Centre of Polymer Systems Novel pH sensitive chitosan -grafted- star shaped polylactide nanoparticles for sustained and controlled release application Antonio Di Martino, Pavel Kucharczyk , Vladimir Sedlarik Centre of Polymer Systems Czech Republic

Drug Delivery Systems (DDS) DDS: approaches, formulations, technologies for the targeted delivery and/or controlled release of therapeutic agents Safe Perform therapeutic function Convenient administration Ease of manufacturing

Why Polysaccharides? Considered one of the most promising materials for drug delivery Various sources : Algae, Microbial, Plants and Animals Abundant Low cost Large number of reactive groups Chemical composition Wide range od Mw Biocompatible Biodegradable Low Immunogenicity Non-Toxic

Polysaccharides Based Nanoparticles Hyaluronic Acid Dextran Heparin Pectic Acid Cyclodextrin Chondroitin Sulfate Chitosan

Bioactive Molecules Antibiotics Steroids Antineoplastics Amoxicillin   Antibiotics Amoxicillin Sultamicillin Methicillin Steroids Progesterone Testosterone Estradiol Antineoplastics Vincristine Paclitaxel Doxorubicin

Large amount of drug released immediately upon placement in the media Burst Effect Large amount of drug released immediately upon placement in the media Advantages Wound treatment Targeted delivery (triggered burst release) Pulsatile release Disadvantages Local or systemic toxicity In vivo short t1/2 Waste of drug Short release profile Frequent administration Difficult to predict intensity Journal of Controlled Release 73 (2001) 121 –136

Burst Effect Causes How to reduce…. Process conditions Surface characteristics Morphology Carrier-Drug interactions Causes How to reduce…. Surface extraction Drug loading distribution Surface modification Polymer morphology and composition preparation steps cost

Aim of the work CS modification by grafting Star Shaped carboxy-terminated Poly Lactic Acid (SSPLA) Reduction of TMZ Burst Intensity

Chitosan (CS) Biocompatibility Biodegradability Non-toxic Not immunogenic Chemical modification Soluble in mildly acidic aqueous media Well-known behaviour

Bioactive molecule Temozolomide (TMZ) Alkylating agent Anaplastic astrocytoma Glioblastoma multiforme Why Use a Carrier for TMZ ? Short half-life Intrinsic tumor resistance Limited access to brain tumors Poor response

Methods

SSPLA & CS-SSPLA synthesis & characterization SSPLA : Star Shaped carboxy-terminated Poly Lactic Acid Polycondensation reaction Pentetic Acid as core molecule MSA as catalyst GPC FTIR-ATR 1H-NMR Characterization CS-SSPLA : Chitosan – g –Star Shaped carboxy-terminated Poly Lactic Acid CS FTIR-ATR 1H-NMR + EDC, NHS 48h , RT SSPLA CS-SSPLA

Nanoparticles preparation PolyElectrolytes Complexation method Fast Low cost Solvent-free NPs size related topolymers w/w Dextran sulfate (DS, Mw 40 kDa) CS-SSPLA / DS (w/w): 2 1 mg TMZ I. Add DS + TMZ solution DS + II. 30 min stirring , RT DS CS-SSPLA TMZ CS-SSPLA TMZ

Nanoparticles characterization Dynamic Light Scattering (DLS) z-potential (mV) Transmission Electron Microscopy (TEM) Swelling in different media Average dimension (nm) Stability over time Morphology Dimension

TMZ Encapsulation & Release Dt = amount of TMZ added (mg/ml) Df = amount of TMZ free after encapsulation (mg/ml) Simulated Intestinal Fluid (SIF) : pH 6.8 – Pancreatin free Preparation Media (PM) : pH 5.5 Simulated Gastric Fluid (SGF): pH 1.8 – Pepsin free Temperature : 37 ˚C 180 rpm shake Release conditions Encapsulation and Release were evaluated by UV-Vis at 325 nm

SSPLA and CS-SSPLA SSPLA CS-SSPLA Mn = 1900 g/mol Mw = 4000 g/mol GPC Mw/Mn = 2.4 GPC 1H- NMR (-COOH/-OH ratio) = I 5.01/ I 4.2 = 3.45 CCOOH = 0.979 mmol/g CS-SSPLA FTIR-ATR = presence of amide bond 1H-NMR

Nanoparticles dimension & charge CS = 120 nm ± 10 nm CS-SSPLA = 170 nm ± 24 nm Average dimension (DLS): CS = 38 ± 0.5 CS-SSPLA = 23 ± 1 z-potential (mV) : TMZ does NOT INFLUENCE NPs dimension and z-potential CS-SSPLA CS Preparation media (pH 5.5) Room Temperature

Encapsulation Efficiency (EE) TMZ was encapsulated during preparation Encapsulation Efficiency (%) CS = 250 ± 3 mg TMZ / mg carrier CS-SSPLA = 266 ± 5 mg TMZ / mg carrier

Release studies I CS CS-SSPLA prolonged release pH Swelling SSPLA Release rate SSPLA Does not significantly influence the swelling

Release studies II : Burst CS CS-SSPLA Increases or decreases release intensity at the initial time pH SSPLA Reduces burst intensity

Conclusions Nanoparticles dimension < 200 nm Nanoparticles are stable up to 1 month at room temperature 250-260 mg TMZ / mg of carrier Prolonged release up to 10 days More than 60% reduction of the initial burst in presence of SSPLA

Future Perspectives Multi-drug encapsulation Combination of hydrophilic and hydrophobic drugs Peptides or proteins Preparation of tablets for oral administration

Acknowledgements This work was financially supported by the Czech Science Foundation (Grant No. 15-08287Y) the Ministry of Education, Youth and Sports of the Czech Republic – NPU I Programme (LO1504) and EUPRO (LE12002) the Internal Grant Agency of TBU in Zlin –Czech Republic (Grant No. IGA/CPS/2015/003)