FORMULATION AND EVALUATION OF CHITOSAN NANOPATICLES OF A BROAD SPECTRUM ANTIBACTERIAL

DEVELOPMENT AND EVALUATION OF POLYMERIC NANOPARTICLES CONTAINING

AN ANTICANCER DRUG

SYNOPSIS FOR

M. PHARM. DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

RAVI KIRAN M.

I M. PHARMACY

Department of Pharmaceutics

Dayananda Sagar College of PharmacY

2010

ANNEXURE-II

PROFAMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

Name of the candidate and address (in block letters)

RAVI KIRAN M.

I M. PHARMACY,

DEPARTMENT OF PHARMACEUTICS,

DAYANANDA SAGAR COLLEGE OF PHARMACY,

KUMARASWAMY LAYOUT,

BANGALORE-560078.

PERMANENT ADDRESS

#3-1/1, 1st Floor,

1st cross, Marappanapalya,

yeswanthapura,

BANGALORE, karnataka.

2.

Name of the institute

Dayananda Sagar College of Pharmacy, Shavige Malleswara Hills,

Kumaraswamy Layout,

Bangalore-560078,

Karnataka.

3.

Course of study and subject

Master of Pharmacy in Pharmaceutics

4.

Date of admission to course

20 July 2010

5.

Title of the project:

DEVELOPMENT AND EVALUATION OF POLYMERIC NANOPARTICLES CONTAINING AN ANTICANCER DRUG

6.

Brief resume of the intended work:

6.1 Need of the study:

Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade other tissues. Cancer is not just one disease but many diseases. There are more than 100 different types of cancers. Cancer is a multi-step process typically occurring over an extended period beginning with initiation followed by promotion and progression. Cancer is responsible for about 18% of all deaths in the world and is a major public health problem in many parts of the world. In developed countries, cancer is the second most common cause of death, and epidemiological evidence points to the emergence of a similar trend in developing countries. Deaths from cancer worldwide are projected to continue rising, with an estimated 12 million cases in 2020.

Treatment of cancer includes chemotherapy, radiation therapy, gene therapy, photodynamic therapy, biologic therapy, surgical removal of tumor cells, etc. Chemotherapy is the most convenient and non-expensive when compared to other modes of treatment. The goal of cancer chemotherapy is to slow, block, or reverses the process of carcinogenesis through the use of natural or synthetic compounds. Varieties of anticancer drugs are available in the market and some of them are under clinical trials. The main problem with anti-cancer drugs is that they not only affect the cancerous cells but also affect the normal cells. These happen due to non-specific targeting to cancerous cells and hence other normal cells get affected.

Recently, drug targeting especially targeting of drugs by nanoparticles have been getting much attention by the researchers for treating cancer. Nanoparticles have been used to successfully smuggle a powerful cancer drug into tumor cells - leaving healthy cells unharmed - is one of the first therapeutic uses for nanotechnology in living animals. A critical advantage in treating cancer with nanoparticles is largely through the inherent leaky vasculature present serving cancerous tissues. The defective vascular architecture, created due to rapid vascularization necessary to serve fast-growing cancers, coupled with poor lymphatic drainage allows an enhanced permeation and retention effect.

Targeting the tumor vasculature is a strategy that can allow targeted delivery to a wide range of tumor types. Tremendous opportunities exist for using nanoparticles as controlled drug delivery systems for cancer treatment. Natural and synthetic polymers including albumin, fibrinogen, alginate, chitosan, and collagen have been used for the fabrication of nanoparticles.

Hence, considering the paramount importance of treating cancer an attempt will be made to target and deliver an anti-cancer drug to cancerous cells so as to minimize dose required for the therapy, adverse effects and also dosing frequency. This will be achieved by formulating polymeric nanoparticles which contains an anticancer drug.

6.2 Review of literature

In a study, three-step tumor targeting of paclitaxel1 using biotinylated polylactic acid-polyethylene glycol (PLA-PEG) nanoparticles and avidin–biotin technology was evaluated in vitro as a way of enhancing delivery of paclitaxel. Paclitaxel was incorporated both in biotinylated and non-biotinylated PEG-PLA nanoparticles by the interfacial deposition method. Small (mean size 110 nm), spherical and slightly negatively charged (-10 mV) biotinylated and non-biotinylated nanoparticles achieving over 90% paclitaxel incorporation were obtained. Biotinylated nanoparticles were targeted in vitro to brain tumor (glioma) cells (BT4C) by three-step avidin–biotin technology using transferrin as the targeting ligand. The three-step targeting procedure increased the anti-tumoral activity of paclitaxel when compared to the commercial paclitaxel formulation Taxol and non-targeted biotinylated and non-biotinylated nanoparticles.

