Effective loading and controlled release of camptothecin by O- carboxymethylchitosan aggregates Liu Nan 4 th semester in Ph. D course Lab. of Biopolymer Engineering

Contents Conclusion Results and discussion Experiment Introduction Object

Introduction

Novel delivery systems Entrapment of drugs, proteins or genes in small vesicles or within polymeric matrices. Colloid delivery system Reduce the unwanted toxic side effect and improve the therapeutic effect. Nanoparticles, liposomes, microemulsions, polymeric self-assemblies, and so on.Introduction

Chitosan The second most plentiful biomass Biocompatible, biodegradable, and nontoxic natural polymer. Application in drug delivery Limitation Limited solubility (pH>6.5) Limited effectiveness as absorption enhancer at neutral pH valuesIntroduction

CH 2 OCH 2 COOH O-carboxymethylated chitosan (OCMCS) A kind of water soluble chitosan derivative. Biocompatibility and antibacterial activity. Carboxyl groups and amino groups in OCMCS macromolecules elicit special physicochemical and biophysical properties. Introduction O O H NH 2 H H H OH CH 2 OH n O O H NH 2 H H H OH n ChitosanOCMCS

Introduction Camptothecin (CPT) is a plant alkaloid and very effective in the treatment of gastric, rectum and bladder tumors. Anticancer drug, which is a plant alkaloid. Scarce water solubility A number of toxic effects, such as neutropenia, thrombocytopenia, anaemia and a number of nonhaematological toxic effect.

Object  Investigate the drug-loading as well as the controlled release behavior of OCMCS aggregates on CPT.  Discuss the release kinetic of CPT from aggregates.

Experiment

Experiment Chitosan powder, Mw6.7×10 5 g/mol; DDA90% was obtained from Lianyungang Biologicals Inc. (China) Camptothecin (CPT) C20H16N 2 O4, Mw348.4 were purchased form Sigma-Aldrich company. The other materials were of commercial grade and were used without further purification. Materials

2g chitosan immersed in 25ml 50% NaOH solution for 24h Monochloroacetic acid 5g dissolved in isopropanol 25ml Reaction for 8h at room temperature. Experiment Synthesis of OCMCS

OCMCS solutions were prepared in a series of concentrations from 0.03 to 3mg/ml. CPT was added in 10ml of OCMCS solution to make saturation state. The amount of CPT dissolved in each OCMCS solution was evaluated using steady-state fluorescence spectroscopy (SSF). Experiment Loading capacity of OCMCS aggregates

5ml of CPT/OCMCS and CPT aqueous saturated solution were put in the dialysis membrane. The seal dialysis membrane placed in PBS solution. Then the solution was measured at wavelength of nm by UV-VIS- NIR scanning spectrophotometer. Experiment In vitro release study

Experiment In vitro cancer cell activity MCF-7 cell were grown in DMEM containing 10% fetal bovine serum supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin at 37 ℃ under a 5% CO2 containing atmosphere Cells were seeded into 96-well plate at 5000/well and grown to 50% confluency. 100ul of CPT/OCMCS was added. After 24h incubation, 20ul combined MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)/PMS (phenazine methosulfate) solution was added to each well. After incubating the plate for 2h, the absorbance of each well at 490nm was recorded by using a 96-well plate reader.

Experiment MTS/PMS Assay MTS is an analog of MTT It has the similar reaction mechanism with MTT. MTS to MTS-formazan. The number of viable cells is correlated to the quantity of the formazan produced.

Results and discussion

Fig. 1. Effect of OCMCS concentration on the fluorescence emission spectra of camptothecin in aqueous solution of OCMCS. The solubility of CPT is significantly enhanced in OCMCS solution. The solubility of CPT does not increase linearly with OCMCS concentration. Drug loading in OCMCS aggregates

Fig. 2. CPT loading as a function of OCMCS concentrations. 1 Up to a concentration of mg/ml, the drug’s solubility increases. 2 CPT’s solubility decreased to minimum of 0.1mg/ml 3 The solubility increases again. Results and discussion Drug loaded in OCMCS aggregates

Fig. 3. CPT loading as a function of OCMCS concentrations. The critical aggregation concentration (cac) of OCMCS is determined to be 0.05mg/ml. (Zhu et al., 2005) Results and discussion Drug loaded in OCMCS aggregates

Fig. 4. Release behavior of CPT from OCMCS in PBS (pH 7.4, 0.01M) solution at 37 ℃ t 50% : the time 50% amount of the drug was released ; t eq : time to achieve equilibrium; F eq : the fraction of CPT released at equilibrium. Saturated CPT int 50% (min)t eq (min)F eq (%) Water mg/ml OCMCS mg/ml OCMCS mg/ml OCMCS Results and discussion The release of CPT from OCMCS aggregates

M t and M ∞ are the absolute cumulative amounts of drug released at time t and infinite time. l is the thickness of the flat sample D is the diffusion coefficient. Diffusion coefficients Saturated CPT inD 1 ×10 7 (cm 2 /s)D 2 ×10 7 (cm 2 /s) Water21.75/ 0.03 mg/ml OCMCS mg/ml mg/ml The release mechanismDiffusion controlled Degradation controlled A mix of them Results and discussion Diffusion coefficients

Fig. 5. the relative anti proliferative activity of cancer cell dependence on the OCMCS concentration. Results and discussion Antitumor activity

Conclusion

Conclusions A novel, biocompatible OCMCS aggregates were developed into a controlled drug delivery system. The amount of drug loaded in OCMCS is strongly dependent on its concentration. Not only the aggregates but also unimer of OCMCS can effectively load the hydrophobic CPT. The maximum amount of CPT loaded in OCMCS was found to be 6 times than that of camptothecin in water at a concentration of OCMCS of mg/ml. The diffusion coefficient of CPT in the presence of OCMCS exhibits low. In vitro measurements confirm the slow release behavior of CPT from OCMCS.

Q & A