| Background:The global health and economic burden of malignant tumors continue to increase largely because of aging population alongside an increasing adoption of smoking. According to the GLOBOCAN 2008 reports, about 12.7 million cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008. Lung cancer is the leading cancer site in males, comprising 17% of the total new cancer cases and 23% of the total cancer deaths. Only 15% of lung cancers could survive for 5 years after diagnosis in America. This poor outcome of longtime survival is a reflection on the disappointing therapy regimens for treating advanced, inoperable and postoperative recurrence non-small-cell lung cancer. Chemotherapy is currently one of the important regimens for treating cancer besides of surgery and radiation. However, these cytotoxic drugs which are currently available for chemotherapy could cause serious side effect, such as myelosuppression, organ toxicity, nausea, and vomiting, allergic reaction and hair-loss which let the patient live in pain. This is mainly because of the unselective cytotoxicity of cytotoxic drug. Despite the progresses achieved over last teen years, the overall rate of response to chemotherapy is still low and there remains a real need to improve cytotoxic drugs for better tumor targeting and reducing unselective cytotoxicity.Hyaluronic acid (HA) is an integral component of extracellular matrix and plays several physiological functions, such as preserving water for the skin. It has been found to be accumulated in the stroma at the invading edge of a range of solid tumors. Tow HA receptors, CD44 and the receptor for Hyaluronic acid-mediated motility (RHAMM) has also been found to be over-expressed in those solid tumors which indicating that HA could activate intracellular signaling cascades associated with tumor growth, tumor cell adhesion and metastasis through binding to its receptors. In malignant tumors, CD44 is in a high affinity state with the capacity to bind and internalize HA and plays a critical role in malignant cells'migration and invasion. In contrast to malignant tumor cells, most normal primary cells also express CD44, but in a low affinity state that does not bind HA. Taking all the relevancies together, CD44 has the potential as a biological target in the treatment of solid tumors and HA, in the other hand, has the potential as a targeted delivery vehicle for chemotherapy agents.Attempts have been made and there are mainly two distinctly different strategies to improve anti-cancer agents based on HA, i)to create pro-drugs based on HA by direct conjugation of active cytotoxic drugs with low MW HA, or by the covalent coupling of HA; ii) to create a novel excipient base on high MW HA for entrainment of active cytotoxic drugs. However, there has been limited success both in vitro and in vivo studies to develop intravenous pro-drugs based on HA. This result was largely because of that the pro-drugs was preferentially uptake by the liver which is involved in the metabolism of HA and has the potential for liver toxicity. In addition, the HA pro-drugs did not result in increasing efficacy, but instead of reducing the cytotoxicity of chemotherapeutic drugs due to chemical modification. As an alternative approach for using HA as a drug carrier, the second strategy did seem to overcome the two limitations of HA pro-drugs and utilized the unique tertiary structure of high MW HA in aqueous solution. Due to poor intra-tumoral lymphatic drainage and vascular leakage, the high MW HA can preferentially accumulate within the malignant site and it could promote passive uptake the anti-cancer drugs entrained in that novel excipient by the malignant cells, a phenomenon known as the "enhanced permeability and retention" (EPR) effect. However, the high MW HA could not act as an active tumor targeted drug-carrier and there is no way to accurately characterize the drug:HA ratios in end-product. Much work is still to be done and the nanoscale drug-carrier device which can be conjugated with several functional molecules simultaneously, including tumor-specific ligands, anti-cancer drugs and excipients promises the possibility to develop an ideal tumor-targeted drug carrier.Among those candidates for drug carrier system, chitosan (CTS) has received much attention due to its non-toxic, biocompatible, biodegradable, controlled release and anti-tumor properties. Chitosan, also known as soluble chitin, deacetylchitin and poly-(D) glucosamine, is produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (crabs, shrimp, etc.). Chitosan is a linear polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. Chitosan and its derivatives have a positive charge, which allows them to bind strongly to negatively charged polymers, macromolecules and even some polyanionic in the liquid medium. And these ionotropic gelation processes make the drug-loaded nanoparticles easily. The size of these drug-carriers can be determined by the ratio of CTS:negatively charged polymers, so as the entrainment efficacy and loading capacity. And these nanoscale drug carriers have been engineered for controlled release. Preparation of chitosan controlled-release formulations which loading chemotherapy drugs, insulin, antibiotics and other drugs, has been achieved good results, some have been applied to clinical. However, there has been found that the chitosan based drug-loaded nanoparticles in vivo were rapidly uptake and cleared by reticulo-endothelial system (RES) and it was difficult to achieve the desired result.If through physical adsorption or covalent binding one layer or multi-layer hydrophilic polymer objects on the surface of the general nano-particles, such as polyethylene glycol (PEG), may form a steric layer, thereby preventing plasma protein opsonin close chitosan and enabling the chitosan nanoparticles to escape from the capture of mononuclear phagocytic system. These multifunctional conjugations of drug carriers have the potential to overcome the disadvantage of the traditional