An attempt was made to formulate busulfan2 nanoparticles using five different types of poly(alkyl cyanoacrylate) polymers. Poly(isobutyl cyanoacrylate) (PIBCA) and poly(ethyl cyanoacrylate) polymers showed good loading efficiency. Molecular modeling along with energy minimization process was employed to identify the nature of the interactions occurring between busulfan and PIBCA. Optimization studies showed PIBCA nanoparticles displaying busulfan loading ratios equal to 5.9% (w/w) with percentage yield to be 71% (w/w). H-NMR spectroscopy was done to show the chemical integrity of the drug was preserved after loading into nanoparticles. The in vitro release studies under sink conditions, in water, or in rat plasma showed a fast release in the first 10 min followed by as slower one over 6 h.

A study investigated the possibility of preparing doxorubicin3-loaded human serum albumin (HAS) nanoparticles. Doxorubicin was loaded to the HAS nanoparticles either by adsorption to the nanoparticles’ surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150 nm and 500 nm with a loading efficiency of 70–95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution.

A study was undertaken to develop cremophor-free lipid-based paclitaxel4 (PX) nanoparticle formulations prepared from warm microemulsion precursors. Two optimized paclitaxel nanoparticles (NPs) were prepared: G78 NPs composed of glyceryl tridodecanoate (GT) and polyoxyethylene 20-stearyl ether (Brij 78), and BTM NPs composed of Miglyol 812, Brij 78, and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Both nanoparticles successfully entrapped paclitaxel at a final concentration of 150 (g/ml with particle sizes less than 200 nm and over 85% of entrapment efficiency. Cytotoxicity studies in MDA-MB-231 cancer cells showed that both nanoparticles have similar anticancer activities compared to Taxol.

A study investigated the in vitro anticancer activity of cisplatin5-loaded PLGA-mPEG nanoparticles on human prostate cancer LNCaP cells. The uptake of the PLGA-mPEG nanoparticles by the LNCaP cells was also studied. Blank PLGA-mPEG nanoparticles exhibited low cytotoxicity, which increased with increasing PLGA/PEG ratio in the PLGA-mPEG copolymer used to prepare the nanoparticles. PLGA-mPEG nanoparticles loaded with cisplatin exerted in vitro anticancer activity against LNCaP cells that was comparable to the activity of free (non-entrapped in nanoparticles) cisplatin. Little differences in the in vitro anticancer activity of the different nanoparticle compositions were found. Visual evidence of nanoparticles uptake by the LNCaP cells was obtained with nanoparticles labeled with PLGA(4165)-PyrBu(274) or dextran-rhodamine B isothiocyanate using fluorescence microscopy.

An attempt was made to evaluate the potential of chitosan nanoparticles as carriers for the anthracycline drug, doxorubicin6 (DOX). They entrapped a cationic, hydrophilic molecule into nanoparticles formed by ionic gelation of the positively charged polysaccharide chitosan. Hence attempt was made to mask the positive charge of DOX by complexing it with the polyanion, dextran sulfate. This modification doubled DOX encapsulation efficiency relative to controls and enabled real loadings up to 4.0 wt % DOX. Separately chitosan and DOX was investigated for any complexes prior to the formation of the particles. No dissociation of the complex was observed upon formation of the nanoparticles. Fluorimetric analysis of the drug released in vitro showed an initial release phase, followed by a very slow release. The evaluation of the activity of DOX-loaded nanoparticles in cell cultures indicated that those containing dextran sulfate were able to maintain cytostatic activity relative to free DOX, while DOX complexed to chitosan before nanoparticle formation showed slightly decreased activity. Additionally, confocal studies showed that DOX was not released in the cell culture medium but entered the cells while remaining associated to the nanoparticles. The preliminary studies showed the feasibility of chitosan nanoparticles to entrap the basic drug DOX and to deliver it into the cells in its active form.

