| Docetaxel(DTX)is a category of broad-spectrum and highly effective antitumor compound obtained through the structural modification of antitumor taxane compound,paclitaxel(PTX).The therapeutic efficiency of DTX is 2-4 times higher than that of PTX.In clinical,DTX plays an important role in the therapy of solid tumor such as breast cancer,non-small cell lung cancer,malignant melanoma and head and neck neoplasm.However,as the more and more widely application of DTX in clinical,a plenty of problems gradually reveal.To begin with,the solubility of DTX in aqueous is pretty low(5-6 ?g/ml),thus,DTX cannot disperse well in body fluid and it is really easy for DTX to be cleared from circulation of body fluid.DTX in preparations commonly applied in clinical such as Taxotere(?)and Duopafei(?)is usually be solubilized with organic solutions such as ethanol and non-ionic surfactants such as Tween 80,but the utilization of these solutions and non-ionic surfactants can result in hemolysis and hypersensitivity reactions,these reactions severely decrease the tolerance of patients towards the preparations,in addition,the toxic and side effects of DTX such as hepatic impairment and neutropenia and multi-drug resistance(MDR)of tumor tissues mediated by pathways such as P-glycoprotein(P-gp)overexpressed on the surface of tumor tissues and autophagy of tumor tissues caused by long-periodic DTX administration also limit the further application of DTX.Thus,it is necessary to design a kind of suitable DTX formulation to solubilize DTX,efficiently delivery DTX to tumor tissues and improve the bioavailability and tumor inhibition ability of DTX.Amphiphilic carrier-drug conjugation is a type of amphiphilic conjugation synthesized through conjugating of hydrophilic carrier and hydrophobic drug via chemical reaction of active chemical groups or specific linkage,compared with conventional polymeric micelles,amphiphilic carrier-drug conjugation based polymeric micelles possess low drug leakage,and the introduction of linkages or chemical bonds with special physicochemical properties into micelles can endow micelles with unique physicochemical properties.However,the covalent binding between carrier material and drug makes drug release become slow and results in low cumulative drug release amount.Thus,hydrophobic drug molecules are incorporated into micelles through covalent binding with carrier and non-covalent encapsulation.In this way,when micelles accumulate in tumor tissues,not only can drugs release from the micelles to tumor tissues through free diffusion and the concentration of drugs in tumor tissues can quickly arrive at the concentration at which the tumor tissues can be effectively inhibited to achieve highest tumor inhibition efficiency,the sustained and controlled release of drugs conjugated with carriers are capable of maintaining the therapeutic efficiency,thus the therapeutic efficiency of chemotherapeutic agents can be effectively improved.Chondroitin sulfate(CS)is a kind of biologically sourced highly hydrophilic linear macromolecular polysaccharides consisted of ?-1,4-linked repeated disaccharide units formed by conjugation of 1 mol D-glucuronic acid and 1 mol N-acetyl-D-galactosamine via ?-1,3 glycosidic bonds.CS shows high bioavailability and unique physicochemical properties.And there are a large number of active groups existing in molecules of CS,for example,hydroxyl and carboxyl groups,which makes CS easy for modification and processing.Thus CS and its derivations are regarded as an ideal kind of carrier material.In this research,hydrophilic skeleton material,CS is modified with hydrophobic DTX to obtain amphiphilic CS-DTX conjugation with self-assembling behavior.In addition,compared with normal tissues,a large amount of reductive glutathione(GSH)that can break disulfide bonds in molecules through reducing disulfide binds into sulfhydryl groups exists in tumor tissues,according to this,in this research,redox responsive DTX loaded CS-DTX conjugation based micelles are prepared through introducing disulfide bonds into micelle system to improve the solubilization and biovailability of DTX and promote the tumor accumulation of DTX for highly effective and lasting tumor treatment with decreased toxic and side effects.This research mainly includes following aspects:1.Synthesis of redox responsive amphiphilic CS-ss-DTX