrogel showed 5 and 4 log
reduction of bacteria respectively (Figure 5.4B).
Figure 5.4. Bacterial inhibition study. (A) Inhibition of bacterial growth on agar plate by
Control negative (i); Gentamicin positive control 50 µg/mL (ii) right upper and 500 µg/mL
(ii) right lower; Control film (iii); TQ hydrogel (iv) and TQ film (v) against Staphylococcus
a b
c
130
aureus; (B) Ex vivo antibacterial activity by Control (i); Control film (ii); Gentamicin
sulfate USP, 0.1% marketed cream (iii); TQ hydrogel (iv); TQ film (v) and Log of bacterial
reduction with different treatment groups (vi). Data represent mean ± SD of four replicates.
***p = < 0.001 and ^^^p = < 0.05.
5.3.8. Scratch assay for wound closure activity
In vitro scratch wound healing assay has been proven as a simple, valuable and
inexpensive experiment to obtain first insights into how plant preparations or their isolated
compounds can positively influence formation of new tissue [137]. This assay has
commonly been applied to measure cell migration, cell proliferation and wound closure in
response to test components. In this experiment a “wound gap” in a cell monolayer of both
HDF and HaCat is created by scratching, and the ability to migrate cells to repopulate the
scratch area over time is monitored. The results of these experiments are shown in Figure
5.5 (A-E). It was found that TQ showed significant positive effects on wound healing
activities of HDF and HaCaT cell lines. TQ (100 ng) showed significant wound closure
activity with HDF compared to control (p<0.05) (Figure 5.5D). 77% of wounds were
closed with 100 ng TQ at 12 hours followed by 100% wound closure at 24 hours. On the
other hand, 43% wounds were closed with control at 12 hours (Figure 5.5C and D).
Additionally, at 4 h, 8 h and 12 h all the different concentration of TQ (1-1000 ng) showed
increased wound closure activity compared to control, suggest the cell migrates faster in
presence of TQ. The number of fibroblast cells in the scratched area was found to
131
132
Figure 5.5. Effect of different treatment groups on the wound healing of keratinocytes and
fibroblasts. (A) Representative micrographs from control, 1 ng/mL and 100 ng/mL of TQ,
showing the original wound and the wound after 6 days; (B) Quantitative analysis of wound
closure as a function of time. The wound area was determined as the wound area at a given
time relative to the original wound area. Data are presented as the means ± SD (n=5-6).
***p<0.001 (control vs 100 ng/mL) and ^p<0.05 (Control vs 1 ng/mL). (C) Representative
micrographs from control, 1 ng/mL and 100 ng/mL of TQ, showing the original wound
and the wound after 24 hour; (D) Quantitative analysis of wound closure as a function of
time. The wound area was determined as the wound area at a given time relative to the
original wound area. Data are presented as the means ± SD (n=6). **p<0.01 and
***p<0.001 (control vs 100 ng) and ^p<0.05 (Control vs 1 ng); (E) Quantitative
measurement of cells number migrating in the corresponding scratched wound areas at
different treatment groups. The values plotted were means of 6 determinations (𝑛 = 6).
***p<0.001 (100 ng/mL vs control/1 ng/mL/10 ng/ml).
increase between 10 ng to 200 ng TQ and then decrease with the increased concentration
of TQ. 100 ng TQ showed a significant increase in the number of fibroblast cells compared
to all other experimental conditions (Figure 5.5E). Using the HaCaT cell line 100 ng TQ
showed 85% wound closure activity at day six which is significantly higher (p = 0.0001)
than the experimental control (Figure 5.5A and B). At all time points (Day 1, Day 2, Day
3 and Day 6) 1 ng, 10 ng, 100 ng, 200 ng and 1000 ng TQ showed increased wound closure
activity compared to the control. Rapid wound closure activity was observed between day
three and day six at all TQ concentrations (1-1000 ng).
5.3.9. Wound healing and anti-bacterial activity of TQ film in vivo
The photographs of the wounds at 0, 3, 7, 14, and 21 days post-wounding are shown
in Figure 5.6A. Almost all of the wound areas were re-epithelialized in different
experimental groups. The percentage of wound closure were determined and compared
among all experimental groups (Figure 5.6C). At day 7 and 10, 25% and 42% wounds
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were closed respectively using TQ film. Whereas, applying gentamicin marketed cream
12% and 39% wound closure activity were observed at day 7 and 10 respectively. Based
on this observation, it can be stated that an accelerated but not statistically significant
wound healing was observed using TQ film at day 7 and 10 compared to the gentamicin
marketed formulation (Figure 5.6C). Additionally, at day 14 similar wound closure activity
was obtained using both TQ film (62%) and gentamicin marketed formulation (63%). At
all time points both TQ film and gentamicin showed higher wound closure activity
compared to the control film experimental group. A higher wound closure activity (though
not statistically significant) was observed at all time points with the bacterial wound
experimental group compared to the TQ film, gentamicin cream and control film. One
possibility may be fewer handling or disturbance of the wounds in this group helped wound
closure. In this case, the bacterial wound with the vehicle control serves a more appropriate
control for the study. As the vehicle control would provide the same wound environment
as the treatment group whereas the bacterial wound without any vehicle would not provide
the same microenvironment of the wound.