A study examined the possibility of preparing polymeric nanoparticles containing methotrexate7 (MTX) using methoxy poly (ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer. MTX-encapsulated polymeric nanoparticles of ChitoPEG copolymer has around 50–300 nm particle size and showed spherical shape when observed by transmission electron microscope. Nuclear magnetic resonance study indicated that MTX was complexed with chitosan and core–shell type nanoparticles was formed in aqueous environment, i.e., MTX/chitosan complexes composed of inner-core and MPEG composed of outer-shell of the nanoparticles. Loading efficiency of MTX in the polymeric nanoparticles was 94% (w/w) of ChitoPEG-1, 91.1% (w/w) of ChitoPEG-2, 90.1% (w/w) of ChitoPEG-3 and 65.2% (w/w) of ChitoPEG-4. Higher the drug feeding ratio, higher is the drug content and lower the loading efficiency. Higher the MPEG graft ratio in the copolymer, lower the drug content and loading efficiency. Drug contents evaluated by NMR were same as found by UV spectrophotometer.

Chitosan nanoparticles containing the anticancer drug paclitaxel8 were prepared by solvent evaporation and emulsification cross-linking method. Uniform nanoparticles with an average particle size of 116 ± 15 nm with high encapsulation efficiencies (EE) were obtained. A sustained release of paclitaxel from paclitaxel-loaded chitosan nanoparticles was successful. Using different ratios of paclitaxel-to-chitosan, the EE ranged from 32.2 ± 8.21% to 94.0 ± 16.73 %. The drug release rates of paclitaxel from the nanoparticles were approximately 26.55 ± 2.11% and 93.44 ± 10.96% after 1 day and 13 days respectively, suggesting sustained drug delivery system. Cytotoxicity tests showed that the paclitaxel-loaded chitosan had higher cell toxicity than the individual paclitaxel and confocal microscopy analysis confirmed excellent cellular uptake efficiency. Flow cytometric analysis revealed two subdiploid peaks for the cells in the paclitaxel-loaded nanoparticles and paclitaxel treated groups, respectively, with the intensity of the former higher than that of the latter. Moreover, the cell cycle was arrested in the G2-M phase, which was consistent with the action mechanism of the direct administration of paclitaxel. These results indicate that chitosan nanoparticles have potential uses as anticancer drug carriers and also have an enhanced anticancer effect.

An attempt was made to prepare hydrophobically modified glycol chitosan (HGC) nanoparticles by introducing a hydrophobic molecule, cholanic acid, to water soluble glycol chitosan. The HGC nanoparticles were easily loaded with the anticancer drug docetaxel9 (DTX) using a dialysis method, and the resulting docetaxel-loaded HGC (DTX-HGC) nanoparticles formed spontaneously self-assembled aggregates with a mean diameter of 350 nm in aqueous condition. Under optimal conditions for cancer therapy, the DTX-HGC nanoparticles showed higher antitumor efficacy such as reduced tumor volume and increased survival rate in A549 lung cancer cells-bearing mice and strongly reduced the anticancer drug toxicity compared to that of free DTX in tumor-bearing mice.

Nano-sized poly (D, L lactide-co-glycolide) (PLGA) particles containing estrogen10 were prepared employing emulsification–diffusion method. Estrogen was chosen as a model drug. The preparation method consists of emulsifying a solution of polymer and drug in the aqueous phase containing stabilizer, previously saturated, followed by adding excess water. Influence of process variables on the mean particle size of nanoparticles were studied. It was clarified that the type and concentrations of stabilizer, homogenizer speed, and polymer concentration determined the size of PLGA nanoparticles. Especially when didodecyl dimethyl ammonium bromide (DMAB) was used as a stabilizer, estrogen containing nanoparticles of smaller than 100 nm was obtained.

In a study, gelatin nanoparticles (GPs) were modified with NeutrAvidinFITC-biotinylated epidermal growth factor11 (EGF) to form EGF receptor (EGFR)-seeking nanoparticles (GP-Av-bEGF). Aerosol droplets of the GP-Av-bEGF were generated by using a nebulizer and were delivered to mice model of lung cancer via aerosol delivery. Analysis of the aerosol size revealed that 99% of the nanoparticles after nebulization had a mass median aerodynamic diameter (MMAD) within the suitable range (0.5–5 mm) for lower airway deposition. The fluorescence images obtained from live mice showed that the GP-Av-bEGF could target the cancerous lungs in a more specific manner. Fluorescence analysis of the organs revealed that the GP-Av-bEGF was mainly distributed in cancerous lungs. In contrast, nanoparticle accumulation was lower in normal lungs. The histological results indicated that the fluorescent GP-Av-bEGF was co-localized with the anti-EGFR-immunostain due to EGFR binding. The GP-Av-bEGF could target to the EGFR-overexpression cancer cells in vivo and may prove to be beneficial drug carriers when administered by simple aerosol delivery for the treatment of lung cancer.

