Polymeric nanoparticles for encapsulation and controlled

release of bioactive compounds

Antonio Di Martino

Supervisor: doc. Ing. Vladimír Sedlařík, Ph.D.

Zlin 26.08.2016

Motivations

Targeting Reduction of side effect Higher drug’s efficacy (Emax)

-Nanoparticles have emerged as important players in modern medicine with clinical application ranging from contrast agents to carriers for drug and gene delivery

Nanoparticles

Motivations

Chitosan

Cationic High charge density at pH < 6.5 Biocompatible Biodegradable Hemostatic Bacteriostatic Fungistatic

Polylactic acid

• Linear PLA (LPLA)

• Carboxy enriched PLA (CPLA)

Drug 2

Drug 3

Amphiphilic carrier

Drug 1

Outline

Section 1 : Polysaccharide-based nanocomplexes for co-encapsulation and controlled release of 5-fluorouracil and temozolomide

Section 2 : Chitosan grafted low molecular weight polylactic acid for protein encapsulation and burst effect reduction

Section 3 : Amphiphilic chitosan-grafted-functionalized polylactic acid based nanoparticles as a delivery system for doxorubicin and temozolomide co-therapy

Section 1 : MotivationsDevelopment of polysaccharides based nanocomplexes for biomedical application;

Multiple encapsulation of anticancer drugs;

Controlled and sustained release of payload molecules;

Protection of the loaded drugs from external environments;

Enhancement of drug’s efficacy (Emax) and reduction of side effects.

Section 1 : Methods Nanocomplexes preparation : Polyelectrolyte complexation method (PEC)

Polycation : Chitosan (CS) : LMW; 75-85% DD

Polyanion : Alginic acid sodium salt (ALG); Mw 12,000–25,000 g/mol Polygalacturonic acid sodium salt (PGA) from oranges; Mw 25,000–50,000 g/mol; > 85% titration

Bioactive compounds:

5-Fluorouracil (5-FU) Temozolomide (TMZ)

Preparation media : Water solution containing 1% of CH3COOH ( pH 5.5)

Section 1 : Methods Nanocomplexes

• Dynamic light scattering (DLS)• z-potential• TEM • SEM Morphology and dimension in solution and dried form

Encapsulation efficiencyand drug stability

Release studies UV-VIS

• UV-VIS (325nm TMZ; 275 nm 5-FU) • LC-MS

• Simulated Gastric Fluid (SGF)• Preparation Media (PM)• Phosphate Buffer (PBS)• Physiological Solution (PS)• Human Serum (HS)

All media respect the European Pharmacopoeia standards

Variables

• Polycation / Polyanion weight ratio (w/w : 0.5 to 5) • pH of the release media• Ionic strength of the media• Single and multiple loading

Section 1 : Results Polycation/polyanion couple Weight ratio (w/w) Concentration Presence of drug(s)

Average diameter and z-potential are influenced by :

200 nm

CS-ALG

Ave

rage

dim

ensi

on (n

m)

unloa

d5-F

UTMZ

5-FU+TMZ

0

50

100

150

200

2500.115

w/w CS-PGA

Ave

rage

dim

ensi

on (n

m)

unloa

d5-F

UTMZ

5-FU+T

MZ0

50

100

150

200

2500.115

w/w

z-pot. shifts from negative to positive

Section 1 : Results Direct correlation between

Polycation/polyanion couple and EE

EE and CS/ALG or CS/PGA weight ratio

Difficult to relate EE to drug structure

In multiple encapsulation the drugs are well balanced

CS-ALG is more suitable for multiple encapsulation

Single Loading

Multiple Loading

Section 1 : ResultsAmount of drug released after 6h of media contact

TMZ

5-FU

TMZ5-FU Polysaccharides couple influence

initial release intensity

Modulation of initial burst intensity by changing environment condition

No correlation between drug structure and burst intensity

Different trend in case of multiple loading

5FU+TMZ5FU+TMZ

Cum

ulat

ive

rele

ase

(%)

