| Chemotherapy is still one of the main strategies for cancer therapy. However,non targeted chemotherapy drugs will accumulate in the whole body, and will cause great damage to the normal somatic cells. Targeted drug delivery system(TDDS), as a new drug delivery method, showed great potential for the delivery of anti-tumor drug. Targeting ligands to the drug delivery system can enhance the drug selectivity,reduce the drug dosage and increase the blood drug concentration in the tumor site,which has great prospects in biomedical and nano-biotechnological applications. How to release drug in specific tumor tissues and cells is another outstanding problem in the effective application of nano systems. With the excellent properties of the nano system prepared by the stimulus responsive materials, which can bypass the biological barrier to achieve targeted intracellular drug delivery. Among them, pH responsive nanoparticles have been paid much attention. In this study, the TDDS,which had the function of targeting and pH response was prepared, and the physical and chemical properties as well as the anti-tumor effect were explored. The main work of this study is as follows:1. Synthesis and characterization of the carrier: amphiphilic block copolymers consisting of polyethylene glycol(PEG) and poly(β-amino esters)(PAE) was synthesized using a Michael-step polymerization. 1H-NMR, gel-permeation chromatography(GPC) and gas chromatography(GC) proved that we synthesized the PEG-PAEs successfully, the average molecular weight of PEG-PAE block copolymer was optimized to 9237 Da with the polydispersity of 1.19, less organic residue, in accordance with the provisions of the pharmacopoeiaand.2. Preparation and characterization of the drug loaded micelles: blank micelles(BMS) and doxorubicin(DOX) loaded nanoparticles(PEG-PAEs/DOX nanoparticles,PDNs) were prepared by dialysis method, and the PDNs/aptamer(Apt) complex nanoparticles(PDANs) were prepared by electrostatic attraction. The critical micelle concentration of BMs was 6.3 μg/mL, and appeared good pH response capability; thePDANs has uniform size and good dispersion, the average particle size and zeta potential were 183.1±27.2 nm and +31.2±6.3 mV respectively, Apt combined with PDNs firmly; the study of drug loading property showed that the drug loading and encapsulation efficiency of DOX were 75.19% and 9.81% respectively; the results of in vitro drug release showed that, with the decrease of pH, the cumulative release rate gradually increased, showing pH dependent release properties.3. Biocompatibility evaluation: the biocompatibility of the carrier was evaluated from the cellular and animal level. The hemolytic ratio and cytotoxicity of BMs was investigated by hemolytic test and CCK-8 assay. The results demonstrated that in the drug concentration range, non hemolytic activity was detected, and the cytotoxicity level was 0 or 1, indicating good biological compatibility; the acute toxicity test showed no obvious toxicity in mice after the injection of BMs.4. Targeted performance and anticancer effects: The ability of targeting and anticancer effects of PDANs was evaluated from the cellular and molecular level. The distribution of intracellular drug showed that PDANs could enter the cells after 4 h,and the fluorescence intensity of PDANs in tumor cells is much higher than that in normal cells, showing good targeting function; cell morphology results showed that the loading drugs can promote the process of apoptosis more effectively; in vitro anti-tumor effect results showed that PDANs showed strong cell toxicity even at low concentration, and revealed better targeting performance to tumor cells; cell cycle distribution results showed that, The cell cycle of MCF-7 cells remained in the late stage of G0/G1 after mixed with PDNs and PDANS; fluorescence quantitative PCR results showed that the PDANs inhibited the expression of Bcl-2 mRNA, played a significant role in gene therapy.In summary, the nanoparticles with active targeting function and pH response properties are expected to be a new type of TDDS, which can be used in the field of targeted breast cancer therapy. |
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