The estradiol12(E2)-loaded chitosan nanoparticles (CS-NPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The CS-NPs had a mean size of (269.3 ± 31.6) nm, a zeta potential of +25.4 mV, and loading capacity of E2 CS-NPs suspension was 1.9 mg/ml, and entrapment efficiency was 64.7% on average. They also investigated the levels of E2 in blood and the cerebrospinal fluid (CSF) in rats. The plasma levels achieved following intranasal administration (32.7 ± 10.1 ng/ml; tmax 28 ± 4.5 min) were significantly lower than those after intravenous administration (151.4 ± 28.2 ng/ml), while CSF concentrations achieved after intranasal administration (76.4 ± 14.0 ng/ml; tmax 28 ± 17.9 min) were significantly higher than those after intravenous administration (29.5 ± 7.4 ng/ml tmax 60 min). The drug targeting index (DTI) of nasal route was 3.2, percent of drug targeting (DTP%) was 68.4%. These results showed that the E2 must be directly transported from the nasal cavity into the CSF in rats. Finally, compared with E2 inclusion complex, CS-NPs improved significantly E2 being transported into central nervous system.

The ammonium glycyrrhizinate13-loaded chitosan nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions. The particle size and zeta potential of nanoparticles were determined by dynamic light scattering and a zeta potential analyzer respectively. The effects including chitosan molecular weight, chitosan concentration, ammonium glycyrrhizinate concentration and polyethylene glycol (PEG) on the physicochemical properties of the nanoparticles were studied. These nanoparticles have ammonium glycyrrhizinate loading efficiency. The encapsulation efficiency decreased with the increase of ammonium glycyrrhizinate concentration and chitosan concentration. The introduction of PEG can decrease significantly the positive charge of particle surface. These studies showed that chitosan can complex TPP to form stable cationic nanoparticles for subsequent ammonium glycyrrhizinate loading.

6.3 Objective of the study:

The objective of the study is to develop polymeric nanoparticles containing an anticancer drug, which is expected to

· Improve site specificity.

· Maintain the therapeutic drug concentration in the site of action for a prolonged period of time.

· Improve the drug’s efficiency

· Reduce the dose related side effects.

6.4 Plan of work

The work will be executed as follows:

· Selection of suitable drug and polymer for the preparation of nanoparticles.

· Preformulation studies.

· Optimizing the procedure for the preparation of nanoparticles.

· Formulation of different batches of nanoparticles of anticancer drug.

· Evaluation of prepared nanoparticles include:

· Process yield

· Particle size analysis

· Percentage of drug loading

· In vitro drug release studies

· Release kinetics

· Stability studies

· Animal studies - optional

7.

Materials and methods

7.1 Source of data:

Official Pharmacopoeia, Standard books, Pharmaceutical databases, internet, etc.

Selection of Drug and Polymer:

Drug will be selected based on its availability and effectiveness for treating cancer. Polymer will be selected based its suitability for preparing the nanoparticles.

Method of preparation of nanoparticles:

A suitable method will be used for the preparation of nanoparticles which depends on the polymer and drug used for the study. The method of preparation will be optimized by optimizing the formulation parameters.

Animal studies:

The animals will be divided into four groups. First group will serve as normal control, second group as the tumor control group, third one as pure drug treated and the fourth group as drug bound to nanoparticles treated. All the animals except the normal control group will be injected with DLA cells (1×106 cells per mouse) intraperitonially. The animals will be observed after treatment for survival rate and estimated for other biochemical parameters.

7.2 Method of collection of the data (including sampling procedure, if any):

The pharmacological details of the drug will be collected from various standard books, journals and other sources like research literature databases such as Medline, Science Direct, etc.

Experimental data will be collected from the evaluation of designed formulation and then subjecting the formulation to different studies such as preformulation, process yield, particle size, percentage of drug loading, release profile, stability studies, etc.