0

20

40

60

80

100 5FU+ TMZ5FUTMZ

Cum

ulat

ive

rele

ase

(%)

0

20

40

60

80

100 TMZ5FU

Cum

ulat

ive

rele

ase

(%)

0

20

40

60

80

100 TMZ5FU

Cum

ulat

ive

rele

ase

(%)

0

20

40

60

80

100 5FU+ TMZ5FUTMZ

CS-ALG CS-PGA

Section 1 : Results – Key point

N

N

N

NN

ONH2

CH3O

N

NH

O NH2

NN NH

CH3N

NH

NH2

O NH2

N+

NCH3

+TMZ MTIC

AIC

Diazomethane cation

H2O

-CO2

TMZ quickly hydrolyse in physiological condition

Improve stability of TMZ is a challenge

TMZ free in PSTMZ loaded in CS-ALG

t1/2 : 35 – 180 min

t1/2 : few minX100 120 140 160 180 200 220 240

0

1

2

3

4

5

6

7

8

9

10

m/z

Cou

nts

x105

TMZPS 6h

100 120 140 160 180 200 220 2400

1

2

3

4

5

6

7

8

9

10

m/z

Cou

nts

x105

TMZPM 6h

TMZ

TMZMTIC

AIC

3h

6h

Section 1 : ConclusionsDimension and ζ-potential in the range of 100–200 nm and − 30 to + 35 mV,

respectively;EE between 20 and 80%; Sustained release (up to one week) and pH controlled release;Modulation of the initial burst intensity;NO interferences between TMZ and 5-FU during loading and release process;NO structural alteration of the drugs;Protection from external environment

Section 2 : MotivationsPreparation of amphiphilic nanocarriers;

Encapsulation of environmental sensible macromolecules;

pH controlled release;

Initial burst reduction.

Section 2 : Methods• FTIR-ATR• 1H-NMR• Conductometric titration

Nanoparticles

PEC using dextran sulphate as polyanionDynamic light scattering (DLS)z-potentialTEM SEM Morphology and dimension in dried form and solution

Release studies • Simulated Gastric Fluid (SGF)• Physiological Solution (PS) UV-Vis (280 nm)

Variables

• Polymer concentration• pH of the release media;• Ionic strength of the media;• PLA side chain Bovin Serum Albumin (BSA)

Section 2 : Results

pH

Sw

ellin

g (%

)

0 2 4 6 8 10 120

100

200

300

400

500

600

700

CS-g-PLACS

Sw

ellin

g (%

)

SGFSIF PS

PBS0

100

200

300

400

500

600

700

800CS

CS-g-PLA

Diffusion-controlled release pH and ionic strength swelling dependence PLA has light influence on swelling

Swelling behaviour

BSA Encapsulation efficiency and Loading capacityp < 0.05 p < 0.05

50% 1-2 mg/mL optimal condition PLA side chain doesn’t reduce EE

Section 2 : Results – Key pointOverall Release

C50; t1/2 C50; t1/2

C50; t1/2

C50; t1/2

pH controls the release rate

PLA side chain prolongs the release in acidic media

PLA reduces release intensity at the initial time

PLA effect is more evident in acidic condition

t t

I I

t = time; I = intensity

SGF PS

Section 2 : Conclusions

High stable amphiphilic nanoparticles based on CS and PLA were obtained;

High BSA loading and pH dependant release kinetic;

Sustained BSA release in different environment;

PLA side chain reduces the release intensity at the initial time.

Section 3 : MotivationsDevelopment of not toxic and biocompatible set of amphiphilic polymers based

on chitosan (CS) grafted by different PLA structures (PLLA; PLACA);

Multiple loading of anticancer drugs;

Controlled and delayed release of the loaded molecules;

Reduction of the initial burst.

