1. Polysaccharides Based Nanoparticles for Drug Delivery Application
Antonio Di Martino
Tomas Bata University in Zlin
Faculty of Technology
-Czech Republic-
Vilnius University
Faculty of Chemistry
-Lithuania-

2. Polysaccharide Based Nanoparticles for Drug Delivery Application

3. Why Nanoparticles ?
Dispersion or solid form with size in the range nm (for drug delivery)
Various morphologies – nanospheres, nanocapsules, nanomicelles, nanoliposomes etc…
Drug (s) can be : dissolved, entrapped, encapsulated or attached to a nanoparticle matrix
High Encapsulation Efficiency
Drug protection
Controlled Release
Decrease side effects
Reverse tumor multidrug resistance
Cell Internalization

4. Why Polysaccharides ?
Polysaccharides have been considered as one of the most promising material for drug delivery
Various Sources : Algae , Microbial, Plants and Animals
Abundant
Low Cost
Large number of Reactive Groups
Chemical composition
Wide range of Mw
Biocompatible
Biodegradable
Low Immunogenity
Not Toxic

5. Most used Polysaccharides in Drug Delivery
Hyaluronic Acid
Pectin
Chitosan
Alginate
Chondroitin Sulfate
Amylose
Pullulan
Cyclodextrin
Heparin
Dextran

6. Polysaccharide Based Nanoparticles preparation
Based on their structural properties Polysaccharides Nanoparticles are prepared by
Covalent crosslinking (CC)
Ionic crosslinking (IC)
Polyelectrolytes complexation (PEC)
Self-assembly (SA)
Glutaraldehyde
Di-tri carboxylic acid ( succinic acid, malic , tartaric and citric acid)
TryPolyPhosphate

7. Aim of the work
Prepare a set of polysaccharides based nanoparticles for encapsulation and controlled release of bioactive molecules (DNA Alkylating Agents, Peptides, Hormones)
Understand the optimal condition to obtain stable nanoparticles in solution with suitable properties for drug delivery application
Optimize the procedures for obtaining nanoparticles in powder

8. Materials - Polycations
Chitosan Low Molecular Weight (CS-LMW) : cps
Chitosan Medium Molecular Weight (CS-MMW) : cps
Chitosan (CS)
D.D %
Chitosan-grafted –PLA
(CS-g-PLA)
Coupling Reaction
Room Temperature
Reaction Time : 48 h
Good Efficiency
Low Molecular Weight PLA Polycondensation reaction using MSA as initiator

9. Materials - Polyanions
Pectin (Pec)
Alginate (AA)
Carboxy Methyl Cellulose (CMC)

10. Methods - Nanoparticles Preparation
Nanoparticles were prepared by Polyelectrolytes complexation method
Mild acidic aqueous media (pH 5.0)
PC / PA (w/w) ratio (range 0.1 to 5)
Room Temperature
Solvent Free
Chem. Soc. Rev., 2012, 41,
Polycations
Polyanions
CS-LMW
CS-MMW
CS-g-PLA
PC-PA couple : AA
Pec-LMW
Pec-HMW
CMC +

11. Methods - Nanoparticles Characterization
Nanoparticles in solution
Dynamic Light Scattering
Dynamic Viscosity
Particles diameter
z-potential
Stability
Shape prediction and evolution
Dried at ͦC
Freeze-Dried at -40 ͦC under vacuum
Freeze ͦC and dried at -40 ͦC under vacuum
Nanoparticles in solid form
Surface analysis
Porosity
Swelling
FTIR-ATR
DSC

12. Results – Nanoparticles in solution - (Particles Diameter and z-pot.)
sample
PC/PA ratio (w/w)
Particles diameter (nm)
PDI
-pot. (mV)
CS-LMW -AA
0.1
197
0.210
-32
0.5
164
0.221
-29 1
153
0.259 -4 2
166
0.355
+30 5
210
0.276
+41
CS-LMW –Pec LMW
188
0.301
-23
189
0.240
-13
175
0.219 -9
0.222
+14
181
0.268
+39
CS-LMW-Pec HMW
176
159
0.330
-24
137
152
0.264
+28
180
0.309
+44
CS-LMW -CMC
230
0.163
-28
221
0.285
-14
172
0.211 +3
196
0.159
+37
0.231
+43
sample
PC/PA ratio (w/w)
Particles diameter (nm)
PDI
-pot. (mV)
CS-LMW -AA
0.1
197
0.210
-32
0.5
164
0.221
-29 1
153
0.259 -4 2
166
0.355
+30 5
210
0.276
+41
CS-LMW –Pec LMW
188
0.301
-23
189
0.240
-13
175
0.219 -9
0.222
+14
181
0.268
+39
CS-LMW-Pec HMW
176
159
0.330
-24
137
152
0.264
+28
180
0.309
+44
CS-LMW -CMC
230
0.163
-28
221
0.285
-14
172
0.211 +3
196
0.159
+37
0.231
+43
sample
PC/PA ratio (w/w)
Particles diameter (nm)
PDI
-pot. (mV)
CS-g-PLA -AA
0.1
180
0.285
-48
0.5
157
0.279
-36 1
132
0.365
-14 2
119
0.250 11 5
181
0.154 28
CS-g-PLA-Pec LMW
221
0.349
-32
195
0.118
-21
182
0.245
-11
111
0.121 8
141
0.223 26
CS-g-PLA- Pec HMW
285
0.378
-39
259
0.231
-26
240
0.163 -5
261
0.275 12
294
0.342 30
CS-g-PLA-CMC
301
0.131
-34
287
0.112
-27
222
0.099 -9
201
0.146 7
234
0.201 32

