Design,Synthesis And Biological Properties Of Nano Drug Delivery System Based On Elastin-Like Polypeptide

With the development of genetic engineering and immune therapy,more and more functional peptides and proteins are applied in the nano-drug delivery system for cancer therapy.Elastin-like polypeptides(ELPs)based on the structural motif found in mammalian tropoelastin consist of a repeated pentapeptide sequence VPGXG.Meanwhile,ELPs have good biocaompatibility,biodegradable,low immunogenicity and a favorable pharmacokinetic profile.More important,ELPs can be easily expressed at high yield from Escherichia coli and rapidly purified by exploiting their phase transition behavior in a non-chromatographic way.Using gene recombination technology,several functional peptide fragments can be fused in genetic level and then expressed as multi-function fusion proteins.In addition,control over the genetic code allows the numbers and locations of reactive sites for small molecular to be precisely specified.Due to their active and rapid dividing and growth,the pathological of cancer cells is different from normal tissues,such as the defective angiogenesis,enhanced permeability and retention of tumor vessels,anaerobic metabolism,cell transformation,growth out of control,weak acid environment outside the cells due to the increased ability of transmembrane pH adjust and up expression of the cancer cell surface related receptor because of the large demand of nutrient.Taking advantage of the specificity of tumor microenvironment,one can achieve selective targeted drug delivery.In this paper,we present the construction and application of nano-drug delivery system based on ELP via genetic recombinant technology and molecular biology method.The detail works are listed below.(1)We prepared phenylboronic acid-incorporated ELP nanoparticles by polymerizing N-3-acrylamidophenylboronic acid in presence of ELP.It was found that the nanoparticles had a spherical shape with a diameter of～100 nm and highly stablity.The nanoparticles could be rapidly internalized by single cell and 3D cells through receptor-mediate mode.After loading an anticancer agent doxorubicin(DOX)into ELP nanoparticles,we investigated the in vitro release and cytotoxicity of the drug-loaded ELP nanoparticles.Finally,the bio-distribution and in vivo anticancer effect were evaluated in H22 tumor-bearing mice.(2)We developed a double targeting fusion peptide comprised of ELP,anti-EGFR nanobody and a cell penetration peptide iRGD expressed from Escherichia coil through genetic engineering technology.The circular dichroism spectrum assay suggested that the fusion protein retained the inherent conformation of each segment.The multi-functional fusion protein showed a better cellular uptake and 3D cells penetration.When coadimistration with DOX,the multi-functional fusion protein showed an enhanced tumor accumulation and anticancer effect comparing to free DOX.(3)We conjugated DOX to the multi-functional fusion protein through an acid-sensitive hydrazone bond.The multifunctional recombinant polypeptide-DOX conjugates showed great uptake in cancer cells than controls.In the H22 tumor-bearing mice,the ELP-anti-EGFR-iRGD-DOX highly reduce the DOX accumulation in the main organs except tumor,thus significant improve the maximum tolerance dose and the finally anti-cancer efficacy comparing to free DOX.