| It is well known that cancer has become one of the most serious threats to human at present, and more than 20 million people die of cervical cancer worldwide each year. In the currently clinical treatments to cervical cancer, chemotherapy drugs play a very important role in killing local tumor tissues and removing systemic cancer cells. However, these drugs, owing to its drastic side effects, often bring cancer patients with great pain. Thus, it is necessary to design and develop a novel anticancer drug.Targeting drug delivery system (TDDS) with effective therapy and lower side effect has been one of the hottest topics in current pharmacology. TDDS is mainly composed of targeting ligand, carrier, anticancer drug and corresponding technique. The targeting effect and binding sites of TDDS is determined by the targeting ligand, and the drug loading and delivery is depended on carrier with biocompatibility, biodegradability and processing feasibility. Anticancer drug gives TDDS specific anticancer effects, and the technology is the core element of TDDS.After reading a large number of literatures, we have designed the research program of this paper. Because of over-expression of folate receptor (FR) on the surface of cancer cells, we selected its corresponding ligand folic acid (FA) as targeting ligand. In order to delivery drug to targeting site and extend its duration of action, we chose poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] with good physical-chemical properties and biological characteristics as drug carrier. The DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA-PEG-P(HB-HO) NPs) were prepared by W1/O/W2 solvent extraction/evaporation method, adopting doxorubicin (DOX) as the model anticancer drug. Their physicochemical properties, targeting effect and pharmacodynamics were also investigated.The DOX/FA-PEG-P(HB-HO) NPs have been prepared in two stages. First, an original FA-PEG-P(HB-HO) conjugate with 86.39% yield was synthesized by amide bonds, and its chemical structure was confirmed by FTIR and’H NMR spectroscopy. The DOX/FA-PEG-P(HB-HO) NPs were then prepared by W1/O/W2 ultrasonic emulsification method. The correlative parameters of the method were optimized by L9(34) orthogonal design, and the optimal conditions were as follows:the concentration of DOX, FA-PEG-P(HB-HO), PVA, Tween80 and Span80 were 10%,6.5%,3%,5% and 1%(W/V), separately. The volume ratio of oil phase/internal water phase was 3:1, and the ultrasonic output and time was 200W and 180s, respectively. The average size, drug loading capacity and encapsulation efficiency of such NPs were found to be 241.6±9.3nm,29.6±2.9% and 83.5±5.7%. The in vitro release profile displayed that nearly 80% DOX was released in the first 11 days and its release formulation fitted to Higuchi equation. There were no great changes in the properties of the NPs when they stored for 6 months at 4℃. Therefore, the DOX/FA-PEG-P(HB-HO) NPs have excellent stability.The cellular compatibility, cellular targeting, cytotoxicity and apoptosis of these NPs to HeLa cells were investigated by in vitro cell line experiments. The results of MTT showed that no toxicity was observed after incubation with blank P(HB-HO) NPs and blank FA-PEG-P(HB-HO) NPs, and the two polymers displayed satisfactory cellular compatibility. The DOX/FA-PEG-P(HB-HO) NPs (IC50=0.87μM) displayed greater cytotoxicity to HeLa cells than other treated groups. The intracellular uptake tests of the NPs in vitro and fluorescence observation showed that the DOX/FA-PEG-P(HB-HO) NPs were efficiently taken up by HeLa cells. In addition, the results of microscope observation and flow cytometry exhibited that the apotosis of HeLa cells treated with 50μM DOX/FA-PEG-P(HB-HO) NPs was 84.25±2.43%, with significant difference (p<0.05). These data in vitro demonstrated that DOX/FA-PEG-P(HB-HO) NPs could target the HeLa cells efficiently and lead to a strong cytotoxicity due to high affinity of FA and FR.The tissue distribution, targeting effect and anti-tumor activity of DOX/FA-PEG-P(HB-HO) NPs were evaluated by in vivo experiments. After building HeLa xenograft tumor models, the BALB/c nude tumor-bearing mice were randomly divided into several groups. The results of tissue distribution and targeting tests showed that the half-life of the DOX was prolonged, its bioavailability was enhanced and its toxicity to heart was greatly reduced when DOX was prepared as NPs, the DOX concentration of DOX/FA-PEG-P(HB-HO) NPs group was much higher than other treated groups, and the DOX concentration in the tumors of this group was 10.81 and 3.33 times higher than free DOX group and DOX/P(HB-HO) NPs group, respectively. Thus, the DOX/FA-PEG-P(HB-HO) NPs have good targeting to HeLa tumors. In vivo anti-tumor activity demonstrated that the tumor weight of DOX/FA-PEG-P(HB-HO) NPs group was much smaller than control group (p<0.01), and the inhibit tumor rate was 76.91%. The final mean tumor load of the NPs was 178.91±17.43 mm3, remarkably smaller than other treated groups. Furthermore, the body weight change of DOX/FA-PEG-P(HB-HO) NPs group was very stable. Histopathology observations of tumors displayed that necrosis areas after DOX/FA-PEG-P(HB-HO) NPs treatment were severe. All these results have illustrated that DOX/FA-PEG-P(HB-HO) NPs are effective in targeting treatment of tumors.In conclusion, the technique of synthesizing FA-PEG-P(HB-HO) was built, and the product was achieved for the first time. Moreover, the technique and technical parameters of preparing DOX/FA-PEG-P(HB-HO) NPs were established, and a novel targeting nano-agent for treatment of cervical cancer was developed. The results of in vitro and in vivo experiments have illustrated that the NPs with little side effects are effective in the ability of active targeting, and are remarkable in treating cervical cancer. The new TDDS may represent a promising prospect in future. |
PDF Full Text Request