Bacterial sample analysis of the wound site at day 1, day 2, day 3 and day 7 showed
significant (p<0.001) bacterial reduction using both TQ film and gentamicin compared to
the bacterial wound and control film (Figure 5.6B). Significant bacterial reduction coupled
with wound closure activity of TQ film that is similar to the gentamicin marketed cream
may be considered as a promising aspect of TQ’s usefulness in treating infected wounds.
Further studies should investigate in more details the benefits of using TQ in the treatment
and management of wound infection.
134
135
Figure 5.6. Macroscopic observations, wound closure and bacterial reduction. (A)
Photographs of wounds in BALB/c mice in which the wounds received TQ loaded film
and Gentamicin. The animal with bacterial wounds and wound with no bacterial served as
control group and animal with control film served as a vehicle control. Representative
photographs of the wounds were taken at 0, 3, 7, 10, 14, and 21 days post-wounding; (B)
Log of bacterial reduction at each time point (Day 1, 2, 3 and 7) using different
136
experimental groups. Data are presented as the means ± SD (n=2-4). ***p<0.001 (Bacterial
wound vs TQ Film) and ^^^p<0.001 (Bacterial wound vs Gentamicin); (C) Percentage of
wound closure in all experimental groups at 0, 3, 7, 10, and 14 days post-wounding.
5.3.10 . Histological examination
Histopathological study was performed by taking 1 cm2 of animal tissue at the end
of the study. The tissues were stained with Masson’s trichrome. Re-epithelization was
observed in experimental groups of control wound, bacterial wound, TQ film and
gentamicin formulation (Figure 5.7). A complete re-epithelization was observed with the
animal received one extra dose of TQ film (+ dose) compared to the group which received
four doses of TQ film (Figure 5.7v and vi). Therefore, future research should be conducted
to find out the appropriate dosage regimen (formulation type, TQ concentration in the
formulation, dosing interval, length of treatment) that would be needed to bring in the
beneficial effect of TQ in wound re-epithelization. We also predict that wound remodeling
was still in progress in animals with TQ film treatment. Whereas, wound remodeling was
may be completed with other animal groups. More studies need to be conducted to confirm
these findings and to understand the wound environments under different conditions in the
future. Future research should also examine the usefulness of other dosage form of TQ like,
cream, gel, topical spray formulation in wound closure activity.
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Figure 5.7. Masson’s trichrome staining of the different samples at day 21 post-wounding
(control wound (i); Gentamicin sulfate USP, 0.1% marketed cream (ii); bacterial wound
(iii); control film (iv); TQ film (v); TQ film + dose (vi)) indicates epidermis and
indicates dermis.
5.4. Conclusions
Open wounds are prone to bacterial infection and if not treated at earlier time point
might also provide an entry point for microbes that cause systemic infections. Additionally,
infected wounds heal less rapidly as infection at the wound site can produce toxins that can
further kill the regenerating cells and can delay the progression of wound healing. Several
topical and oral antibiotics are presently being used to treat wound infections in humans.
However, due to their adverse effects and the presence of antibiotic-resistant organisms,
researchers are now investigating different bioactive compounds of plant origin for their
antibacterial activity to offer an innovative treatment strategy. The use of TQ to treat
138
infected wound is justified by this work, as TQ exhibited commendable activity against
Staphylococcus aureus.
In this study, novel TQ loaded polymeric films and hydrogels were developed using
different polymers. The application of these TQ containing films and hydrogels showed in
vitro skin permeation and a strong antibacterial activity against Staphylococcus aureus
since no bacterial growth was observed with TQ loaded film and hydrogel formulations. In
vitro scratch wound healing assay revealed wound closure activity of TQ. Moreover,
preclinical mice model of Staphylococcus aureus wound infection demonstrates a
significant reduction in bacterial population using TQ film and wound closure activity.
Based on this result it can be stated that, significant bacterial reduction coupled with wound
closure activity of TQ film might be able to provide a new treatment strategy specially for
those who has developed resistance towards the commonly used antibacterial agents. In
summary, TQ has potential to be used as an agent for wound healing and to treat bacterial
skin infections. TQ/PVP films and hydrogels developed in this study have potential for the
treatment and management of wound and Staphylococcus aureus related bacterial skin or
wound infections. This study would provide the supplementary evidence of TQ’s
significant potential in the area of microbial control and wound healing. But further
research is needed to confirm this novel finding.
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Appendix A. Development of lidocaine loaded EUDRAGIT® RLPO transdermal
patch application
A.1. Introduction
Transdermal route has attracted most of the formulation scientists owing to its
advantages such as prevention of hepatic first pass metabolism, avoidance of
gastrointestinal degradation, higher bioavailability at lower dose, direct transport into
systemic circulation, non-invasive, easy administration, higher patience compliance, lower
risk of overdose etc. [13, 138, 139]. Transdermal drug delivery systems in the form of
patches have been available on the market for several decades. Patches can be classified as
matrix (drug-in-adhesive) systems, or reservoir, or membrane-controlled systems [140].