Section 3 : MethodsPLLA

PLACA (2%-5%)

CSLMW 75-85% DD

• CS-g-PLLA• CS-g-PLACA (2-5%)

Polymer characterization: FTIR-ATR; 1HNMRNanoparticles preparation : PECPolyanion : CS; CS-g-PLLA; CS-g-PLACA(2%-5%)Polyanion : Dextran sulphate; Mw 50kDaBioactive compounds : Doxorubicin (DOX) ; Temozolomide (TMZ)

DOX TMZ

PLLA and PLACA were kindly provided by Ing. Pavel Kucharczyk, Ph.D

Section 3 : Methods IINanoparticles

• Dynamic light scattering (DLS)• z-potential• TEM • SEM Morphology and dimension in dried form and solution

Encapsulation efficiency

Release studies UV-VIS

• UV-VIS (325nm TMZ; 480 DOX)

• Simulated Gastric Fluid (SGF)• Preparation Media (PM)• Phosphate Buffer (PBS)• Physiological Solution (PS)• Human Serum (HS)

All media respect the European Pharmacopoeia standards

Variables

• Polycation / Polyanion weight ratio (w/w : 0.5 to 5) • pH of the release media• Ionic strength of the media• Single and multiple loading• PLACA side chain

Section 3 : Results Polycation to polyanion w/w from 0.05 to 5

average diameter rise from 10 to 40 % ( max : 300 nm CS-g-PLACA5%) z-potential shifts from negative to positive (25-35 mv) aggregation phenomena

Polycation to polyanion w/w = 2

Determination of the best w/w between the polymers is highly important

Ave

rage

dim

ensi

on (n

m)

unloa

dDOX

TMZ

DOX +TMZ

0

100

200

300

400CS-g-PLA

CS-g-PLACA2%

CS-g-PLACA5%CS

z-po

t. (m

V)

unloa

dDOX

TMZ

DOX +TMZ

0

10

20

30

40

50CS-g-PLACA

CS-g-PLACA2%

CS-g-PLACA5%CS

Section 3 : Results

50%

Single Loading

• Polyions w/w = 2• 50% = 500mg• 50% = 166 mg drug/ mg carrier

Double Loading (DOX + TMZ)

• Reduction of EE• Max : 60mg DOX and 60 mg TMZ per mg carrier• Drugs are well balanced ( close to 1:1)

pH

Enc

apsu

latio

n ef

ficie

ncy

(%)

3.5 5.5 7.4 90

20

40

60

80

100CS

CS-g-PLA

CS-g-PLACA2%

CS-g-PLACA5%

pH

Enc

apsu

latio

n ef

ficie

ncy

(%)

3.5 5.5 7.4 90

20

40

60

80

100CS

CS-g-PLA

CS-g-PLACA2%

CS-g-PLACA5%

pH

Enc

apsu

latio

n ef

ficie

ncy

(%)

3.5 5.5 7.4 90

10

20

30

40

50 CS

CS-g-PLA

CS-g-PLACA2%

CS-g-PLACA5%

Section 3 : Resultsk (h-1)

0.15DOXTMZ 0.59

0.110.54

CS CS-g-PLA0.0350.037

CS-g-PLACA2% CS-g-PLACA5%

0.055 0.041

CS CS-g-PLA CS-g-PLACA2% CS-g-PLACA5%

t50 (h)

4 5 24 202 2 12 72

PLA side chain reduces k (h-1) and prolongs t50 (h)

Percentage of COOH groups clearly influence k (h-1) and t50 (h)

Difficult to correlate drug structure - release kinetic

DOX TMZ

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40

50

60

DOX

TMZ

CS

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40

50

60DOX

TMZ

CS-g-PLA

40%

30%

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40DOX

TMZ

CS-g-PLACA2%

lag time

20%

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 1 2 3 4 5 60

10

20

30

40DOX

TMZ

CS-g-PLACA5%

lag time10%

Sustained-release Delayed-release

Section 3 : Results – Key point

Section 3 : Conclusions

Nanoparticles dimension, temperature response, EE and release rate are influenced

by the amount of –COOH groups along the side chain;

Diameter falls in the range 150-300 nm and ζ-potential between 12–34 mV;

Encapsulation and co-encapsulation efficiency > 50 μg/mg polymer;

PLACA side chain causes a delay in the release;

DOX and TMZ are well balanced inside the system.

