Establishment And Application Of Targeted Drug Delivery System Using Maleimide Functionalized Biopolymer

With the growing of environmental pollution and the increase of the pressures of modern life, cancer has become a common disease of modern society. Surgery, radiation therapy, chemotherapy, Chinese medicine treatment and targeted therapy are the major methods for cancer treatment. Among them targeted therapy catches more and more attention of people for its therapeutic effect and small damage to human body.γ-poly(glutamic acid)(γ-PGA) is a natural polymer which can be prepared by bacterial fermentation. It is non-toxic, biodegradable and rich in active sites, which makes it be widely used in agriculture, medicine, chemical industry and other fields. In this article, we used Bacillus subtilis strain DL for producingy-PGA, and prepare a derivative ofy-PGA (PGA-Asp) by connecting aspartic acid on it. We called this derivative as biological platinum and it can be used as a vector of cisplatin. In order to increase the capacity of targeting tumor tissue of biological platinum, we first connected a six carbon maleimide linker(NME) to it by amidation reaction, and then connected a tumor targeting peptide GNGRAHAC by addition reaction between linker and targeting peptide. This new vector is also loaded with cisplatin. We called it tumor targeting biological platinum(NGR-Mal-PGA-Asp-Pt).Our studies are summarized as follows:1. Preparation and characterization of Maleimide functionalized biopolymer (Mal-PGA-Asp)The γ-PGA yield of Bacillus subtilis strain DL could reach33g/L with average molecular weight of570kDa and purity of98.36%. Then we degraded it by high temperature and pressure to obtain small molecular weight γ-PGA, which molecular weight ranged from30to60kDa. The optimized degradation condition is pH3.0and10minutes. We prepared PGA-Asp by amidation reaction and analysed its amino acid composition. The result showed that the composite of glutamate via aspartic acid is2.68:1. Then we connected NME to PGA-Asp to form Mal-PGA-Asp and this action was confirmed by SDS-PAGE.2. Preparation and characterization of tumor targeting biological platinum (NGR-Mal-PGA-Asp-Pt) Mal-PGA-Asp connected tumor targeting peptide GNGRAHAC by addition reaction and loaded cisplatin by substitution reaction. Under the inverted fluorescence microscope, we could see that both FITC marker peptide FITC-GNGRAHAC and FITC-NGR-Mal-PGA-Asp-Pt had a specific interaction between SKBR-3cells (high expression of CD13) and Hela cells (low expression of CD13). The content of cisplatin of NGR-Mal-PGA-Asp-Pt measured by UV spectrophotometry with o-phenylenediamine(OPD) was385.8μg/ml. The cisplatin loading rate was calculated as23.8%.3. Cytotoxicity evaluation of tumor targeting biological platinum in vitroWe incubated SKBR-3and Hela cells with Pt, PGA-Asp-Pt or NGR-Mal-PGA-Asp-Pt for two hours and five minutes, respectively, and tested their survival rates by MTT assay. The result showed that when incubated for two hours, Pt had more cytotoxicity to either SKBR-3or Hela cells than PGA-Asp-Pt and NGR-Mal-PGA-Asp-Pt, and when incubated for five minutes, NGR-Mal-PGA-Asp-Pt had more cytotoxicity to SKBR-3cells than PGA-Asp-Pt and Pt, while all three of them showed no significant difference in cytotoxicity to Hela cells. Flow cytometry indicated that NGR-Mal-PGA-Asp-Pt had21%higher binding rate to SKBR-3cells than to Hela cells.In summary, the present study constructed a tumor-targeting drug delivery system based on poly(glutamic acid). The system showed both passive and active targeting ability in vitro. It is expected to screen new tumor targeting drugs by connecting different tumor targeting motifs and drugs on this system.