PSAs (Pressure-Sensitive Adhesives) are traditionally used in patch production. The main
families of PSAs present several drawbacks related to their chemical structures. Therefore,
nowadays acrylics and silicone-based PSAs have been largely replaced by
polyisobutylenes (PIBs) due to the reduced allergenicity. Poly(methyl methacrylate)s are
commonly used in various pharmaceutical preparations including in transdermal patches
because of their well-recognized biocompatibility and safety even if additives or chemical
cross-linking agents are required to provide them with required adhesive properties [141-
143].
EUDRAGIT® RL is referred to as ammoniomethacrylate copolymers in the
USP/NF [144]. This copolymer is synthesized from acrylic acid and methacrylic acid esters
[145]. Unlike natural cellulose derivatives the synthesis of methacrylic copolymers via
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free-radical polymerization is highly reproducible. Firstly, using various acrylic acid or
methacrylic acid derivatives long polymer chains are formed by chain growth reactions. In
a subsequent step, the functional properties of the final (meth)acrylic copolymers can be
adjusted by selection from a variety of monomers. The functional co-monomers are
responsible for adjusting the solubility profile. Whereas, the non-functional co-monomers
are responsible for steering the polymer properties. Various grades of EUDRAGIT® can
be obtained by varying the chain length through various termination and transfer reactions.
Different grades of EUDRAGIT® has different physicochemical properties and their
applicability in pharmaceutical industries also differ. EUDRAGIT® RLPO is insoluble at
physiological pH and able to swell to form permeable films as it contains a higher number
of functional quaternary ammonium groups (10%). This ammonium groups are present as
salts which allow water molecules to penetrate freely into EUDRAGIT® RLPO and can
further influence the drug flux. Additionally, EUDRAGIT® RLPO is a polymer with
moderate glass transition temperature of 63°C and it has a pH of 6 that is close to the human
skin pH. Moreover, this polymer is stable, possess good film making characters and act as
crystallization inhibitors [146]. All of these properties can be useful to solve some of the
existing issues with transdermal patch delivery, such as; 1) a pronounced tendency to drug
crystallization with silicon-based PSAs (Pressure-Sensitive Adhesives) [147]; 2) the
disadvantage of using (PIBs) in place of PSAs are related to their easy oxidation and low
air and water vapor permeability that can interfere with drug flux through the skin and can
cause skin maceration.
Hydrophobic polymers of EUDRAGIT® classes including EUDRAGIT® RLPO,
EUDRAGIT® RSPO, EUDRAGIT® NE 30D, EUDRAGIT® EPO have been widely used
141
in pharmaceutical formulations. The high release and flux of lipophilic drugs can be
achieved by combining the hydrophobic and hydrophilic polymers in a suitable ratio. The
previous study showed the use of EUDRAGIT® RL 100, EUDRAGIT® RS 100 and
HPMC (Hydroxy propyl methylcellulose) in the preparation of matrix patches of lipophilic
drugs [148]. HPMC is a water-soluble cellulose ether. It has been widely used in
pharmaceutical products due to its hydration and gel forming abilities. Therefore,
EUDRAGIT® RLPO and HPMC were chosen to develop lidocaine transdermal patches.
In the current study, lidocaine was chosen as a model drug due to its lipophilic
characteristic (log P = 2.3) and its applicability as a topical/transdermal anesthetic. It was
also used in this study because of its tendency to become crystalline in PSAs. The aim of
this work was to develop and characterize the prolonged release transdermal patch using
EUDRAGIT® RLPO and lidocaine as a model drug to improve its adhesive and cohesive
strength, to study the drug release of lipophilic drug using the hydrophobic polymer, to
examine the lidocaine crystallization, to evaluate the water vapor transmission rate
(WVTR) of the developed transdermal patch system and to compare the rheological
properties of the formulation.
A.2.Materials and Methods
A.2.1. Materials
Lidocaine was purchased from MP Biomedicals (Solon, OH, USA). Chitosan was
purchased from Alfa Aesar (Haverhill, MA, USA), Hydroxy propyl methylcellulose
(HPMC) was purchased from Ashland (Parlin, NJ, USA), Triethyl Citrate (TEC) was
142
purchased from Jungbunzlauer (Newton Centre, MA, USA) and Acetone was purchased
from J.T. Baker (Phillipsburg, NJ, USA). Eudragit® RLPO was a gift from Evonik
(Piscataway, NJ, USA). Drierite was purchased from W.A. Hammond Drierite Company
Ltd (Xenia, OH, USA). Glacial acetic acid, 1N sodium hydroxide, Acetonitrile, Monobasic
potassium phosphate were purchased from Fisher Scientific (Bridgewater, NJ, USA).
Nonwoven polyester layer CoTran™ 9695 and fluorosilicone coated polyester film
Scotchpak™ 9709 were gifts from 3M Drug Delivery Systems (St. Paul, MN, USA).
A.2.2. Preparation of lidocaine-loaded transdermal patches
Transdermal patches containing lidocaine were prepared by solvent evaporation
techniques (Figure A.1) using composition given in Table A.1. Lidocaine and Eudragit®
RLPO polymer were first dissolved in an acetone solvent then HPMC and Chitosan were
incorporated into the lidocaine containing Eudragit® RLPO dispersion with constant
stirring. The obtained uniform dispersion was casted on nonwoven polyester layer coated
with a hypoallergenic acrylate adhesive (3M CoTran™ 9695; 3M Drug Delivery Systems,
St. Paul, MN) and allowed for air drying at laboratory ambient temperature for overnight.
