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Chitosan grafted carboxy functionalized polylactide nanoparticles for multidrugs controlled and sustained release Antonio Di MartinoCenter of Polymer SystemsTomas Bata University in ZlinCzech Republic

dimartino@ft.utb.cz

National ResearchTomsk Polytechnic UniversityRussiaDrug Delivery Systems (DDS)Traditional drug delivery systems

Oral Injection basedInhalational/PulmonaryTransdermalWhy new delivery systems?

Targeted drug deliveryMaximum efficacy with minimum side sffectsControlled drug deliveryOptimize drugs therapeutic effects, convenience and doseEnhance product life-cycleImprove patient complianceControl over all healthcare cost

Systems for the delivery of drugs to target sites of pharmacological actionsNanoparticles as DDS

I) Dispersion or solid form with size in the range 10-700 nm

II) Various morphologies nanospheres, nanocapsules, nanomicelles, nanoliposomes etc

III) Drug (s) can be : dissolved, entrapped, encapsulated or attached to the nanoparticle matrix

IV) High Encapsulation Efficiency

V) Drug protection

VI) Controlled Release

VII) Decrease side effects

VIII) Reverse tumor multidrug resistance

IX) Cell Internalization

Why Nanoparticles ?Polysaccharides based NanoparticlesPolysaccharides 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

Chitosan

Hyaluronic Acid

Alginate

Cyclodextrin

Dextran

PectinChitosan (CS)

Physico-chemical properties

Molecular weightDeacetylation degreeViscositySolubility Antibacterial MucoadhesiveReactivityComplexation ability

Conventional formulationsCompression tabletsWet granulationGelsFilms EmulsionsWetting agentCoating agentMicrospheres Microcapsules

Novel applicationsBioadhesionDrug deliveryVaccine deliveryDNA deliveryAim of the workPreparation and characterization of amphiphilic nanoparticles based on chitosan grafted carboxy functionalized polylactide

Encapsulation of Doxorubicin , Temozolomide, 5-Fluorouracil

Influence of environment on the release trend

Doxorubicin (DOX)Temozolomide (TMZ)

5-Fluorouracil (5-FU)Polylactide-citric acid synthesis (PLACA)Carboxy functionalized PLA has been obtained through direct melt polycondensation of Lactic acid (LA) and Citric acid (CA)Methanesulfonic acid (MSA) as initiator. More effective compared to metal saltsMSA is adapt to obtain low Mw PLA

Lactic acidCitric acidCarboxy functionalized polylactide (PLACA)Kucharczyk et al. Journal of Applied polymer Science 2011, 122,1275-1285CS-g-PLACA synthesisCoupling reaction between CS amino groups and PLACA carboxy groupsEDC as COOH activatorR1-NH2+HOOC-R2 R1-NH-CO-R2+H2O

Improve chitosan stability in solutionEncapsulate hydrophobic and hydrophilic drugs simultaneouslyDi Martino, Sedlarik, Int. J.Pharm . 2014 Oct 20, 474, 1-2, 134-45CS-g-PLACA Nanoparticles preparationPolyelectrolytes complexation methodEasyFastLow costSolvent-freePossibility to modulate size and surface charge of nanoparticles Dextran sulfate ( Mw 50 kDa ) as polyanion

Polymer to DS ratio (w/w) : from 0.1 to 5 TMZ DOX 5-FU : Encapsulation and Release EE(%) = (DtDf / Dt)100 Dt = total theoretical amount of drug added (mg/mL) Df = concentration of free drug after encapsulation (mg/mL)Release kinetic mathematical models

Zero-order

First order

Higuchi

Hixson-Crowell

Kosmeyer-Peppas

UV-Vis spectroscopy

DOX 480 nm TMZ 328 nm 5-FU 525 nm

ResultsResults : CS-g-PLACA nanoparticles

Influence of polymer / DS ratio (w/w) on nanoparticles size and z-potentialpH 5.5DLS analysis

Dependence of nanoparticles dimension on polymer/DS ratio (w/w)Best compromise between size and z-potential is presented at w/w ratio 2Change in polymers chains arrangement

Size (nm)Results : SEM and TEM analysis

CS-g-PLACADextran Sulfate+-+++++++------Results : CS-g-PLA nanoparticles

CSCS-g-PLACA Stability studies : nanoparticles dimension VS timeInfluence of pH T : 37 CPolymer / DS (w/w) : 2PLACA side chain increases nanoparticles stabilityIIIIIIResults: Single LoadingCSCS-g-PLACARoom temperaturePolymer / DS (w/w) : 2pH 5.5 represents the optimal conditionDrugs are well balanced in both systemsCS-g-PLACA shows higher encapsulation efficiency than CS CSCS-g-PLACA5%Co-Encapsulation Efficiency (%)Co-Encapsulation Efficiency (%)Results: Multiple LoadingResults: CS-g-PLACA- Release kineticsDOX, TMZ and 5-FU loaded separatelyPhysiological solution (pH 7.4)T : 37 CNo initial burst effect is observed in all formulationsRelease begin after 5 hours

tlagCommon trendResults: CS-g-PLACA-Release kineticsDOX, TMZ and 5-FU loaded simultaneouslyPhysiological solution (pH 7.4)T : 37 C

DOX, TMZ and 5-FU are released concurrentlyNo initial burst effect is observed in all formulationsRelease begin after 4 hours

tlagRelease Kinetic mathematical analysisZero-order(R2)First-order(R2)Higuchi (R2)Hixson-Crowell (R2)Korsmeyer-Peppas (R2)TMZ0.730.830.970.970.90DOX0.880.850.980.980.915-FU0.790.810.980.910.92Physiological solution pH 7.4T : 37 CCS-g-PLACAHiguchi model shows the best fitDrugs are released by diffusionR2 values in Korsmeyer-Peppas models indicates that the Fickian diffusion represents the controlling factorResults related to Hixson-Crowell model demonstrate that a slight change in the nanoparticles surface area is induced by the mediaConclusionsCS-g-PLACA based nanoparticles show dimension in the range 100-200 nm

Nanoparticles show good stability as far as 10 days

Up to 70% of encapsulation efficiency of TMZ, 5-FU and DOX

No initial burst effect at physiological condition

Balanced release of drugs when loaded simultaneously

OutlooksCell uptakes studies

Evaluation of citotoxicity in different cancer cells lines

In vivo testsResults : PLACA

UV-VisFTIR-ATR

1H-NMR300 MHzConc : 1% (w/w) in DMSO-d690 pulse angleZn-Se crystalResolution : 2cm-1

Conc: 0.1 mg/mL in CHCl3Resolution : 0.5 nmPath 10mm

Results: CS-g-PLACA

FTIR-ATR1H-NMR