and characterization of its structure and self-assembling behavior:Disulfide bonds containing CS-Cys and carboxyl DTX(DTX-COOH)were successfully synthesized and CS-ss-DTX was successfully obtained through conjugating the two kinds of products together,the structure of intermediate products CS-Cys,DTX-COOH and final product CS-ss-DTX were characterized with FT-IR,MS,1H-NMR and other types of spectroscopy methods,determining the successful synthesis of targeted products.The substitution degree of CS-ss-DTX(11%)could be calculated through calculating the ratio of area of correlative peak in 1H-NMR.In order to characterize the self-assembling behavior of CS-ss-DTX in aqueous solution,the critical micelle concentration(CMC)of CS-ss-DTX was determined with pyrene fluorescence method.The result showed that the CMC of CS-ss-DTX(0.040?g/ml)is relatively low.2.The preparation of free DTX loaded CS-ss-DTX based micelles and the characterization of physicochemical properties of the micelles:DTX loaded CS-ss-DTX micelles were successfully prepared with dialysis method,the amount of DTX encapsulated in micelles was determined with high performance liquid chromatography(HPLC),it could be determined that the drug loading contend(DLC)of free DTX loaded CS-ss-DTX based micelles was 14%.The morphological properties,size distribution and zeta potential of this kind of micelles were characterized with Dynamic light scattering(DLS)and transmission electron microscope(TEM)respectively.The prepared micelles were spherical nanoparticles with homogeneous size distribution and proper surface charge,the average particle size and zeta potential of micelles were(259.67 ±2.06)nm and(-27±0.43)mV respectively.The comparison of size distribution of micelles in normal and reductive environment could prove redox response of micelles.The drug release behavior of free DTX loaded and blank CS-ss-DTX based micelles in medium with or without D,L-dithiothreitol(DTT)was characterized with dialysis method.It could be found from the results that in both mormal and reductive medium,the cumulative DTX release amount of DTX loaded CS-ss-DTX based micelles was higher than that of blank CS-ss-DTX based micelles(58.9%vs 26.8%,49.5%vs 19.2%),while the cumulative DTX release amount of two kinds of micelles in medium with DTT was much higher than that in medium without DTT(58.9%vs 49.5%,26.8%vs 19.2%).It could be confirmed from these results that the cumulative drug release amount of free DTX loaded CS-ss-DTX based micelles was pretty high in a long period of time and the drug release behavior of this kind of micelles showed significant redox response.3.The evaluation of in vitro and in vivo antitumor efficiency of CS-ss-DTX based micellesHuman breast cancer cell line(MCF-7)was applied to evaluate the in vitro antitumor efficiency of CS-ss-DTX based micelles.Because DTX molecule was not fluorescent,thus fluorescent coumarin-6(C-6)was encausulated into CS-ss-DTX based micelles to evaluate the cell uptake of CS-ss-DTX based micelles.The cell uptake of C-6 loaded CS-ss-DTX based micelles was characterized with fluorescence microscope.The results showed that the amount of C-6 loaded Cs-ss-DTX based micelles uptaken by MCF-7 cells was much higher than the amount of free C-6 uptaken by MCF-7 cells,demonstrating that CS-ss-DTX can accumulate in tumor tissues to sufficiently exert tumor inhibition efficiency.The in vitro cytotoxicity of free DTX loaded CS-ss-DTX based micelles to MCF-7 cells was evaluated with MTT(Methyl thiazolyl tetrazolium)method.The results showed that compared with free DTX,DTX loaded CS-ss-DTX loaded micelles showed enhanced cytotoxicity to MCF-7 cell line.After incubating MCF-7 cell line with free DTX loaded CS-ss-DTX based micelles for 48h,it could be observed with fluorescent microscope that the cell nucleus of MCF-7 cells showed significant collapsing,chromatin condensation and fragment.The density of cells decreased significantly and the growth of cells was greatly inhibited.Mouse melanoma cell(B16)-bearing female Kunming mice with suitable body weight were applied to evaluation the in vivo antitumor efficiency of free DTX loaded CS-ss-DTX based micelles.The results showed that compared with DTX solution,DTX loaded CS-ss-DTX based micelles showed improved tumor inhibition efficiency. |
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