The patches were then covered with Fluorosilicone Coated Polyester film (3M
Scotchpak™ 9709), cut into appropriate sizes and stored at room temperature.
143
Figure A.1. Schematic representation of solvent evaporation method.
Table A.1. Lidocaine-loaded patch composition % (w/w) at different drug loading ranging
from 4% to 20% (Formulation A-D).
Formulation
Code
Eudragit®
RLPO
HPMC Chitosan TEC Lidocaine Acetone
A 10 7 7 16 4 56
B 10 7 7 16 5 55
C 10 7 7 16 10 50
D 10 7 7 16 20 40
Solvent evaporation
144
A.2.3. Patch characterization
A.2.3.1 Thickness
Patch thickness was determined using a digital caliper (Mitutoyo America
Corporation, Aurora, IL, USA) and three measurements were performed on each patch to
obtain the average thickness.
A.2.3.2 Weight variation
To ensure the weight uniformity, 3 randomly selected patches from each
formulation were subjected to individual weighing.
A.2.3.3. Content uniformity
The lidocaine-loaded patch was cut into 7 x 5 inch2 pieces and accurately weighted.
The patch was then dissolved in 500 mL of 50:50 acetonitrile and water solvent and filtered
through a syringe filter (0.45 µm). Drug concentration was quantified using HPLC method.
A.2.4. High Performance Liquid Chromatography (HPLC)
Lidocaine was quantified using a Shimadzu Prominence PDA HPLC system
(Empower 3 software). A C18 column (Waters, Xbridge) with dimensions of 250 X 4.6
mm and 5 µm packing was used. Acetonitrile and Solution A (20:80) was used as a mobile
phase. Solution A was prepared by mixing 50 mL of glacial acetic acid with 930 mL of DI
water and finally adjusted the pH to 3.4 with 1N sodium hydroxide. The column
temperature was set to 25 °C. The flow rate used was 1.5 mL/min with injection volume of
145
20 µL and run time for 15 minutes. Lidocaine peak was detected at 254 nm, 4 min retention
time. The method was linear at a concentration of 0.17–1.7 mg/mL with R2 value of 0.99.
A.2.5. Mechanical properties
Mechanical properties in term of tensile strength (TS) and percent elongation at
break (E/B) were determined using Tinius Olsen Material testing equipment (H50KT,
Horsham, PA, USA) working with a 100 N loaded cell. The patch strip between two
clamps was positioned at a distance of 50 mm and the test speed was set to 5 mm/s. Tensile
stress (N/m²) is the force applied to induce film deformation. Tensile stress was calculated
using the following equation-
Stress = Force applied (N)/Area (m²) (1)
The elongation at break is the distance traveled by the upper plate up to the point where
the film separates compared to the origin and was calculated as follows-
% 𝐸𝑙𝑜𝑛𝑔𝑎𝑡𝑖𝑜𝑛=(𝐿𝑒𝑛𝑔𝑡ℎ 𝑎𝑡 𝑏𝑟𝑒𝑎𝑘𝑖𝑛𝑔 𝑝𝑜𝑖𝑛𝑡−𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑓𝑖𝑙𝑚)/(𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙
𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑓𝑖𝑙𝑚) X 100 (2)
A.2.6. Loop tack test or adhesive strength study
Tinius Olsen Material testing equipment (H50KT, Horsham, PA, USA) equipped
with software (Horizon) was used to study the adhesive properties of the transdermal patch.
Stainless steel plate was used due to its low surface energy like the human epidermis.
146
Adhesive strength in terms of peak adhesive force (N) was determined. The force required
to detach the film on the upper platen from the stainless-steel plate known as the adhesive
force. The upper platen with a 1-inch width patch attached was lowered to the surface of
the stainless-steel, allowed to secure contact with a load of approximately 100 N for 10 s
and then raised at a constant rate of 300 mm/min.
A.2.7. Rheology
Rheological characterization was performed on rheometer (Anton Paar, Graz,
Austria) equipped with a 25 mm flat stainless-steel plate. All tests were done at 32ºC and
a gap of 1 mm. Following tests were carried out-
A.2.7.1. Oscillation stress sweep
The samples were subjected to increasing stress (0.1 - 100% strain) at a constant
frequency of 1 Hz. This test allows determination of the liner viscoelastic region (LVR) of
the sample, and therefore the consequent choice of the stress value to use in the subsequent
oscillation test.
A.2.7.2. Frequency sweep
Frequency sweeps were done by oscillating the samples at angular velocity (ω)
range of 0.1 – 100 rad/sec at a constant stress obtained from LVR. Effect of stress on elastic
modulus (G') and viscous modulus (G'') were recorded.
147
A.2.8. In vitro release study
Vankel VK 7000 dissolution system was used to obtain the release profile of
Eudragit patches. The release studies were performed according to USP apparatus 5, paddle
over disc method. One liter of phosphate buffer pH 7.4 at 32ºC was used as the dissolution
medium. The paddle rotation speed was adjusted to 50 rpm. Two milliliter samples were
withdrawn at predetermined time intervals. At 24 hours the paddle rotation speed was set
to 100 rpm for an hour for infinity sample. At the end of the study the samples were
analyzed using a UV-Visible spectrophotometer at 254 nm.
