Studies On Docetaxel-loaded PH-sensitive Liposomes

Cancer always cause serious damage to human health. In our country the number of new cancer about300millions every year, deaths from cancer each year reached more than270million, equivalent to every day there are more than7300people died of cancer. Chemotherapy has been one of the three majoytreatment methodologies for malignant tumors.Docetaxel (DTX) is a semisynthetic taxiod derived from European yew tree, Taxus baccata, and it is a mitotic spindle posion that accelerates the microtubule assembly from tubulin and blocks the depolymerization of the microtubule. It has been demonstrated extrordinary anticancer effects both in vivo and in vitro aganist variety of tumors including breast cancer, ovarian cancer, lung cancer, melanoma etc. However, DTX as a fat-soluble drugs, the use of DTX causes several side effects including neutropenia, musculoskeletal toxicity, peripheral neuropathy and which have limited its clinical application. DTX was easy cause allergic reactions because of using tween-80as solubilizer. In order to overcome these problems and to improve the drug efficacy and reduce side effects caused by nonspecific delivery, so delivery of therapeutic and diagnostic agents is one of the biggest challenges of chemotherapy and medicine in general.Targeted cancer therapy was using a specific carrier directed to the antiturnor effect of the tumor cells without affecting normal cells or tissue function. In contrast to conventional cytotoxic chemotherapy, targeted cancer therapy possess specific anticancer effects and less toxicity. Therefore, in this study, two novel docetaxel-loaded liposomes careiers were prepared to improve solubility and bioavailability. The first system was the docetaxel pH-sensitive liposomes (DTX-PLPS); the second one was RGD modified docetaxel pH-sensitive liposomes (DTX/RGD PLPS). pH-sensitive liposomes can be targeted to the low pH of tumor tissues, play the role of physical chemistry targeting; and targeting factor RGD can targeting the overexpression tumor cell surface integrin receptors, play the role of active targeting. This paper studies the pharmacy and biological characteristics of the two liposomes, discusses its prospects as a cancer drug delivery. The main methods and results were as follows.1. Studies on the docetaxel pH-sensitive liposomesThe drug content of DTX was determined by HPLC method. We separte free DTX and the DTX-PLPS using solution-ultracentrifugation method. In our study, DTX was taken as the model drug to be wrapped up into pH-sensitive liposomes which were prepared by phosphatidylethanolamine by thin-film dispersion method. The entrapment efficiency was taken as the index to evaluate the effects of the temperature of rotary evaporation, stirring time, amount of CHOL, amount of CHEMS and the ratio of drug and lipid on the characteristics of liposomes. The amount of CHEMS, temperature of hydration, hydration time and the ratio of drug and lipid as the most influential factors, which were optimized by orthogonal design method concerning the entrapment efficiency. The final optimized formulation was35mg of PE,15mg of CHOL,15mg of CHEMS,6mg of DTX,60℃of hydration temperature,1.5h of hydration time,800rpm of hydration speed. The encapsulation efficiency and drug loading were86.40%,7.31%, respectively.On the basis of optimized formulation, DTX-loaded pH-sensitive liposomes were prepared by adding sufficient quantum of tlactose into liposomes suspension, and the obtained lyophilized pH-sensitive liposomes showed good redispersibility as well as homogeneity. The optimized liposomes were sphere and homogeneous in size with average partical size of203.32±3.2nm, and the polydispersity is small; Zeta potential is-31.17±2.20mV. Through the study of the stability, encapsulation efficiency and drug content of freeze-dried liposome have good stability, coform with the requirments of preparation.2. Studies on the RGD-modified pH-sensitive liposomes loaded wtih docetaxel In this study, we used the solid-phase synthesis of peptides attached the RGD toCHEMS, synthesis RGD-CHEMS. The resulting product and CHOL, PE in appropriate proportions were produced RGD-modified pH-sensitive liposomes loaded with docetaxel. The particle diameter measured by dynamic light scattering method is221.12±1.7nm and the polydispersity is small; Zeta potential is-30.24±2.3mV.Compared with Doupafei(?), in vitro release results of two DTX-loaded pH-sensitive liposomes in different pH value (pH were7.4,6.0,5.0,4.0) phosphate buffer which contain0.5%tween-80, indicated that the drug release slowly from liposomes. We also found that the drug release in acidic medium was faster than that in physiological environment of pH7.4. It can be seen that the release of the drug from the liposomes preparation shows a certain pH-sensitivity.3. In vitro antitumor evaluation of the DTX-loaded liposomesIn this study, we use MTT colorimetric method to evaluate the antineoplasmic activity in vitro of docetaxel solution, DTX-PLPS and DTX/RGD-PLPS on the tumor cells of MCF-7, A549and HepG2. The results show that the cytotoxicity of two DTX-loaded liposomes agnist three cencer cell lines was superior to docetaxel solutions. The antineoplasmic activity of DTX in vitro:DTX/RGD-PLPS>DTX-PLPS>DTX-solution. And the IC50value of DTX/RGD-PLPS, DTX-PLPS and DTX-sol ware as follows:MCF-7:2.02+0.08,3.79±0.17,8.57+0.51μg/mL; A549:2.03±0.11,2.38±0.42,6.39±3.92μg/mL; HepG2:1.65±0.05,3.12±0.15,5.04±0.62μg/mL. The sudies on cellular uptake indicated that the liposomes can increased the concentration of drug in tumor greatly, and the degree of uptake of RGD-modified liposomes were significantly stronger than the unmodified liposomes. Therefore, we conclude that the liposomes have good cell compatibility, could be used to deliver docetaxel to the tumor effectively. And targeting factor RGD-modified liposome can actively targete tumor cells, and expected to achieve high efficiency and low toxicity of targeted cancer therapy purposes.4. In vivo studies of DTX-loaded liposomes delivery systemThe pharmacokinetic experiment of Doupafei(?), DTX-PLPS, DTX/RGD-PLPS was carried out on rabbit. Compared with Doupafei(?), the time of DTX-loaded liposomes in vivo are prolonged. DAS2.0pharmacokinetic procedure was applied to calculate parameters and chose the best compartment model, the results show that three preparations all folllowed the two compartment model. The AUC of DTX-PLPS and DTX/RGD-PLPS are19.51、16.31ug/L·h, respectively, Significantly greater than the AUC of Doupafei(?). The results indicated that DTX-PLPS and DTX/RGD-PLPS could significantly lenghten the retention time of drugs in vivo and had a well-sustained release efficacy.In this study, water insoluble DTX was successfully incorprated into liposomes. DTX-loaded liposomes had high entrapment efficiency and good stability. It can physical chemical and active targeted to the tumor tissue and significantly increased the amount of drugs into the cells. Therefore, the liposomes presented in this paper could significantly enhance therapeutic efficacy and decrease the toxicity. The RGD modified liposomes might be a promising targeting formulation for cancer therapy.