| Cancer remains the major disease threating human life and health, though the recent advances of early diagnosis technique and therapeutic intervention technique. Cancer therapy is one of the most challenging problems worldwide. Chemotherapy is one of the main treatments for cancer, especially for the metastatic tumors and those which could not be treated by topical therapy such as surgery or radiation therapy. However, some side effects of chemotherapy drug were inevitable, owing to the non-specific drug distribution in vivo.Polymeric micelles, self-assembled from amphiphilic block copolymers in aqueous media, is a nano-drug delivery carrier with core-shell structure. Polymeric micelles exhibit many advantages, such as improving the solubility of insoluble drug, avoiding the absorption of plasma protein, protecting drugs when delivered, prolonging the systemic circulation time, passive targeting in the tumor tissue by EPR effect and lowering the toxicity to normal tissues. Polymeric micelles have been extensively investigated to deliver antitumor drugs since 1990s. In 2007, PEG-PLA/PTX micelle (Genexol-PM(?)) came into the market and was used to treat recurrent or metastatic breast cancer and non-small cell lung cancer. Genexol-PM(?) showed obvious accumulation in tumor tissues, better therapeutic effects with less hypersensitivity and other side effects compared to Taxol(?).Physical encapsulation and chemical conjugation were the common drug-loading patterns for polymeric micelles. The advantages of physical encapsulation were simplified preparation process and controllable conditions, while lower drug loading and burst release were the drawbacks. Less burst release and accurate loading efficiency were the advantages of chemical conjugation. While this method suffered from the possibility of slower drug release and decreased drug activity after micelles were accumulated in the tumor tissues, since the chemical bond between drug and carrier was ruptured slowly.In this work, two pH-sensitive conjugates, Pluronic P123-single-hydrazone-Docetaxel (P123-s-hyd-DTX) and Pluronic P123-double-hydrazone-Docetaxel (P123-d-hyd-DTX), prepared based on previous methods, were employed as carrier to further encapsulate docetaxel (DTX). Dual drug-loading stategy was employed simultaneously to improve the drug loading. In addition, controllable drug release was another advantage of double drug-loading: encapsulated DTX was released firstly to achieve therapeutic concentration and then conjugated DTX was released to maintain therapeutic concentration. Owing to the pH-sensitivity of two conjugates, DTX release was less when micelles were in blood circulation. While the hydrazone bonds would be ruptured and DTX was released rapidly after micelles were accumulated into tumor tissues.The research content of this work included three main parts:(1) The establishment of determination method in vitro of DTX content, (2) The preparation and characterization of Pluronic P123-single-hydrazone-Docetaxel/Docetaxel (P123-s-hyd-DTX/DTX) micelles and Pluronic P123-double-hydrazone-Docetaxel/Docetaxel (P123-d-hyd-DTX/DTX) micelles, (3)The in vitro and in vivo evaluation of P123-s-hyd-DTX/DTX micelles and P123-d-hyd-DTX/DTX micelles. The main methods and results were as follows:1. The determination method of DTX contentThe determination method in vitro of DTX content was established by ultraviolet-visible spectroscopy. The standard curve, precision within-day and between-day, method recovery and recovery were assessed, respectively. There was a good linear relationship between concentration and absorbance when the concentration of DTX was between 5μg/mL and 40μg/mL. In addition, the precision and recovery met the analysis requirement.2. The preparation and characterization of P123-s-hyd-DTX/DTX micelles and P123-d-hyd-DTX/DTX micellesPolymeric micelles were prepared by the thin-film hydration method. The amount of conjugates and DTX, the volume of normal saline, hydration time and hydration tempreture were selected for single-factor studies. Then orthogonal experiment was designed to achieve optimal formulations and parameters based on the results of the single-factor studies. Three batches of P123-s-hyd-DTX/DTX micelles and P123-d-hyd-DTX/DTX micelles were prepared respectively, according to the optimal prescription and technology. The morphology, particle size, zeta potential, encapsulation efficiency (EE) and drug loading (DL) were measured, respectively. The dynamic light scattering (DLS) was employed to determine the particle sizes of two micelles when incubated with plasma.The morphology of both micelles was uniform and round without adhesion. The particle size of P123-s-hyd-DTX/DTX micelles was (121.63±3.72) nm, PDI was 0.32, Zeta potential was (-7.50±0.55) mV, the EE was (88.30±2.11)％and DL was (14.87±0.10)％ respectively. While the particle size of P123-d-hyd-DTX/DTX micelles was (110.37±9.03) nm, PDI was 0.39, Zeta potential was (-4.27±0.34) mV, the EE was (83.06±2.73)％and DL was (12.64±0.12)％, respectively. The results of plasma stability test showed that the two micelles could remain stability in plasma for 12 h under laboratory conditions.3. The in vitro and in vivo evaluation of P123-s-hyd-DTX/DTX micelles and P123-d-hyd-DTX/DTX micellesThe pH-sensitivity of the two micelles was investigated by in vitro release study. In addition, the pH responsiveness of the two micelles was assessed. In vitro cytotoxicity on B16F10 melanoma cell of Pluronic P123 and two micelles was studied by MTT assay and the effects were compared with DTX solution (dissolved in DMSO). The tumor-bearing mices were employed to evaluate the in vivo anti-tumor effects of two micelles, with Docetaxel Injection as positive control. The changes of tumor celles in each group were observed by HE staining. The in vitro hemolytic test of two micelles was conducted in this work. After in vivo anti-tumor test, HE staining of normal tissues was conducted to observe the physiological change.The results showed that the release profiles of the two micelles exhibited pH sensitivity. Especially under pH 6.5, the release amount of P123-d-hyd-DTX/DTX micelles was much more than P123-s-hyd-DTX/DTX micelles, indicating that P123-d-hyd-DTX/DTX micelles showed higher pH responsiveness than P123-s-hyd-DTX/DTX micelles. The results of MTT assay indicated that the two micelles showed dose-dependent antiproliferative activity. When the concentration of DTX was low, the cytotoxicity of DTX was much higher than two micelles (p<0.05). With the increase of DTX concentration, both micelles exhibited comparable cytotoxicity with free DTX. Compared to NS, both micelles showed significantly obvious antitumor effects (p<0.01) without weight lose. The results of HE staining for tumor tissues showed massive tumor cells necrosis after accepting Docetaxel Injection or P123-s-hyd-DTX/DTX micelles or P123-d-hyd-DTX/DTX micelles, while the anti-tumor effects of the two micelles were much superior. The results of hemolytic test and HE staining for normal tissues indicated the two micelles could be used for intravenous administration without hemolysis or normal tissues damages.In summary, the two polymeric micelles based on dual drug-loading stategy exhibited excellent anti-tumor activity and less tissue toxicity, which laid test foundation for the further study of novel DTX micelles. |
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