A.2.9. Water vapor transmission of transdermal patch system
The water vapor transmission rate (WVTR) is the amount of moisture transmitted
through a unit area of film in a given duration and at specified temperature. All tests were
done at 32ºC and 90% RH chamber. Water vapor cups were filled to within 3 mm of the
opening with Drierite and was assured that it will not make contact with the adhesive of
the test specimen. The test specimens were applied adhesive face down over the opening.
The test adhesives were brought into intimate contact with the flange using pressure. The
assembly was placed in the humidity cabinet for 24 hours conditioning period. After 24
hour it was removed from the cabinet, cooled for 15 minutes at standard conditions (24ºC
and 50% RH) and weighted on an analytical balance. This weight is the W1. After the
initial weighing the assembly was returned to the humidity cabinet for 72 hours. Removed,
conditioned for 15 minutes and weighed. This weight was used as W2. Finally, the WVTR
for the prepared patches was calculated using the following equation-
148
WVTR = W2- W1 X 2400/T X A (1)
Where,
W1 = weight (in grams) before exposure period
W2 = weight (in grams) after exposure period
T = exposure time (in hours)
A = area (in square inches) of opening in dish
A.2.10. Shower resistance study
A 10 minutes shower test was performed on the marketed and experiments patches.
Both of the patches were cut into same sizes (10 X 7 cm) and were brought into intimate
contact with the stainless-steel plate using pressure. A water flow of 40 psi was applied on
the patches for 10 minutes. This cycle was repeated until the sign of detachment was
observed.
A.2.11. SEM-EDS (Scanning Electron Microscopy-Energy Dispersive Spectroscopy)
The surface morphology of the transdermal patch was examined using a scanning
electron microscope (TM3000, Hitachi, USA) at 500 magnification.
149
A.2.12. Differential scanning calorimetry studies
Patches used for studying the crystallization of the drug were kept at laboratory
ambient temperature in a closed container for 30 days. A Perkin-Elmer Pyris-6 differential
scanning calorimeter (DSC) was used to study the crystallinity of the drug in the
transdermal patches and the physical mixtures of the polymers used in this study.
Approximately 6-8 mg of each sample of Ex patch 10%, Ex patch 20%, pure drug, pure
polymers (Eudragit® RLPO, HPMC, Chitosan), 1:1 physical mixtures of polymers and 1:1
physical mixtures of polymers, drug and TEC were hermetically sealed in a flat bottomed
aluminum pan with aluminum cover and heated over a temperature range of -30ºC to 150ºC
at a linear heating rate of 10ºC/min.
A.2.13. Data and statistical analysis
Results are reported as mean ± SD (n=3). The statistical analysis of the data was
performed by using one-way Anova, the Tukey post-hoc tests and Student’s-t test, and p-
values < 0.05 were considered significant.
A.3. Results and discussion
The transdermal patches of lidocaine were prepared according to Table A.1. The
patches were the combination of hydrophilic polymer (HPMC and Chitosan) and
hydrophobic polymer (EUDRAGIT®) with varying amounts of lidocaine. Solvent casting
150
technique used to prepare the patches was satisfactory. The developed patches were thin,
flexible and smooth.
A.3.1. Appearance and patch thickness
A matrix dispersion transdermal patch was prepared. Lidocaine-loaded patch had a
smooth surface with transparent color. The thickness, weight and drug content assay of
lidocaine patches were shown in Table A.2. Thickness of all five experimental patches was
found to be in the range of 0.85±0.02 to 1.31±0.005 mm. The marketed lidocaine patch
thickness was 1.17±0.04 mm. Each formulated patch provided the low value of SD,
indicating the uniformity of its thickness. The average weights of these formulations ranged
from 2.03±0.05 to 3.80±0.13 gm. Each formulation showed the low value of SD, indicating
the uniformity of the developed patch.
A.3.2. Content uniformity
To determine the content uniformity other parameters were kept constant
(EUDRAGIT® RLPO, HPMC, Chitosan). Drug loading was varied from 4% to 20%
(Table A.1). Drug content (%) of formulations varied from 94±1 to 107±3.5 indicating
drug was dispersed uniformly throughout the patches. The small SD also indicates that the
solvent evaporation method was efficient to fabricate lidocaine-loaded patches with good
uniformity of dosage unit and minimal variation.
151
Table A.2. Physical and mechanical properties of transdermal patches containing
lidocaine. Data represents N=3, mean ± SD.
A.3.3. The effect of EUDRAGIT® RLPO in adhesive and cohesive strength
The adhesion of patches to the skin is a prerequisite for maintaining drug release.
The loop tack tests define tack as the force required to separate a loop made by clamping
the ends of a patch strip at a specified time that has been brought in contact with a specified
area of a defined surface. Formulation B and C showed significantly higher adhesive
strength compared to the other formulations possibly due to the varying concentration of
drugs (Figure A.2). All the formulations have the same concentration of HPMC, Chitosan,
EUDRAGIT® RLPO and TEC. The control positive formulation doesn’t contain any
EUDRAGIT® RLPO. The presence of a large number of hydroxyl groups in HPMC and
152
Chitosan may have been played a role in adhesion [149]. Though control formulation
without EUDRAGIT® RLPO showed adhesive properties but the adhesive layer of the
control patch did not strip cleanly from the plate, leaving noticeable residues, which was
an evidence of cohesive failure (Figure A.3). This could be due to the observed lower
cohesive properties of the patches made of just hydrophilic polymer. EUDRAGIT® RLPO
may have function as an adjunct adhesive in the patch containing hydrophilic polymers.