Summary Remarks Polymeric nanoparticles as drug delivery systems represents a promising strategy in biomedical field as

increase the bioavailability, solubility, stability of different class of drugs;

A set of amphiphilic polymers based on chitosan grafted with different polylactic acid have been prepared and characterized;

Nanoparticles displayed suitable characteristics in terms of dimension, z-potential, shape and stability for drug delivery application;

Obtained nanoparticles were able to encapsulate, simultaneously, and release, following different kinetics, bioactive compounds widely used in cancer therapy;

Protection and preservation of the chemical structure and activity of the loaded drugs were demonstrated.

List of Activities and Outputs

Training abroad :

09.2014 – 11.2014 - Vilnius University, Faculty of Chemistry, Department of Polymer Science.Vilnius, Lithuania – Preparation of polysaccharides based nanocomplexes for drug delivery application

01.2015 – 4.2015 - National Research Tomsk Polytechnic University, Department of High Physics Technology. Tomsk, Russian Federation – Surface modification of Iron based NPs for MRI application

03.2016-05.2016 - National Research Tomsk Polytechnic University, Department of Technology of Organic Substances and Polymer Materials – Russian Federation - Preparation of Iron-Gd MRI contrast agent

List of publications :I. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2014, 460: 184-190.

II. International journal of pharmaceutics, 2014, 474.1: 134-145

III. Biotechnology letters, 2015, 37.3: 557-565.

IV. Synthetic Metals, 2015, 202: 16-24.

V. International journal of pharmaceutics, 2015.496,912-921

VI. European Journal of Pharmaceutical Sciences, 2016 – In Press Accepted Manuscript

 

List of Activities and Outputs

I. Nanocon 2013, Brno, Czech Republic.

II. Plastko 2014, Zlin, Czech Republic.

III. Siberian Youth Conference "Current Topics in Organic Chemistry” 2015 Sheregesh, Russian Federation.

IV. Nanocon 2015, Brno, Czech Republic.

V. 4th EPNOE International Polysaccharide Conference 2015, Warsaw, Poland.

VI. 2nd International Young Scientist School ‘Nanostructured naterials 2016, Tomsk, Russian Federation.

VII. 7th International Conference ‘Nanoparticles, Nanostructured coatings and microcontainers: technology, properties, applications ’.2016,

Tomsk, Russian Federation.

VIII. 17th international scientific conference “chemistry and chemical engineering in XXI century”. 2016. Tomsk, Russian Federation.

 

 

 

 

Conference proceedings

List of Activities and OutputsProjects:

I. CZ.1.05/2.1.00/03.0111: European Fund of the Research and Development for Innovations programme, namely the Priority Axis 2 - Regional R&D centres, Ministry of Education, Youth and Sports of the Czech Republic (MEYS) 2013-2015

II. GJ15-08287Y : Czech Science Foundation 2015-2017

III. QJ1310254 : Ministry of Agriculture of the Czech Republic (MZe). 2013-2017

IV. LE12002 : Ministry of Education, Youth and Sports of the Czech Republic (MEYS) 2012-2015

V. IGA/FT/2014/012 : Internal Grant Agency of Tomas Bata University in Zlín 2014

VI. IGA/CPS/2015/003 : Internal Grant Agency of Tomas Bata University in Zlín 2015

VII. IGA/CPS/2016/003 : Internal Grant Agency of Tomas Bata University in Zlín 2016

Reviewers questions

Antonio Di Martino

Supervisor: doc. Ing. Vladimír Sedlařík, Ph.D.

Zlin 26.08.2016

Reviewer : Assoc. prof. Ing. Adriana Kovalcik, Ph.D

1) How the ranges of molecular weight of PLA influences its suitability for using PLA as polymer drug carrier in the form of nanoparticles? Is it for preparation better to use PLA with a higher or a lower molecular weight and why?