nanoparticles that most of them are rapidly cleared by RES in vivo. HA is an amphiphilic polymer and the conjugation of HA coupled chitosan nano-particles promises an ideal tumor-targeted drug delivery system.In this study, we chose CTS as a raw material, using ionotropic gelation method, to prepare nano-particles which containing the mode drug and coupled with low MW HA. And all these nanoparticles products were characterized. The nanoparticles were marked by FITC and nanoparticle cell uptake was monitored by fluorescent microscopy. The cytotoxicity of HA coupled nanoparticles loading DTX was evaluated by MTT assay using A549 non-small cell lung cancer cell lines.Objective:1. To explore the feasibility of HA coupled chitosan-TPP nanoparticles (HACTNPs) as a targeted anti-cancer drug delivery system for non-small cell lung cancer.2. To prepare the docetaxel (DTX) loaded chitosan-TPP nanoparticles (DTX-CTNPs), and then to couple the drug-loaded nanoparticles with HA. The characterizations and in vitro release properties were evaluated.3. To monitor the cell uptake ability for HA coupled nanoparticles and non-coupled nanoparticles using fluorescent microscopy.4. To evaluate the tumor cell proliferation inhibitory effect of drug-loaded nanoparticles in vitro by an MTT assay and to prove that the drug-loaded chitosan nanoparticles can exert the anti-tumor effect of the loaded drug. And to verify that the HA coupled drug-loaded nanoparticles are better than the non-coupled drug-loaded nanoparticles due to their CD44+ tumor targeted ability which promise that the HACTNP as an ideal targeted carrier for chemotherapeutic drugs.Methods:1. Through ionotropic gelation method which combine the negatively charged ion on TPP with positively charged amino on chitosan to form chitosan-TPP nanoparticles (CTNPs) and the docetaxel (DTX) was encapsulated at the same time to form drug-loaded nanoparticles (DTX-CTNPs). And then the blank nanoparticles and drug loaded nanoparticles were both coupled with HA to form HA coupled blank chitosan-TPP nanoparticles (HACTNPs) and HA coupled drug-loaded chitosan-TPP nanoparticles (DTX-HACTNPs). All these nanoparticles were characterized by transmission/scanning electron microscopy, laser scattering and Fourier transform infrared spectroscopy. The encapupsulation efficiency as well as loading capacity was investigated by ultraviolet spectrophotometry. Dynamic dialysis study of drug-loaded nanoparticles was also carried out in vitro to evaluate their abilities of controlled release.2. The HA coupled and non-coupled blank nanoparticles were both marked with FITC. And the uptake studies of HA coupled and non-coupled nanoparticles were carried out using fluorescent microscopy. In order to verify the involvement of CD44 receptors in nanoparticles uptake, studies were conducted in presence and absence of free HA and the effect of cell uptake was observed.3. The in vitro cytotoxicities of free DTX, DTX loaded HA coupled nanoparticles and non-coupled nanoparticles against A549 cell lines were compared, using MTT assay. And the IC50 of different formulations were calculated.4. Using SPSS 13.0 statistical software for data processing, all data was expressed by mean±standard deviation (x±s). The crossover effect of grouping and drug concentration on the cell inhibitory rate was tested by two-way classification ANOVA. Depending on the outcome of the test of homogeneity of variances, the one-way ANOVA or Welch Test was also applied. And the difference between the means of 2 groups was assessed by the LSD or Dunnett's T3 multiple comparisons test. In all cases, a value of P<0.05 was accepted as significant.Results:1.The drug-loaded HA coupled and non-coupled nanoparticles were round or oval in shape, good dispersion when the CTS/TPP mass ratio was 5.3:1, DTX concentration was 0.5mg/ml, the HA/NPs mass ratio was 1:10. Their average particle sizes were 228nm,200nm, respectively. Their drug loading capacities were 16.5%, 22.4%, respectively. And the encapsulation efficiency of uncoupled drug loaded nanoparticles was 59.2%. With 46.0% release of DTX from drug loaded coupled nanoparticles and 51.4% release of DTX from drug loaded uncoupled nanoparticles after 48 h and further zero order release profits were observed up to 7days. And in all cases the drug release was found to be over 80% at the end of 7 days. For the Fourier transform infrared spectroscopy of drug-loaded HA coupled nanoparticles, the characteristic peaks of HA, chitosan, TPP and DTX were observed.2. The uptake of FITC marked nanoparticles by A549 cell lines were visualized under fluorescence microscopy. The fluorescence intensity was found to be more in case of HA coupled blank nanoparticles compared to non-coupled nanoparticles.3. The cell inhibitory rates of all the formulations were drug concentration-dependent. And the differences of cell inhibitory rate between the 3 groups(DTX-HACTNPs, DTX-CTNPs and Free-DTX) at each DTX concentration were significant (F=123.445, P=0.000; F=193.792, P=0.000; F=477.704, P=0.000; F=123.349, P=0.000). And the IC50 of HA coupled drug loaded nanoparticles, non-coupled drug loaded nanoparticles and free DTX were (15.06±0.94)μg/ml, (25.73±3.37)μg/ml, (5.35±0.61) u g/ml, respectively(F=73.871, P=0.000).The in vitro cytotoxicity of drug-loaded chitosan nanoparticles were much lower than that of free DTX as the cytotoxicity was defined by the IC50 (P=0.000). And the HA coupled drug loaded nanoparticles have performed better anti-tumor effect as compare to non-coupled drug loaded nanoparticles (P=0.001).Conclusion:1. HA coupled and uncoupled chitosan nanoparticles bearing DTX have been engineered via ionotropic gelation method. And these nanoparticles have good shape and good size distribution, as well as high encapsulation efficiency and better drug controlled release properties.2. The HA coupled chitosan nanoparticles are anticipated to be promising alternate carriers for targeting of CD44+ tumor cells. |
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