| With the increase of the morbidity and mortality rate of cancer in recent years, it has become a major health problem in the world wide. Surgery, radio therapy and chemotherapy are major therapeutics in treatment of cancers and chemotherapy is the most common therapeutic approach. Docetaxel (DTX), one kind of the taxanes, could restrain the depolymerization of microtubule and block the proliferation of tumor cells, which has been widely used in treatment of metastatic breast cancer, non-small-cell lung cancer, prostate cancer, ovarian cancer and so on. DTX has a 2-4 times higher activity than that of pactaxel (PTX).Despite the favorable anti-tumor effects, many limitations such as poor aqueous solubility and systematic toxicity because of non-specificity in distribution have restricted the clinical use of DTX. The use of Tween 80 in the clinical formulations of DTX had caused severe side effects. What’s worse, the cancer developed resistance to DTX due to the increasing usage of taxanes, which became another serious issue. The major mechanism of cancer cell resistance is the excessive expression of transported protein p-gp on cell membrane.With the development of polymer science, using the high molecular polymer as drug carriers to load hydrophobic drugs to improve their aquous solubility and anti-tumor activity has been the focous. The amphiphilic block polymer could self-assemble into micelles with hydrophobic core and hydrophilic shell, which could carry hydrophobic drugs in the core. There are two kinds of drug load modes of polymer micelles, physically loading and chemically conjugating. The later one is covalently conjugating drugs to polymers to form polymer-drug conjugate, and hydrophobic drug takes part in the assembly of micelles. Chemically conjugating could improve the drug loading amount and stability. What’s more, covalently conjugating drugs to polymer by the environment sensitive bond could concentrate drugs in tumor tissues and improve therapeutic effect, which is one of the innovations in drug delivery system. Based on the special environment in tumor tissues such as the lower pH, enzymes and redox condition, some chemical bonds could be used to prepare environment sensitive drug delivery system.In this article, we aimed to synthesized one reduction sensitive polymer-drug conjugate that can self-assemble into micelles in water and carry drugs. The low-molecular weight biodegradable methoxy poly (ethylene glycol)-poly (D, L-lactide-co-glycolide) (mPEG-PLGA, PP) is chosen as polymeric skeleton to be conjugated with docetaxel (DTX) by disulfide bond (PP-SS-DTX) to construct the reduction-sensitive polymer-drug conjugate. The synthesized conjugate will be utilized to entrap DTX or verapamil (VRP) to design the multi-fuctional drug delivery system.The research includes the following aspects.1. Synthesis and characterization of PP-SS-DTX conjugate.The PP-SS-DTX conjugate was synthesized via three steps taking a disulfide-containing agent (3,3’-dithiodipropionic acid, DTDP) as linker between PP and DTX. The structure of PP-SS-DTX was characterized by 1H-NMR and FT-IR. In addition, the critical aggregation concentration of PP-SS-DTX determined by pyrene fluorescence probe technique was 10.20μmol/L.2. Evaluation of the reduction-sensitive and dual-mode drug loading PP-SS-DTX/DTX micelles.The dual-mode drug loaded PP-SS-DTX/DTX micelles were prepared via membrane-dialysis method. The morphology and the size of PP-SS-DTX/DTX micelles were further determined by TEM and DLS. The PP-SS-DTX/DTX micelles were spherical in morphology with size of 112.3 nm. PP-SS-DTX/DTX micelles had higher drug loading amount of (14.65±0.71)%. The hemolysis assay reveals the good blood compatibility of PP-SS-DTX/DTX micelles. In order to investigate the reductive sensitivity of PP-SS-DTX/DTX micelles, dithiothreitol (DTT) is added into the release medium. The drug release assay declared that compared with DTX solution, the PP-SS-DTX/DTX micelles possessed many merits, including sustained drug release mode, reduction sensitive drug release property and programmed drug release process. The human breast cancer cell MCF-7 and melanoma cell B16F10 were chosen to carry the in vitro cytotoxicity assay and cellular uptake assay. The PP-SS-DTX/DTX micelles are more cytotoxic than that of free DTX solution for both MCF-7 and B16F10 cancer cells. Since the DTX are nonfluorescent, the hydrophobic fluorescent dye coumarin-6 (C-6) was used as a probe to imitate DTX to prepare the PP-SS-DTX/C-6 micelles. The cellular uptake of micelles was assessed by fluorescent inverted microscope (ECLIPSE-Ti, Nikon) in a qualitative way and flow cytometer (BD FACSAria III) in a quantitative way. The cellular uptake rate of PP-SS-DTX/C-6 micelles by both MCF-7 and B16F10 cancer cells was higher than that of C-6 solution. The high cellular uptake rate by cancer cells ensured that the drug was concentrated in pathological tissue, which was the main factor for improvement of anti-tumor activity.3. Evaluation of the dual-mode and dual-drug loading PP-SS-DTX/VRP micelles on circumventing multi-drug resistance.The thin film dispersion-probe untrasonic method was used to prepare the PP-SS-DTX/VRP micelles. Dual-mode refers to the two kind drug loading mode, covanlently conjugating and physically loading. Dual-drug loading indicates the codelivery of two kinds of drugs, docetaxel (DTX) and verapamil (VRP). VRP was the inhibitor of p-gp, which could combine with p-gp and inhibite its transporting function.In the preparation of micelles, the ultrasonic time was evaluated based on the drug loading amount (DL) and entrapment efficiency (EE) of VRP. The DL and EE of VRP was decrease with the increase of ultrasonic time. The morphology and the size of PP-SS-DTX and PP-SS-DTX/VRP micelles were determined by TEM and DLS. The PP-SS-DTX and PP-SS-DTX/VRP micelles were spherical in morphology with size of (79.3±1.2) nm and (78.3±4.6) nm, respectively. The drug loading amount of DTX and VRP in PP-SS-DTX/VRP micelles was (8.94±0.72)% and(5.66±1.60)%. The entrapment efficiency of VRP was (53.49±10.96)%. In order to investigate the reductive sensitivity of PP-SS-DTX/VRP micelles, dithiothreitol (DTT) is added into the release medium and both the DTX and VRP are found the reduction sensitive release property. The human breast cancer cell MCF-7 and drug resistant breast cancer cell MCF-7/ADR were chosen to study the anti-drug resistance property of PP-SS-DTX/VRP micelles. The in vitro cytotoxicity assay, cellular uptake assay and the cell apoptosis assay were carried out on the two cell lines. The PP-SS-DTX and PP-SS-DTX/VRP micelles were more cytotoxic than that of DTX solution on the two cell lines. In addition, the PP-SS-DTX/VRP micelles were more cytotoxic than that of PP-SS-DTX micelles on MCF-7/ADR cells. Because the DTX and VRP were nonfluorescent, one hydrophobic fluorescent dye rhodamine 123 (RH 123), the substrate of p-gp, was used to sign micelles to conduct the cellular uptake assay. The MCF-7 cells had a similar cellular uptake rate for PP-SS-DTX micelles and PP-SS-DTX/VRP micelles, and both were higher than that of RH 123 solution. However, MCF-7/ADR cells had barely uptake to RH 123 solution. The cellular uptake rate of PP-SS-DTX/VRP micelles by MCF-7/ADR cells was higher than that of PP-SS-DTX micelles. The cell apoptosis assay revealed that more apoptosis cells were found after incubated with PP-SS-DTX micelles and PP-SS-DTX/VRP micelles than that of DTX solution. In conclusion, the PP-SS-DTX/VRP micelles possess many merits including inhibiting the function of p-gp, improving the cellular uptake rate by resistant cells, accelerating the apoptosis process, and improving the anti-tumor activity of DTX.4. In vivo pharmacokinetic study of PP-SS-DTX micelles.In this study, the wistar rats were chosen as the model animals and HPLC method was selected to determine the concentration of DTX in plasma after tail vein i.v. administration. The in vivo pharmacokinetic property of PP-SS-DTX micelles was represented via the plasma drug concentration-time curve by utilizing DTX solution as control. Appropriate compartment model and pharmacokinetic parameter was determined by DSA 2.0 software. The result showed that plasma drug concentration-time curve of PP-SS-DTX was more durable than that of DTX solution. The pharmacokinetic characters of both the PP-SS-DTX micelles and DTX solution were consistent with two-compartment model. The PP-SS-DTX micelles had a lower clearance rate (CL), longer half elimination life and residence time compared with that of DTX solution, which assured the sustained drug release in vivo and high activity.In summary, one reduction sensitive polymer-drug conjugate, PP-SS-DTX, was firstly synthesized, which can self-assemble into micelles to load DTX or VRP to improve water solubility and anti-tumor activity of DTX, and even overcome multi-drug resistance of cancer cells. Therefore, this study has a practical significance for improving solubility of hydrophobic drugs and overcoming multi-drug resistance of cancer, and provided some research foundation in overcoming multi-drug resistance of cancer. |
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