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

Polysaccharide Based Nanoparticles for Drug Delivery Application

Why Nanoparticles ? Dispersion or solid form with size in the range 10-700 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

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

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

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

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

Materials - Polycations Chitosan Low Molecular Weight (CS-LMW) : 20.000 cps Chitosan Medium Molecular Weight (CS-MMW) : 200.000 cps Chitosan (CS) D.D. 75-85% 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

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

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, 6888-6901 Polycations Polyanions CS-LMW CS-MMW CS-g-PLA PC-PA couple : AA Pec-LMW Pec-HMW CMC +

Methods - Nanoparticles Characterization Nanoparticles in solution Dynamic Light Scattering Dynamic Viscosity Particles diameter z-potential Stability Shape prediction and evolution Dried at + 60 ͦC Freeze-Dried at -40 ͦC under vacuum Freeze -198 ͦC and dried at -40 ͦC under vacuum Nanoparticles in solid form Surface analysis Porosity Swelling FTIR-ATR DSC

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

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

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

Results – Nanoparticles in solution - (Dynamic Viscosity) Temperature : 18 ͦC ± 0.5 ͦC Volume : 30 mL ± 1 mL Measuring time : 60-120 sec

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

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 0.005148 25.4569 -40 oC + dried under vacuum 2.1058±0.0854 3.2586 0.011733 27.3504 N2 (liq.) + dried under vacuum 2.8959±0.1050 4.5639 0.027028 43.0528   CS-MMW-CMC 2 +60oC(not frozen) 2.1044±0.7268 2.9445 0.004700 7.9493 2.5902±0.3223 4.1425 0.010954 7.8145 2.6047±0.3083 4.2009 0.004956 8.1760 CS-g-PLA PecLMW 2 2.1126±0.0494 3.2525 0.005045 12.0111 2.7432±0.2189 4.3801 0.004338 6.6111 3.1135±0.0289 4.8883 0.007913 12.8515 Mismatch between particles diameter and surface area No substantial differences among the various freeze and dry treatments Low BET surface area values “ Glue effect ”

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

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

Conclusions CS-LMW – PecHMW ; CS-MMW – CMC and CS-g-PLA – PecLMW represents the optimal polyelectrolytes couples in term of particles dimension (110-140 nm) and PDI (0.121 - 0.276) 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

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

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