Studies On A54 Peptide Functionalized Chitosan-stearic Acid Conjugate Micelles For Tumor Targeting Therapy

The delivery of all of administrated chemotherapeutics into tumor cells by drug delivery system is an extreme object for tumor targeting therapy to enhance the curative effect and eliminate the side effect. However, until now, the targeting delivery have only partial been realized by passive targeting, which was called "enhanced permeability and retention (EPR)" effect, and only few targeting delivery system was commercialized. Here, we designed and synthesized a hepatocarcinoma-binding peptide (A54 peptide, which was identified from a phage-display random peptide library) functionalized and PEGylated stearic acid grafted chitosan (A54-PEG-CS-SA) micelles for tumor active targeting therapy of doxorubicin.In our previous research, stearic acid grafted chitosan (CS-SA) micelles were synthesized, which could target to tumor tissue by EPR and uptake by tumor cells rapidly. The CS-SA micelle delivery system presented high cytotoxicity than itself of drug, however it indicated same high cytotoxicity in normal cells and faster elimination in vivo. The PEGylated CS-SA (PEG-CS-SA) micelles could significantly reduce the uptake by macrophage, while the antitumor activity was lowered. Herein, the active targeting of PEG-CS-SA micelles was designed. A hepatocarcinoma-binding peptide, AGKGTPSLETTP peptide (A54) was used as homing peptide, which was identified from a phage-display random peptide library by in vivo panning, and the most effective peptide specific to the human hepatoma cell line BEL-7402. The synthesized A54-PEG-CS-SA could self-assemble to form nano-scaled micelles in aqueous water, the particle size was 77.85±8.31 nm, and the zeta potential was 40.4±1.9 mV. After loading DOX, the particle size was still sub-100 nm while the zeta potential reduced. Moreover, the drug encapsulation efficiency was above 79.44%. In vitro DOX release from A54-PEG-CS-SA micelles could be prolonged to 72 h. Compared with CS-SA and PEG-CS-SA micelles, the particle size of A54-PEG-CS-SA micelles, whether the blank micelles or drug-loaded micelles, increased slightly and were all sub-100 nm. Moreover, the drug encapsulated ability and in vitro DOX release behavior have no significant difference. Using the human liver tumor cell lines (BEL-7402 and HepG2 cells) and nomarl liver cells (BRL-3A cells) as the model cell lines, the cytoxicity and the cellular uptake of the micelles were investigatived. The resulted indicated that the A54-PEG-CS-SA micelles with relatively high biocompatibility presented special internalization ability into human hepatoma cells (BEL-7402) when the cells were co-incubated with normal liver cells. And the quantitative cellular uptake of the micelles on BEL-7402, HepG2 and BRL-3A cells shown that the cellular uptake of A54-PEG-CS-SA micelles increased by 2 folded in BEL-7402 cells compared with the other two micelles. Furthermore, with the functionalization of A54 peptide, the cytotoxicities of A54-PEG-CS-SA/DOX against BEL-7402 cells increased by approximately 3.2-3.4 folded compared with CS-SA/DOX and PEG-CS-SA/DOX micelles. Using the BEL-7402 human hepatoma bearing nude mice as the model animal, the in vivo bio-distribution of DiR-loaded micelles and anti-tumor activities of drug-loaded micelles were studied. The resulted showed that A54-PEG-CS-SA micelles had high distribution ability to liver and hepatoma tissue in vivo, which suggested that it may be employed as a promising hepatoma active-targeting carrier via A54 peptide mediation. In vivo anti-tumor activity results showed that A54-PEG-CS-SA micelles loading doxorubicin treatments suppressed tumor growth more effectively and reduced toxicity compared with commercial adriamycin injection. Overall, the study suggests that, with the mediation of homing peptide A54, the A54-PEG-CS-SA has promising potential to cure the tumor for most efficient and special tumor targeting, anti-tumor effects enhancement and systematic toxicity reduction.