Study Of Intelligent Tumor Targeted Drug Delivery Systems Based On Chitosan

Many anticancer drugs used in the past decades have shown conspicuous cytotoxicity to tumor cells. However, due to their anticancer mechanism, the highly proliferativecells of tumor and normal tissues are both affected by thesedrugs. This nonselective cell killing has been a long stumbling block in the development of new anticancer drugs. Hence, there has been an intense request to develop efficient carriers that can deliver lipid soluble anticancer drugs exquisitely to the target site while provoking less adverse reactions, which makes these drugs more safe and effective in clinical therapy.In recent years, the development of drug delivery systems facilitating site-specific therapy has achieved significant progress. Safe and nontoxic carriers for delivering the cytotoxic drug to the target tumor cells are the hot topics in this field. In recent years, the stimuli-responsive hydrogel systems have shown great potentials due to their active response to external stimuli such as temperature, pH, specificion, and electric field. Among all intelligent hydrogels studied, temperature and pH-responsive systems have drawn much attention. Because temperature and the pH value are important environmental factors in body, some disease states manifest themselves by a change intemperature and/or pH. The acidic pH of extracellular fluid of solid tumors has extensively been documented for a few decades and combined with biological backgrounds of this observation. Most solid tumors and inflammatory regions in the body have lower extracellular pH than thesurrounding tissues and blood. In particular, the intracellular environment sensitive polymeric micelle, which can release the loaded drug through sensing pH decreases in the acidic endocytic compartments such as endosomes (pH 5-6) and lysosomes (pH 4-5), and has recently been reported to minimize non-specific systemic spread oftoxic drugs while maximizing tumor directed drug delivery efficiency. It has been reported by Kataoka's research group that the anticancer drug Adriamycin (ADR) was linked to the polymer by a pHsensitive hydrazone linker. By this polymer design, the micelles can stably preserve drugs under physiological conditions (pH 7.4) and selectively release them by sensing the intracellular pH decrease inendosomes and lysosomes (pH 5-6). However, how do such environment-sensitive nanoparticles move into the cell interior?Thus, cell internalization is also essential for effective drug delivery by nanocarriers. This will help high accumulation of the drug in the target sites. To attain active targeting of a drug carrier, particulates are derivatized with ligands which bind to specific receptors expressed on the target cells such as antigen/mAb, ligand/receptor, aptamer/counterpart, and a peptide selected from the phase display method/counterpart, etc. Folate behaves as a ligand because its receptors, folate-binding proteins (FBP), are selectively over expressed on the surface ofcancer cells. However, there are seldom quantitative analyses of such specific interactions for active endocytosis in clinical settings. Is the expression level sufficient for active endocytosis or really effective enough? Can it solve problems such as lack of tumor selectivity, and multidrug resistance (MDR)?In recent years, much effort has been made to identify agents called chemosensitisers that are able to overcome MDR, in order to improve chemotherapeutic treatment. It has been demonstrated by Nakanishi and Brantley that a NF-kB inhibitor could enhance the sensitivity of tumor cells to apoptosis induced by chemotherapeutic agents such as taxol, doxorubicin (DOX), tamoxifen, and cisplatin. Pyrrolidinedithiocarbamate (PDTC), a NF-kB inhibitor, was studied as a sensitizer of the anticancer drug. It was revealed by Wijngaarden et al. in the research results that PDTC could overcome anticancer drug resistance and enhance DOX efficacy.A pH-sensitive drug targeting system for solid tumors was established based on N-isopropylacrylamide (NIPAAm) and chitosan conjugates. The mass ratio of NIPAAm and chitosan was adjusted to obtain super pH-sensitive characteristic and the structure was studied by using Fourier transform infrared spectroscope to confirm the successful synthesis of the nanoparticles.The pH-sensitive and drug release characteristics in vitro were studied as well. Human lung cancer cells A-549, Human colon carcinoma cells SW480 and human fibroblast were used to test the biocompatibility of blank and Podophyllotoxin (POD), Camptothecine (CAMP), and Paclitaxel (PTX) loaded nanoparticles further to certificate the reliability of targeting acidic tumor extracellular pH. Results revealed that when charge ratio between NIPAAm and CS achieve 4:1(w/w), the drug-loaded nanoparticles, which diameters ranged from 50 to 150 nm, exhibited super pH-sensitive responses to tumor pH. Encapsulation and loading efficiencies were 73.7%, 63.7%, 85.7% and 8.4%, 2.54%, 9.6%, respectively. The cumulative release rate of drug, which significantly enhanced at pH 6.8 while decreasedrapidly either below pH 6.5 or above pH 6.9 at 37℃. At pH 6.8, drug-loaded nanoparticles showed cytotoxicityin MTT test and fluorescence microscopic study, comparableto that of free drug at the same drug concentrations, whereas at pH 7.4 there was little cytotoxicity at the tested concentration range. Thereby, the atoxic PNIPAAm-g-chitosan nanoparticle has the potentiality as a novel anticancer drugs carrier.Micellar nanoparticles self-assembled from copolymer folate-chitosan (FA-CS) were employed ascarriers to co-deliver Pyrrolidinedithiocarbamate (PDTC) and doxorubicin (DOX) to achieve targeted DOX delivery, with a pH responsive drug release, and to overcome DOX multidrug resistance (MDR). The successful synthesis of FA-CS was determined by NMR. Average particle size was small enough to achieve longevity during systemic circulation. Lower CACs in neutral and alkalescent conditions rather than an acid pH may lead to maintenance of good stability of the micellar nanoparticles in the blood stream. DOX and PDTC encapsulating efficiencies of the micellar nanoparticles were 77.64 and 86.54 wt% while loading content was 12.34 and 15.32 wt%, respectively. The release of DOX at neutral or alkalescent pH was slow and sustained, however, in the weak acidic environment, was much faster with close to 75-95% of its total drug content being released within the first 2 h. Lower IC50 of DOX-loaded micellar nanoparticles suggested that FA-CS micelles greatly enhanced the cellular uptake efficiency. Fluorescence microscopy micrographs further verified that DOX released from CS-FA micelles could be pHsensitive and achieved intracellular targeting. It was confirmed by flow cytometry analysis that codelivery of PDTC and DOX may further overcome the MDR of DOX besides the folate receptor mediated endocytosis process. This co-delivery system may have important clinical implications against liver cancers.Chitosan and FA-CS nanocapsules were prepared for intracellular negative gold nanoparticles delivery. The particles have been characterised by 1H-NMR, dynamic laser light scattering (DLS), and transmission electron microscopy (TEM), the size of nanocapsule with spherical morphology is about 100 nm. To validate the choice of Hela folate free cell lines as a positive control over expressing FR-α, the expression of FR-αwas investigated by Western blot. The inhibitory concentration of 50% (IC50) was calculated for cytotoxicity tests involving C5-Au-C11-COOH NPs. The amounts of Au uptake were measured by ICP-MS in Hela, Hela folate free and A549 cells. The results revealed that using CS and FA-CS as delivery vehicles, the intracellular uptake of negatively charged particle is enhanced to a greater extent.