That further may have been contributed in improving the cohesive properties of the
patches.
Figure A.2. Peak adhesive force of different formulations containing Eudragit® RLPO
and different concentration of Lidocaine. Data represents mean ± SD (n=3).
0
1
2
3
4
5
6
Controlnegative
From A Form B Form C Form D MarketedPatch
Controlpositive
Pe
ak a
dh
esiv
e f
orc
e (
N/c
m²)
***
***
153
Figure A.3. Cohesive properties (a) formulation with EUDRAGIT® RLPO, (b)
formulation without EUDRAGIT® RLPO.
A.3.4. The effect of drug loading and EUDRAGIT® RLPO on the mechanical
properties of transdermal patches
The mechanical properties are useful indications of patch strength that might be
useful to hold up to the rigorous friction from day to day patch use. The results of
mechanical properties in terms of tensile strength and percentage of elongation at break are
shown in Table A.2, Figure A.4 and Figure A.5. All formulations showed tensile strength
with the range of 131- 143 N/m2 and provided percentage of elongation at break in the
range of 107-139%. Formulation A, B, C and D has varying amounts of lidocaine ranging
from 4% to 20%. There was almost no difference in the tensile strength and % elongation
at break values among different drug loaded patches. These results revealed that the
presence of hydrophobic polymer EUDRAGIT® RLPO in formulations may delivered a
patch with higher tensile strength compared to the marketed lidocaine patch (Figure A.4).
a b
154
On the other hand, the % elongation at break was higher with the marketed patch compared
to the all other experimental patches (Figure A.5). This is may be the effect of different
liners used in marketed and experimental patches.
Figure A.4. The effect of different formulations on tensile stress. Data represents mean ±
SD (n=3), ***p < 0.005.
Figure A.5. The effect of different formulations on % Elongation. Data represents mean
± SD (n=3), ***p < 0.005.
0
20
40
60
80
100
120
140
160
Controlnegative
Form A Form B Form C Form D Marketedpatch
Controlpositive
Te
nsile
str
ess (
N/m
²)
0
50
100
150
200
250
Controlnegative
Form A Form B Form C Form D Marketedpatch
Controlpositive
Str
ain
(%
Elo
nga
tio
n)
***
155
A.3.5. The effect of drug loading on rheological behavior of the formulations
The adhesive properties of PSAs are strictly related to the solid and liquid like
behaviors and are dependent on frequency of the applied stress at a given temperature. In
this study, the rheological analysis of the different formulations was used to determine the
viscoelastic parameters such as elastic modulus (G´) and viscous modulus (G´´). If G´ >
G´´, then the material is more solid than liquid as G´ value is the representative to the solid
like behavior. The converse is also true. Initial bonding with the skin usually occurs at low
frequencies and the PSA liquid like nature predominates since it must wet the substrate and
therefore low values of G´ is desirable [150]. Debonding process requires the formulation
to behave like solid and occurs at high frequencies. It also requires high cohesive strength
and is associated with a larger G´´. Figure A.6 shows the rheological behavior of different
drug loaded patch formulations. The result demonstrates that there is a decrease in G´ and
G´´ values with the increase of drug concentrations. Comparing the G´ and G´´ values of
different formulations it is observed that G´ ´> G´. That might be useful in bonding and
debonding process.
156
[A]
[B]
Figure A.6. Rheological behavior of different drug loaded patch formulations in terms of
oscillation frequency sweep data (A) the elastic or storage modulus and (B) the viscous or
loss modulus were plotted against angular frequency.
157
A.3.6. In Vitro release study
All the experimental EUDRAGIT® RLPO patch formulations provided the most
controlled release of lidocaine with 7% being released after 2 h and 100% after 24 h (Figure
A.7). On the other hand, 36% lidocaine was released from the marketed patch after 2 h and
65% drug was released at 4 h (Figure A.7). This result indicated that, due to the presence
of EUDRAGIT® RLPO there was more sustain and controlled release of lidocaine from
the experimental patch compared to the marketed and control patch without EUDRAGIT®
RLPO. Experimental control patch showed 53% drug release at 2 h followed by 84% drug
release at 4 h. This result demonstrated the usefulness of EUDRAGIT® RLPO in providing
a sustain and controlled drug release over a longer period of time in the transdermal patch
formulations.
Figure A.7. In vitro release profile of lidocaine from experimental and marketed patch
formulations in phosphate buffer at pH 7.4 (N=3).
