Studies On Cationic Graft Polymer Micelles As Drug Delivery Systems

Pharmaceutical science has entered into a new era with the coming of 21 century. The third generation, defined as controlled or sustained release formulation, and the fourth generation, defined as targeting preparation, are the focus of pharmaceutical research. Particulate carrier systems have received much attention due to its unique various advantages, also it is a rather quickly developed field in pharmaceutical science. Polymeric micelles own particulate characteristics, such as size distribution, surface charge, hydrophilic or hydrophobic properties of the surface, controlled or sustained release, and biocompatible feature. Hence, these polymeric micelles are widely employed in the field of Pharmaceuticals.Polymeric micelles are formed by self-assembly of amphiphilic copolymer in aqueous media with a nanoscaled size (less than 100 nm). Polymeric micelles possess a special core-shell-type architecture. The core of micelles is formed from the hydrophobic segment while shell is formed from the hydrophilic segment. With this unique structure, polymeric micelles are colloidal drug delivery carriers for various drugs with different properties. The electrostatic interaction drives the formation of complex nanoparticles between polymeric micelles and electronegative macromolecular protein or polypeptide drugs. This drug delivery system can control or sustain drug release and enhance the stability of macromolecular drugs. In addition, the core of polymeric micelles can solubilize water insoluble drugs by physical entrapment. So that the problem in the delivery of poorly water-solubleanti-tumor drugs with clinically useful bioavailability can be solved. Polymeric micelles loading anti-tumor drugs can target to tumor cells through initiative or passive manner and reduce the toxicity to normal tissues, finally, to increase the cure effect. In conclude, polymeric micelles are promising carriers for macromolecular drugs and anti-tumor drugs.Chitosan is biodegradable materials with low toxicity. Chitosan oligosaccharide (CSO) was obtained by enzymatic degradation and ultrafiltration separation. The molecular weight (Mw) of CSO employed was determined by gel permeation chromatography (GPC). In order to choose a better carrier material, chitosan oligosaccharide grafted stearic acid (CSO-SA) was prepared by l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction of SA and CSO backbones. The graft CSO-SA was purified by dialysis with distilled water, alcohol washing, and finally dissolving in pH 12 sodium hydroxide solutions. The purity of CSO-SA was identified by high performance of capillary electrophoresis analysis. The degree of amino substitution was determined by 2,4,6-trinitrobenzene sulfonic acid (TNBS) method. CSO-SA can yield self-aggregates micelles in aqueous media with different pH values. The critical aggregation concentration (CAC) of CSO-SA was determined by measuring the fluorescence intensity ratio of pyrene as a fluorescent probe. Size and Zeta potential of CSO-SA micelles were measured by dynamic light scattering. Luteinizing hormone-releasing hormone (LHRH) was chosen as typical model drug. Under an acidic environment, CSO-SA micelles were protonated by the residual amine groups of CSO. The electrostatic interaction drives the formation of CSO-SA/ LHRH complex nanoparticles between micelles and electronegative polypeptide. The nanoparticles were characterized by TEM. The properties and release profiles and stability of CSO-SA/LHRH complex nanoparticles were investigated. The possibility of CSO-SA micelles as carrier for polypeptide drugs was investigated. Mitomycin C (MMC) was chosen as a typical anticancer drug. Size and Zeta potential of CSO-SA micelles before and after loading MMC were determined. The properties and release profiles of MMC-loaded CSO-SA micelles were investigated. In the human lung cell line A549 and cervicalcarcinoma Hela cell line, pure MMC solution as control, the inhibitory effects of CSO-SA micelles, MMC-loaded CSO-SA micelles on the growth of cells were measured by MTT assay respectively. Apoptosis of tumor cells was determined by Hoechst 33342/PI fluorescent staining. Cellular uptake of pure MMC and MMC-loaded CSO-SA micelles in cytosol was determined by HPLC at different intervals. MMC was labeled by FITC. Siting of FITC-labeled MMC