Studies On A Novel EGFR Small Peptide Ligand And Coupling With Nanostructured Lipid Carriers

Monoclonal antibodies and fragments that serve as ligands have specific targets;however,they are limited in terms of their immunogenicity.The strong mitogenic and neoangiogenic activities of the natural ligand is a major bottleneck as the targeting ligand.In contrast,small peptides are pursued as targeting moieties because of their small size,safe,stability,and their ability to be incorporated into certain delivery vectors.In this paper,we researched deeply the targeting of small peptides from autophosphorylation sites of EGFR C-terminal.For the first time,the small peptide AEYLR(has applied for invention patent in China)from EGFR C-terminal was screened as a targeting ligand of drug carriers,which conjuncted PEG-nanostructured lipid carrier(pNLC).A comprehensive evaluation research in vitro and in vivo was done about active targeting,improving the efficacy,reducing toxicity of AEYLR-conjuncted pNLC.Firstly,we designed small peptides(EY1068INQ,AEY1073LR,PDY1148QQD)derived from three major autophosphorylation sites,then FITC was used to lable these peptides.The binding affinity and internalization capacity of the FITC-labled peptides was tested with high EGFR expressed cells(NCI-H1299,A549)and no EGFR expressed cells(K562)employing LARLLT as a positive control which bound to putative EGFR selected from a virtual peptide library by computer-aided design and the independent peptide RALEL as a negative control,the result revealed that AEYLR exhibited high EGFR specificity.The targeting ability and security of the AEYLR in vitro was further confirmed by ELISA?immunochemistry and MTT assay.A549 xenograft tumor model was set up and the AEYLR peptide was labled by Cy7 for the study of the AEYLR peptide targeting in vivo.The group of Cy7-AEYLR indicated stroger fluorescence than that of Cy7-RALEL and Cy7 after injection 3h?7h,and the targeting ability of AEYLR in vivo was proved.The EGFR targeting NLC vector was constructed by coupling with pNLC and AEYLR peptide(AEYLR-pNLC),the coupling rate of the small peptides were prepared at 1%,3%and 6%of the pNLC by optimizing the coupling conditions.FITC labled pNLC was constructed by Biotin-Avidin-System(BAS),DiR-pNLC and HCPT-pNLC were prepared using NIFR dye Dir and anticancer drugs hydroxycamptothecin(HCPT)as a model drug.All the NLCs were conjuncted small peptide,the particle size of NLCs was between 100.28 nm±2.95nm and 133.9 nm±2.95 nm.The particle size and particle size distribution increased slightly after conjuncting small peptide,no significant change in potential and morphological.The releasing trend of DiR in DiR-pNLC,RALEL-DiR-pNLC and AEYLR-DiR-pNLC and HCPT in HCPT-pNLC,RALEL-HCPT-pNLC and AEYLR-HCPT-pNLC was consistent,the cumulative release amount reached more than 80%in 72h.The targeting of carrier in vitro was test by flow cytometry and internalization capacity using FITC as probe.Binding rate of FITC-AEYLR-pNLC and NCI-H1299 and A549 cells after incubating 37 ? for 3h was 83.71%and 84.08%,respectively,and strong fluorescence showed in microscope.However,binding rate of FITC-RALEL-pNLC,FITC-pNLC and the two cells was 25,70%?31.53%,and weak fluorescence showed in microscope.The results indicated that AEYLR conjuncted pNLC had targeting obvious in vitro.Small animal in vivo imaging experiments were done by injecting the DIR-pNLC,RALEL-DiR-pNLC and AEYLR-DiR-pNLC in A549 xenograft tumor bearing mice tail vein.By DiR distribution characteristicsin,the targeting in vivo of AEYLR conjuncted pNLC was verified by the concentration of the target site improved and the distribution of non-target parts reduced.The IC50 values was determinated by MTT of HCPT,HCPT-pNLC,the different coupling rate RALEL-HCPT-pNLC and AEYLR-HCPT-PNLC using HCPT as a model drug.Compared with the control group,the rate of 6%for AEYLR coupling showed significant cytotoxicity.The pharmacodynamic evaluation in vivo was made by tail vein injection HCPT,the HCPT-the PNLC,coupling rate of 6%RALEL-HCPT-pNLC and AEYLR-HCPT-pNLC in A549 xenograft tumor bearing nude mice.Examine tumor size,nude body weight changes and other indicators,the relative tumor volume of AEYLR-HCPT-pNLC and the magnitude of weight loss were significantly lower than the control group,which proved the AEYLR conjuncted PEG modified NLC of EGFR active targeting mechanism has better the anti-cancer properties.