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