and FITC-labeled MMC-loaded CSO-SA micelles in cells was studied both in A549 cell line. Siting of FITC-labeled MMC and FITC-labeled MMC-loaded CSO-SA micelles in A549 cell line treated with digitonin was studied to investigate the nuclear transport capacity of MMC. The possibility of CSO-SA micelles as carrier for anti-tumor drugs was investigated.Chitosan usually has high molecular weight, high viscosity and insoluble at pH 7.2 and may restrict the uses in vivo. After enzymatic hydrolysis and ultrafiltration by various molecular weight cut off membrane, the Mw of CSO is 22.4 Kda obtained by GPC. This CSO can dissolve in aqueous phase with pH less than 7.4. The hydrophobic chains provided by SA are randomly bound to a portion of the hydrophilic amino of CSO by EDC mediated coupling reaction to form the graft CSO-SA. The impurity percent of CSO-SA after purification is 0.76%. The degree of amino substitute is controlled by the mole ratio of CSO/SA. Three types of CSO-SA with amino substitute degree 3.09%, 5.40%, 9.52% respectively are chosen as materials. The CAC value, size and Zeta potential are increased with the decreasing pH values of dispersed medium.LHRH is chosen to act with CSO-SA micelles with 5.4% amino substitute degree. When LHRH is mixed with CSO-SA micelles, the electrostatic interaction drives the formation of complex nanoparticals, which are formed by many CSO-SA micelles and polypeptide drug. The size is in the nanometer range and more than 100 nm, so the complexes can be called "nanoparticles". When pH values of dispersion medium decline from 7.2 to 5.7, the size of nanoparticles is decreased, while the Zeta potential and LHRH encapsulation efficiency are increased. The particles size performed by TEM is similar to the results from a Zetasizer. The release of LHRH invitro conformed to Higuchi Equation. LHRH release from stearic acid modified CSO nanoparticles is decreased when the pH values of the delivery media decreased, in the range from 7.2 to 5.8. The nanoparticals keep good stability when placed in constant temperature for 48 h. It is indicated that the polymeric micelles CSO-SA is a promising carrier for the delivery of polypeptide drugs.Three types of CSO-SA with amino substitute degree 3.09%, 5.40%, 9.52% respectively are used for the solubilization of water insoluble drugs MMC within the interior region of micelles. The mean diameter of micelles is greater after loading MMC, while Zeta potential is lower. Encapsulation efficiency and release profile in vitro of MMC-loaded CSO-SA were determined by ultrafiltration centrifugation. The encapsulation efficiency is about 54%-65%. The release of MMC in vitro can finish in 10 hours. The release velocity is decreased with the increase of amino substitution degree of CSO-SA micelles.In particular, CSO and SA are chosen as hydrophilic and hydrophobic segments due to their biodegradable nature with lower toxicity. MTT assay is performed to determine the cytotoxicity of graft CSO-SA. The IC50 of CSO-SA micelle is above 300 lag-mL"1, which indicate that the micelles have superiorities of security and low toxicity to be a satisfied carrier.However, MMC-loaded CSO-SA micelles display much greater in vitro cytotoxicity and induce apoptosis against human cancer cell line model when comparing with pure MMC. The cytotoxicity of MMC-loaded CSO-SA micelles can enhance 10 to 81 fold than pure MMC. Uptake of MMC in cell line test shows that MMC distribution in whole cell is much higher in the form of MMC-loaded CSO-SA micelles than pure MMC.The intracellular uptake of CSO-SA micelles is confirmed by fluorescence microscopy. It is observed that the fluorescence from FITC-labeled MMC in cell produced a saturated signal. When the cell membrane is intact, the entry of FITC-labeled MMC in the form of CSO-SA micelles into the cells and finally reach nuclei quickly, while the mere FITC-labeled MMC solution are much slower. Theformer can reach equilibrium within 2 hours in nuclei while the latter distributes in cytoplasma and shows none signal in nuclei. When the cell membranes are treated with digitonin, the FITC-labeled MMC distribute in nuclei is similar to the free MMC. Both can reach the nuclei in 15 minutes. This reveals CSO-SA micelles might assist MMC to enter cell membrane barrier and reach nuclei. CSO-SA micelles could be used as the carrier for the delivery of anticancer drugs.