Targeting Ability Of Tegafur-Loaded Polymer Microparticles Modified With DR5mAb To Hepatoma Cells In Vitro

Hepatocellular carcinoma is one of the most common tumors in the world, and the incidence and mortality in China are the highest around the world, for these reasons, research on liver cancer treatment is important and essential.Tegafur is one of the commonly used chemotherapy drugs for transarterial chemoembolization, with an extensive poisonous side effect, which is lack of specifical targeting. In recent years, ultrasound contrast agent as the drug delivery, applied widely in tumor treatment. Ultrasoud-targeted microbubble destruction can achieve local release for drug. Polymer microbubbles has a good stability in vivo, which can remain drug-loaded microbubbles, and provide a reliable sustained drug release. TNF-related apotosis inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily, found by Wiley, et al. It can induce apoptosis of tumour cells via combination with the cell membrane receptor. At present, there are five types of TRAIL receptor, death receptors (DR4 and DR5) contain death domains, which deliver signal of apoptosis, while the other three lack of death domains and block the apoptosis induced by TRAIL. In physiological conditions, the affinity of TRAIL with DR5 is significantly greater than with DR4, which can promote the apoptosis of tumor cells selectively. DR5 is highly expressed in most solid tumors, such as liver cancer, breast cancer, pancreatic cancer, prostate cancer, but not in normal tissues. It may provide a feasible theoretical basis for TDPM in therapy of liver cancer. In this research, biotin-avidin method was used to conjugate DR5mAb with TLM to observe the targeting performance to SMMC-7721. The study consists of the following two parts:PART I The preparation and general properties of Tegafur-loaded polymer microparticles modified with DR5mAbObjective To prepare a type of Tegafur-loaded and DR5mAb -targeted PLGA-COOH microparticles (TDPM) and explore its physico-chemical properties. Methods Tegafur-loaded microparticles(TLM) were prepared by using double emulsion, and the generated microparticles with DR5mAb were conjugated by carbodiimide method (EDC/NHS) and biotin-avidin method respectively. Light microscope and laser particle size analyzer were used to determine the physicochemical properties of the Tegafur-loaded microparticles. Flow cytometry was used to qualify and compare the connection rates of DR5mAb. Results TLM had a concentration of approximately 2.0×109～3.0×109/ml, a mean size of 676.38±90.93nm and a zeta potential of-(23.50±4.52)mV. Size distribution was 457.80nm-955.40 nm. The average efficiency of drug entrapment was 2.54%±±0.05%, and the mean rate of drug loading was 63.51%±1.26%. There was no significant difference in general properties between before and after sterilizing with 60Co irradiation. The connection rate of Biotin-avidin method was significantly higher than the EDC/NHS method (P＜0.05). Conclusions In this study, Tegafur-loaded and DR5mAb-targeted PLGA-COOH micropart--icles(TDPM) were successfully prepared, and this type of microparticles had a high drug entrapment efficiency, a well-distributed size and stable properties.PART Ⅱ Study on targeting ability of Tegafur-loaded polymer microparticles modified with DR5mAb to hepatocellular carcinoma in vitro.Objective To study the cultivation parameters on hepatoma cell line SMMC-7721 and hepatocyte line LO2 in vitro. The targeting performance of TDPM was verified by being incubated with SMMC-7721 cell lines and LO2 cell lines in vitro. Methods SMMC-7721 cells and LO2 cells were cultured in vitro, cells growth characteristics and the suitable cell concentrations were determined by cell counting. SMMC-7721 cells and LO2 cells were plated in culture dishes for 12h, then divided into 3 groups. The two cells were divided into 3 groups:TLM-7721 group, TDPM-7721 group, and TDPM-L02 group. In the TDPM-7721 group, a large number of microparticles conjugated to the SMMC-7721 cells, while no specific binding was found in the TLM-7721 group and the TDPM-L02 group.Result During the course of cultivation, SMMC-7721 cells and LO2 cells were inoculated with a concentration of 1×104/ml. No apparent microparticles were seen in TLM-7721 group and TDPM-L02 group, while a large number of microparticles gathered on the surface and to the surround of SMMC-7721 cell under fluorescence microscope in TDPM-7721 group. Conclusion Tegafur-loaded and DR5mAb-targeted microparticles can target to SMMC-7721 cells effectively in vitro, which may provide a new approach for the targeted treatment of hepatocellular carcinoma.