0
20
40
60
80
100
120
0 1 2 4 6 8 10 12 18 24 25
% R
ele
ase
Time (h)
Marketed Patch
Ex Patch 4%
Ex Patch 5%
Ex Patch 10%
Ex Control Patch
Ex Patch 20%
158
The zero-order model, the first-order model and the Higuchi square root law model has
been used to describe the drug release kinetics from the polymer matrix systems [151]. The
zero-order, first-order and Higuchi models were applied to the release data. The R2 value
of both zero-order and first-order model indicated less fit. Whereas, the drug release kinetic
data fitted the Higuchi model indicating the release of lidocaine from the experimental
patch was controlled by the diffusion process (Figurer A.8). This study is in agreement
with the study conducted by Doungdaw et al. where the piroxicam release kinetic data from
the EUDRAGIT® RL100 and EUDRAGIT® RS100 fitted with the Higuchi model [145].
In another study, Mohamed et al. also demonstrated that the release of tizanidine
hydrochloride from a EUDRAGIT® RL100 and EUDRAGIT® RS100 bioadhesive buccal
patch occurred by diffusion through the film matrix [152]. Therefore, it can be stated that
the drug molecules are released from the polymer metrices by the following mechanisms:
(a) the presence of higher amounts of quaternary ammonium groups in the EUDRAGIT®
RLPO influenced the water to enter more easily into the polymer matrix and provided
hydration, (b) diffusion of drug molecules through the hydrated polymer layer. It should
be also noted that the significance of these mechanisms will depend on the drug
characteristics and the polymer combinations.
159
Figure A.8. Higuchi release kinetics, in phosphate buffer at pH 7.4 after 25 h (N=3).
A.3.7. Scanning electron microscopy
Drug crystallization is a critical issue in transdermal patch formulations as it is an
indication of formulation instability and can also impact the drug delivery [153]. The
prepared patches were evaluated for their surface morphology and for the indication of
drug crystallization. At the end of 30 days the prepared patches were examined using SEM.
Patches containing higher concentrations of lidocaine (10%) did not show any signs of
crystallization (Figure A.9B) after 30 days of storage at ambient laboratory temperature
suggesting that 10% (w/w) lidocaine was under saturation level as crystallization is likely
to initiate in supersaturated systems with the formation of a nucleation of drug molecules
that is difficult to re-dissolve [154].
0
10
20
30
40
50
60
70
80
2 2.4 2.8 3.1
Cum
ula
tive %
dru
g r
ele
ased
Time½ (h1/2)
Ex Patch 4% Ex Patch 5% Ex Patch 10% Ex Patch 20%
R² = 0.9974
R²= 0.9913
R² = 0.9915
R²= 0.9968
160
[A]
[B]
161
[C]
Figure A.9. SEM-EDS photographs of (A) lidocaine 4% transdermal patch (B) lidocaine
10% transdermal patch after 30 days and (C) marketed 5% lidocaine patch.
A.3.8. Differential scanning calorimetry
Differential scanning calorimetry (DSC) studies of the pure drug produced an
endotherm representing its melting point at 69ºC indicating that lidocaine is the crystalline
drug (Figure A.10A). In the physical mixtures- EUDRAGIT® RLPO, HPMC and chitosan
produced a single melting peak at 60ºC. The melting peak was shifted to 46ºC with the
addition of lidocaine in the 1:1 physical mixture of polymers (Fig. A.10C). No peak
representing lidocaine was observed in the experimental lidocaine 10% and 20% patch
stored for 30 days. This may be explained by the fact that lidocaine is still being solubilized
162
[A]
[B]
163
[C]
[D]
164
[E]
Figure A.10. Differential scanning calorimetry profiles of different components in
transdermal patches: (A) Pure lidocaine; (B) 1:1:1 physical mixture of EUDRAGIT®
RLPO, HPMC and chitosan; (C) 1:1:1:1:1 physical mixture of EUDRAGIT® RLPO,
HPMC, chitosan, TEC and lidocaine; (D) Ex lidocaine patch 10% and [E] Ex lidocaine
patch 20%.
in the patch and even after 30 days of storage lidocaine didn’t change its physical state to
appear as crystalline form.
A.3.9. WVP evaluation
The WVP of marketed patch was 2.21 ± 0.19 g/100 sq inches/24h, whereas the
WVP measured for experimental 4% patch was 0.98 ± 0.04 g/100 sq inches/24h. The
differences in backing liner and polymer composition might have played a role in WVP.
165
The marketed and experimental patch has different backing liner, additionally, the
marketed patch has hydrophilic polymer whereas the experimental patch has hydrophobic
polymer that could have influenced the WVP of both patches.
A.3.10. Evaluation of shower effect on the patches
As transdermal patches are intended to be used for longer periods of time,
evaluating their ability to withstand water force equivalent to usual shower time is logical.
The marketed lidocaine patch was able to stick on the stainless-steel plate for 10 minutes
under 40 psi water pressure but showed complete detachment from the plate at 15 minutes
(Figure A.11). On the other hand, experimental patch was able to withstand the continuous
flow of 40 psi water pressure for 60 minutes (Figure A.11). This is may be due to the reason
that lidocaine marketed patch is composed of hydrophilic polymer like polyvinyl alcohol,
carboxymethylcellulose that tends to swell in water. This water uptake and swelling
tendency reduced the bonding between the patch and stainless-steel plate. Whereas, the
experimental patch has a combination of hydrophilic and hydrophobic polymer that might
have reduced the water uptake of the polymer during the shower test. Which further played
a role in maintaining the bond between the patch and stainless-steel plate.
