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

Malignant tumors are severely influent to human life and healthy, recent studies have been paid close attention to the mechanism in tumor initiation and progression. Cisplatin (CDDP) was one of the most popular chemotherapeutics in clinics, which could effectively inhibit many tumor cell lines'proliferation and has higher efficiency. However, CDDP had many disadvantage of short half-life, low solubility, none target therapy, and especially severe side effects like nephrotoxicity, neurotoxicity, myelosuppression and stomach-intestine toxicity, those weakness restricted its application in the clinics.Polymers used as carriers in drug delivery were more and more popular in recent year, they can interact with drugs to increase its solubility, improve the pharmacokinetics, passive target delivery by the enhanced permeability and retention effect (EPR effect). Aspartylated PGA (PGA-Asp) was biodegradable, nontoxic, non-immunogenic, biocompatible, and could be used as a potential drug delivery carrier, CDDP was loaded into the carrier by complexation with pendant carboxylic ions. However, the polymer-CDDP conjugate was not positively targeted drugs, since drug delivery carrier does not exhibit any targeting properties.The design of smart targeted drug delivery systems that deliver drugs to specific cancer cells will give rise to cancer treatments with better efficacy and lower toxicity levels. The epidermal growth factor receptor (EGFR) targeting peptide (TP13) was conjugated to Maleimide functionalized biopolymer (Mal-PGA-Asp) to obtain the targeting carrier (TP13-Mal-PGA-Asp). Cisplatin was finally loaded by complexation to form a biocompatible and tumor targeted therapeutic drug (TP13-Mal-PGA-Asp-Pt). We investigated the toxicity and antitumor activity of TP13-Mal-PGA-Asp-Pt both in vitro and in vivo indicated that it can significantly inhibit tumor proliferation in vivo, while dramatically decrease side effects. Our studies are summarized as follows:1. N-(maleimidohexanoyl)-ehylenediamine (NME) as the linker molecule is characterized by the end of the maleimide groups while the other end of the amino. Mal-PGA-Asp was synthesized from an amidation reaction between PGA-Asp and N-(maleimidohexanoyl)-ehylenediamine (NME). The Asp loading, repeat units (glutamic acid residue), the mean molecular weight (Mw), RMS radius (Rw) and polydispersity index (Mw/Mn) of PGA-Asp were82%,51,1.14x104Da,31.1nm, and1.792±0.021, respectively. The1H NMR spectrum of Mal-PGA-Asp showed two characteristic peaks, one attributed to PGA-Asp (δ=2.31ppm) and the other to NME (δ=6.75ppm). Mal-PGA-Asp was a versatile drug delivery carrier and can conjugate to a variety of ligands in a site-specific manner.2. The epidermal growth factor receptor targeting peptide (TP13) was designed according to the sequence reported in the literature, which cysteine was added at amino terminal.The conjugation efficiency and CDDP-loading content of various NME loadings of Mal-PGA-Asp were analyzed, the results showed that Mal-PGA-Asp3was suitable as a drug carrier for targeted therapeutic drug. The NME loadings, TP13conjugation efficiency, CDDP-loading content, particle sizes and the half-maximal release time of TP13-Mal-PGA-Asp-Pt were24%,45%,18±2%,87±28nm and15h. The sustained release kinetics of CDDP from TP13-Mal-PGA-Asp3-Pt ensures that the therapeutics will not completely leach out of the drug carrier during circulation and, therefore, can increase therapeutic efficacy.3. Inverted fluorescence microscope and flow cytometry confirmed that a specific interaction existed between SMMC-7721cells (high expression of EGFR), the results indicate that TP13-Mal-PGA-Asp3is a promising targeted drug carrier. Confocal microscopy and transmission electron microscopy confirmed that TP13-Mal-PGA-Asp3-Pt had a specific interaction with SMMC-7721cells, therefore TP13-Mal-PGA-Asp3-Pt could be used as a targeting therapeutic drug for EGFR-positive tumors. TP13-Mal-PGA-Asp3-Pt inhibited SMMC-7721cells Bcap-37cells, HL-7702cells and SH-SY5Y cells growth in a dose-dependent manner and it can induce morphological changes, which suggesting that the killing mechanism may be the same with CDDP. The IC50value of TP13-Mal-PGA-Asp3-Pt (58.54±16.92) against SMMC-7721cells was significantly lower than PGA-Asp-Pt (92.75±8.60) incubated at24h, the cytotoxicity of TP13-Mal-PGA-Asp3-Pt against SMMC-7721cells were higher than that of CDDP and PGA-Asp-Pt incubated at2h; this indicates that the specific binding between the TP13-Mal-PGA-Asp3-Pt and SMMC-7721cells have a significant role to improve its cytotoxicity.4. The in vivo toxicity investigation in KM mice showed that when the cumulative administration concentration of12mg/kg, CDDP treatment had significant system toxicity: mortality rate was73%, average body weight, the erythrocyte and WBC were dramatically decreased; while the toxicity of PGA-Asp-Pt and TP13-Mal-PGA-Asp3-Pt was not detected. The in vivo anti-tumor activity evaluation in human hepatoma cell SMMC-7721-grafted mice showed that when the cumulative administration concentration of50mg/kg, PGA-Asp-Pt and TP13-Mal-PGA-Asp3-Pt treatment group after42days had significant higher anti-tumor activity than the control; the average body weight of PGA-Asp-Pt treatment group after36days was dramatically decreased, the average body weight of TP13-Mal-PGA-Asp3-Pt treatment group increased about17%；AST and UREA of PGA-Asp-Pt treatment group were significantly increased,biochemical parameters of TP13-Mal-PGA-Asp3-Pt treatment group had no significant changes. Histological renal pathology sections showed that normal architecture of kidney with treatment PGA-Asp-Pt was changed and normal architecture of kidney with treatment TP13-Mal-PGA-Asp3-Pt was maintained.Most interestingly,a comparison between tumor volume and body weight change indicated that TP13-Mal-PGA-Asp3-Pt displayed more antitumor activity with the least effect on mouse body weight change than PGA-Asp-Pt.5.Human hepatoma cell SMMC-7721-grafted mice were treated intraperitoneally with FITC-TP13-Mal-PGA-Asp3-Pt,sections of tumor showed a green fluorescence image,which indicated TP13-Mal-PGA-Asp3-Pt could be used as a targeting drug for EGFR-positive tumors in vivo. Platinum recvery of Kunming mouse kidney and liver with or without treatment drug by graphite furnace atomic absorption spectrophotometer was91.74%and96.22%,109.48%and103.48%,respectively；this shown that the method of determination of platinum content in organs with high accuracy.Drug biodistribution of biological platinum and targeted biological platinum was detected by the method.CDDP distribution rate in the kidney when delivered as TP13-Mal-PGA-Asp3-Pt was49%,while the distribution rate of PGA-Asp-Pt was41%； CDDP accumulation in the tumor when delivered as TP13-Mal-PGA-Asp3-Pt had higher than that of PGA-Asp-Pt.This was the reasons that targeted biological platinum enhanced antitumor activity with low rates of toxicity.Due to active targeting,targeted biological platinum had higher antitumor activity with low rates of toxicity.Targeted biological platinum may have a potential targeting therapeutic drug for hepatocellular carcinoma.