2) It is known that PLA is a semi-crystalline polymer. The values of the crystallinity influence its hydrolysis-degradation kinetic development. The submitted work does not show the values of crystallinity of PLA. However, it would be interesting to discuss about an effect of the crystallinity value of PLA matrix on drug release kinetics.

Answer to question 1

Size Morphology Surface chemistry

Application of polymeric nanoparticles SpheresRectangular

disks Rods Worms

Oblate ellipses Elliptical disks UFOs Circular disks PNASJuly 17, 2007 vol. 104 no. 2911901-11904

Molecular weight (Mw)

High molecular weight = larger particles

PARTICLES SIZE

Loading capacity Release kinetic Interactions with biomolecules Cell uptake Half life / clearance

PLA Mw influences CS solubility Assembly mechanism Interaction with polyanions Stability Nanoparticles dimension Loading efficiency of hydrophilic drugs

Answer to question 2

Kucharczyk, et al. Polymer degradation and stability, 2013, 98.1: 150-157

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 25 50 75 100 125 1500

20

40

60

80

100

DOX

TMZ

Release trend from CS-g-PLA in PBS at 37° C

Cmax : 78-82 %t50 : 12h

This is only indicative because degradation of PLA as side chain of CS should be investigated differently The amount of PLA linked to CS in the prepared materials is up to 15% (D.D.)

Mn and mass changes (%) of PLA in PBS at 37 °C.

Di Martino & Sedlarik. Int. Journal of Pharmaceutics,  2014, 474, 1–2, 20,134-1453 weeks

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 25 50 75 100 125 1500

20

40

60

80

100

PLLA

PDLLA 10:90

PDLLA 25:75

PDLLA 40:60

Simulated Intestinal FluidSimulated Gastric Fluid

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 25 50 75 100 125 1500

20

40

60

80

100

PLLA

PDLLA 10:90

PDLLA 25:75

PDLLA 40:60

Time (h)

Cum

ulat

ive

rele

ase

(%)

0 25 50 75 100 125 1500

20

40

60

80

100

PLLA

PDLLA 10:90

PDLLA 25:75

PDLLA 40:60

Simulated Blood

Answer to question 2 PLLA and PDLLA NPs : conc. 5mg/ml DOX release

Cmax t50 (h) k(h-1) R2

PLLA 80-85 12-25 0.01-0.05 >0.99

PDLLA 10:90 85-92 8-12 0.03-0.08 >0.99

PDLLA 25:75 87-90 6-10 0.06-0.07 >0.99

PDLLA 40:60 87-91 5-10 0.09-0.14 >0.99

Reviewer : prof. Dr. Mohamed Bakar

1) It is stipulated that swelling is affected by the pH which attained maximum values at pH 5.5. Explain why tests were carried out at different pH

2) Different mathematical models were presented and discussed to evaluate and predict the release kinetic of drugs. For which reasons, none of them was used with your obtained data?

3) Figure 19 (page 83). Why encapsulation efficiency of 5FU and TMZ is higher in media than without media? Could you give further explanations. Results of Fig.19c and Fig.19 do not show any clear trend.

4) Which system show the best behaviour and for which specific applications of drug release do you suggest it?

Answer to question 1

The pH of individual cellular organelles and compartments in a prototypical mammalian cell

Nature Reviews Molecular Cell Biology 11, 50-61, January 2010

Answer to question 2Release kinetics data have been processed using three models :

Zero order

formulation that do not disaggregate and release the drug slowly concentration independent

𝑄𝑡=𝑄0 −𝐾 0𝑡

= amount of drug dissolved (mg/mL) at time t = initial amount of drug in solution (mg/mL; =0) = kinetic constant (conc./ time)t = time (h; days)

Higuchi model

drug release as diffusion process based on Fick s law release of water soluble and poorly soluble drugs from various matrices