The outline of such methods that would be adopted includes:

1. Selection of drug and polymer for the development of nanoparticles.

2. Pre-formulation studies standard to development of nanoparticles.

3. Selection of suitable drug polymer ratio for the study.

4. Development of nanoparticles based on studies in step 2 and 3.

5. Optimization of the formulations.

7.3. Does it require any investigation or interventions to be conducted or patients or other humans or animals? If so please describe briefly:

Yes – Mice

Animal are required to find out the effectiveness of the prepared formulations.

7.4. Has ethical clearance been obtained from your institute in case of 7.3

Yes

8.

List of references:

1. Pulkkinen M, Pikkarainen J, Wirth T, Tarvainen T, Haapa-aho V, Korhonen H, Seppala J and Jarvinen K. Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin–biotin technology: Formulation development and in vitro anticancer activity.

Eur J Pharm Biopharm. 2008; 70: 66–74.

2. Layre A-M, Couvreur P, Chacun H, Aymes-Chodur C, Ghermani N-E, Poupaert J, Richard J, Requier D and Gref R. Busulfan loading into poly(alkyl cyanoacrylate) nanoparticles: Physico-chemistry and molecular modeling. J Biomed Mater Res B Appl Biomater. 2006; 79B (2): 254-62.

3. Dreis S, Rothweiler F, Michaelis M, Cinatl Jr., Kreuter J and Langer K. Preparation, characterization and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles. Int J Pharm. 2007; 341: 207–214.

4. Dong X, Mattingly C A, Tseng M, Cho M, Adams V R and Mumper R J. Development of new lipid-based paclitaxel nanoparticles using sequential simplex optimization. Eur J Pharm Biopharm. 2009; 72: 9–17.

5. Gryparis E C, Hatziapostolou M, Papadimitriou E and Avgoustakis K. Anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells. Eur J Pharm Biopharm. 2007; 67(1): 1-8.

6. Janes K A, Fresneau MP, Marazuela A, Fabra A and Jose M, Chitosan nanoparticles as delivery systems for doxorubicin. J Control Release. 2001; 73(2-3): 255-67.

7. Seo D-H, Jeong Y-II, Kim D-G, Jang M-J, Jang M-K and Nah J-W. Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan. Colloids Surf B Biointerfaces. 2009; 69(2): 157-63.

8. Li F, Li J, Wen X, Zhou S, Tong Z, Su P, Li H and Shi D. Anti-tumor activity of paclitaxel-loaded chitosan nanoparticles: An in vitro study. Mater Sci Eng. 2009; 29(8): 2392-97.

9. Hwang H Y, Kim I S, Kwon I C and Kim Y H. Tumor targetability and antitumor effect of docetaxel-loaded hydrophobically modified glycol chitosan nanoparticles. J Control Release. 2008; 128: 23–31.

10. Kwon H-Y, Lee J-Y, Choi S-W, Jang Y and Kim J-H. Preparation of PLGA nanoparticles containing estrogen by emulsification-diffusion method. Colloids Surf A Physicochem Eng Asp. 2001; 182(1-3): 123-30.

11. Tseng C L, Wu S Y H, Wang W H, Peng C L, Lin F H, Lin C C, Young T H, Shieh M J. Targeting efficiency and biodistribution of biotinylated-EGF-conjugated gelatin nanoparticles administered via aerosol delivery in nude mice with lung cancer. Biomaterials. 2008; 29: 3014–3022.

12. Wang X, Chi N and Tang X. Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting. Eur J Pharm Biopharm. 2008; 70: 735-740.

13. Wu Y, Yang W, Wang C, Hu J and Fu S. Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate. Int J Pharm. 2005; 295: 235-45.

9.

Signature of the candidate

(RAVI KIRAN M.)

10.

Remarks of the guide:

Forwarded and recommended for research and submission of dissertation.

11.

Name and Designation (in block letters)

11.1. Guide

11.2. Signature

dr. b. wilson,

PROFESSOER & head,

Department of Pharmaceutics,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078.

11.3. Co-guide if any

Not applicable

11.4. Signature

11.5. Head of the department

11.6. Signature

dr. b. wilson,

Professor & Head,

Department of Pharmaceutics,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078.

12.

12.1. Remarks of the principal

12.2 Signature

Dr. V. Murugan,

Principal,

Dayananda Sagar College of Pharmacy,

Kumaraswamy Layout,

Bangalore-560078