166
Figure A.11. Effect of shower or 40 psi water pressure on marketed (A-C) and
experimental patches (D-F).
A.4. Conclusions
Lidocaine-loaded transdermal adhesive patch was successfully developed using
EUDRAGIT® RLPO, 3M CoTran™ 9695 nonwoven layer and Fluorosilicone Coated
167
Polyester film (3M Scotchpak™ 9709) as a backing liner. DSC and SEM studies
demonstrated that lidocaine even in higher concentration (20%) is in amorphous or
dissolved state in the patch formulation with EUDRAGIT® RLPO. All experimental
patches showed sustained release of lidocaine over time and the release kinetics followed
the Higuchi model. All the patch formulations showed higher adhesive strength and tensile
strength compared to the marketed patch that might be useful to hold up to rigorous friction
from day to day patch use. Additionally, the experimental patch was able to withstand 40
psi shower pressure till 60 minutes and did not show any sign of detachment from stainless
steel plate. The development of these patches with EUDRAGIT® RLPO would be relevant
as a potential dosage form for potent drug with crystalline tendency to deliver through
transdermal route. Further, investigation should be conducted to confirm the results.
168
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177
Thesis summary and future perspectives
The scope of this thesis was the development of a novel topically applied polymeric
film system to deliver the bioactive compound thymoquinone (TQ) to the targeted dermal
site for the treatment and management of wound infections. Firstly, this research aimed to
identify different physicochemical and solubility parameters such as, ingredient active gap
(IAG), ingredient skin gap (ISG), solubility of active in the formulation (SolV) and the
formulation solubility in the skin (SolS) to understand the physicochemical interaction of
the active and the penetration enhancer. Based on the study results, we proposed for the
first time a Solubility-Physicochemical-Thermodynamic (SPT) theory to define the action
of penetration enhancers in a given formulation with a specific drug. These studies suggest
that - there is an inverse relationship between measured flux and IAG values given that
there is an optimum ISG, SolV and SolS ratio; The larger the difference in solubility
between the formulation and the skin the greater the driving force for partitioning of the
active into the stratum corneum; the flux is actually proportional to a gradient of
thermodynamic activity rather than the concentration and maximum skin penetration and
deposition can be achieved when the drug is at its highest thermodynamic activity.
Secondly, we have applied the knowledge of SPT theory to evaluate the interaction of our
experimental drug TQ with the skin and various penetration enhancers. The study
concluded that transdermal permeation and adequate skin deposition of TQ can be obtained
by using penetration enhancers and different vehicles. Azone, oleic acid and Transcutol®
P (Tc) at a concentration of 5% was able to provide measurable TQ flux. Additionally,
these penetration enhancers were also able to generate TQ reservoirs in the skin that may
be useful to exert sustained release of TQ from the stratum corneum over longer period of
178
time. This fact provided information to deduce that, Azone, oleic acid or Tc can be the
penetration enhancer of choice to further develop a novel transdermal formulation of TQ.
Thirdly, we have used TQ to synthesize and characterize a biocompatible novel topical
polymeric film and hydrogel system. The developed system showed to be very useful and
efficient in controlling Staphylococcus aureus infection and promoting wound closure. The
presence of TQ-containing films and hydrogels completely wiped out Staphylococcus
aureus in a 10 cm in diameter TSA (Tryptic Soy Agar) plates while 500 µg/mL gentamicin
containing filters gave 10 mm of ZOI. In an ex vivo model, the presence of TQ-film
eradicated the bacterial colonization on human cadaver skin. Furthermore, in the BALB/c
mice wound model, TQ-films showed significant activity in controlling Staphylococcus
aureus wound infection without compromising the healing process.
This research indicates that, TQ has the potential to become an active of interest for
use in both the pharmaceutical prescription/OTC area but also for personal care and
cosmetic uses. It’s antioxidant, anti-inflammatory and anti-neoplastic properties can be
applied together to treat various diseases like breast cancer, cervical cancer etc. and
neurodegenerative diseases e.g., Alzheimer’s and Parkinson’s disease. Since TQ has not
been commercially used before as an antibacterial agent, it has the potential to offer a new
treatment strategy specially for those who has developed resistance towards the commonly
used antibacterial agents. In future work, investigating different concentrations of TQ to
find the minimum inhibitory concentration that might exert efficacy against both gram-
positive and gram-negative microorganisms might expand the scope of this research. It will
be important that future research investigate the combination therapy of TQ with other
known antibacterial agents like, gentamicin, fusidic acid, metronidazole etc. to evaluate the
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potential benefit of combination therapy in different disease conditions such as, rosacea,
psoriasis, skin and soft tissue infection etc.. Further studies could investigate the different
strategies to improve TQ’s photostability and different delivery systems (antimicrobial
topical spray, microbial sealant, wound dressing etc.) for successful delivery of TQ. The
information obtained in this study for the thesis leaves further questions to be answered
such as how TQ promotes wound healing; which immune cells are recruited in the process
of the antibacterial effect of TQ in wound infections; is there any effect of TQ in promoting
angiogenesis during wound healing? Therefore, further studies are needed to provide TQ
related topical drug delivery system in the treatment and management of wound infections.