13. Results – Nanoparticles in solution - (Shape prediction at pH 5.0)
Rh (nm)
Specific Volume (mL/g)
Mw (g/mol)
Input parameters
Relevant factor in Cell – Polymer interaction

14. Results – Nanoparticles in solution - (Estimation Number )
Rh (nm)
Concentration (mg/mL)
Volume (mL)
Mw (g/mol)
Input parameters
Important for in-Vitro and in-Vivo Tests

15. Results – Nanoparticles in solution - (Dynamic Viscosity)
Temperature : 18 ͦC ± 0.5 ͦC
Volume : 30 mL ± 1 mL
Measuring time : sec

16. Results –Nanoparticles in solid form – (Swelling Index)
0.2 g of dried nanoparticles were allow to swell in water acidified with acetic acid (pH 5.0 , V = 20 mL)
CMC
CMC
CMC

17. Results – Nanoparticles in solid form – (Surface Area and Porosity)
Sample name
Freezing and drying procedure
BET surface area (m2/g)
t-plot external surface area (m2/g)
BJH adsorption cumulative volume of pores (cm3/g)
BJH adsorption average pore width (4V/A) (nm)
CS-LMW-PecHMW 1
+60o (not frozen)
1.3748±0.0618
2.0080
-40 oC + dried under vacuum
2.1058±0.0854
3.2586
N2 (liq.) + dried under vacuum
2.8959±0.1050
4.5639
CS-MMW-CMC 2
+60oC(not frozen)
2.1044±0.7268
2.9445
7.9493
2.5902±0.3223
4.1425
7.8145
2.6047±0.3083
4.2009
8.1760
CS-g-PLA PecLMW 2
2.1126±0.0494
3.2525
2.7432±0.2189
4.3801
6.6111
3.1135±0.0289
4.8883
Mismatch between particles diameter and surface area
No substantial differences among the various freeze and dry treatments
Low BET surface area values
“ Glue effect ”

18. Results : Nanoparticles in solid form – (FTIR-ATR)
Ge crystal
64 scan
Resolution 4 cm-1
Only electrostatic interactions occur
Typical polysaccharides peaks
Main peaks related to NH2, COOH and CO-NH2 appear
CS-g-PLA more hydrogen bonds occur

19. Results : Nanoparticles in solid form – (DSC)
Temperature Range : 10 – 200 ͦC
Heating Trend : 10 ͦC / min
Sample
ΔCp (J/g ͦC)
Onset ( ͦC)
End ( ͦC)
Half Width ( ͦC)
CMC
3.286
97.31
102.70
100.01
Pec HMW
2.389
88.33
101.78
95.81
Pec LMW
5.999
52.62
87.79
70.20
CS-MMW
4.078
66.06
93.85
75.94
CS-LMW – Pec HMW
3.611
63.95
90.91
77.43
CS-MMW – CMC
3.770
63.67
86.41
75.04
CS-g-PLA – Pec LMW
3.719
73.66
85.67
79.56
Extend the Temperature Range from 10 to 400 ͦC

20. Conclusions
CS-LMW – PecHMW ; CS-MMW – CMC and CS-g-PLA – PecLMW represents the optimal polyelectrolytes couples in term of particles dimension ( nm)
and PDI ( )
Nanoparticles shape is Rh dependents. When Rh increases a “disk-like” form seems
to be favorable
A relationship between SI and Dynamic Viscosity of nanoparticles solution
with the PC / PA ratio (w/w) was found
Surface Analysis reveals that Freeze-Drying treatments cause a “Glue- Like” effect
independently from PC-PA couple and ratio
4,3 cm

21. What is next ? Encapsulation of DNA Alkylating Agents
Release Studies in Physiological Environment
(Human Serum, Simulated Gastric Fluid, Simulated Intestinal Fluid)
Cell Uptake and Interaction Studies
Nanoparticles Shape Evolution in Physiological Compartments

22. Thank you for your attention
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