𝐶𝑡=√𝐷×(2𝐶−𝐶 𝑠)×𝐶𝑠×𝑡

= drug released at time t ( mg/ cm2 )D = diffusivity (cm2/h)C = drug concentration at initial time (mg/cm3)= drug solubility in the matrix (mg/cm3)

First Order

LogC = - Kt) / 2.303

C0 = initial concentration (mg/mL)C = concentration at time t (mg/mL)K = Kinetic constant (con/time)T – time (h; days)

release of water soluble drugs from porous materials difficult to understand the release mechanism

Cumulative release(%) VS timeLog of drug remaining in the system in % VS time

Cumulative release(%) VS SQRT time

< 0.70

< 0.90 > 0.95

CS/PGA weight ratio (w/w)

Enca

psul

atio

n ef

ficie

ncy

(%)

0.1 1 50

20

40

60

80

1005FU

TMZ

A

CS/ALG weight ratio (w/w)

Enca

psul

atio

n ef

ficie

ncy

(%)

0

20

40

60

80

1005FU

TMZ

B

CS/PGA weight ratio (w/w)

Enca

psul

atio

n ef

ficie

ncy

(%)

0.1 1 50

20

40

60

805FU+ TMZ

5FU

TMZ

C

CS/ALG weight ratio (w/w)

Enca

psul

atio

n ef

ficie

ncy

(%)

0

20

40

60

80

1005FU+ TMZ

5FU

TMZ

D

Answer to question 3

Fig. 19 Relationship between encapsulation efficiency and polycation/polyanion weight ratio. A, B) single loading and C, D) multiple loading

TMZ 5-FU

- drug structure- drug-drug interactions- drug-carrier interactions- drug allocation

Answer to question 4

Not toxic Stable Multiple drug loading Drug protection Delayed and pH controlled release Reduction of initial burst effect

CS-g-PLACA NPs

TARGETED DRUG DELIVERY

are has

Systemic release Prolong concentration of drug in the bloodstream Reduction of administration frequency

CS-g-PLACA

Reviewer: RNDr.Jiri Zednik,Ph.D

1) Page 65. EDC is a zero-length crosslinking agent used to couple carboxyl or phosphate groups to primary amines. One of the main advantages of using EDC, instead of another carbodiimide, is its water solubility, which facilitates carrying out the reaction without the use of solvents. Moreover, reagents and by-products can be easily removed… The sentence is partly confusing. Could you clarify this point please?

2) The following statement is confusing ‘to construct an universal calibration’ Kuhn-Mark-Houwing-Sakurada constants are known for this type of polymers? It is written in the results chapter that hydrodynamic radius depends strongly on pH in the case of chitosan nanoparticles. And viscosimetric detector is not mentioned in the experimental part. Could you clarify this point?

Answer to question 1 Carbodiimides are agents used to activate carboxylic acids  towards amide or ester  formation. N-hydroxysuccinimide is added to increase yield

N,N'-Diisopropylcarbodiimide

N,N'-Dicyclohexylcarbodiimide

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Sulfo-NHS maintaines or improves the solubility of the intermediates NHS can decreases the solubility Unreacted intermediates are water soluble, easy to remove

Answer to question 2 Pullulan is widely used as standard for polysaccharides in GPC

Mark-Houwink parametersa = 0.665K = 0.000201 Mw range 108 - 708,000 g/mol PDI : 1.09-1.13

Hydrodynamic radius of CS nanoparticles is influenced by pH, ionic strength, complexing agent, concentration and temperature

Influence of pH :

-NH2 groups ( 75-85 %), pKa = 6-6.5

soluble insoluble

pH 6-6.5

• monochromatic light LED 850 nm• RI range 1.00 to 1.75 RIU• flow rate range 0.1 to 10.0 ml/min• inner cell temperature 30°C to 50°C

RI detector Waters 2414

Refractive Index detector was used for the presented work

Polygalacturonic acid

Alginic acid