Elastin-like Peptide-based Drug Delivery

Elastin-like polypeptides(Elastin-like polypeptides,ELPs)are elasti and environmentally responsive biopolymers.Due to their characteristics of reversible phase transition,Elastin-like polypeptides(ELPs)have been used for a number of applications,such as protein purification,drug delivery,and tissue engineering.Transition temperature is a key attribute of ELPs and central to ELPs researches.To better predict transition temperature of elastin-like peptide,we seek to establish a convenient and reliable predictive model by employing simple parameters.In this study,two pH-sensitive ELPs were expressed,purified and determined for temperature transition across a range of pH.The pH,concentration and molecular weight(MW)as well as isoelectric point(PI)and pseudo amino acid(PseAA)of these two ELPs were adopted as input parameters.Support vector machine(SVM)and back propagation neural network(BPNN)were performed respectively.We evaluated predictor model by jackknife test that combined with Uniform Design(UD).According to the results of BPNN and SVM,whose mean absolute error(MAE)of BPNN model jackknife test were 4.80 and 4.95 respectively,the predictive ability of BPNN is a minor improvement over SVM.Applying Mackay's data,MAE of BPNN jackknife test was 2.02,while the MAE between experimental and predicted transition temperature was 2.30 in Mackay's predictor model.Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is a promising anticancer agent with tumor-selective apoptotic activity.Formation of aggregates as trimer or polymers is the prerequisite for TRAIL's function as an apoptosis inducer.However the polymerization property of TRAIL has also brought difficulties for its production.RGD-TRAIL is an integrin-targeting TRAIL mutant with enhanced apoptotic induction activity towards tumor cells both in vitro and in vivo.When expressed in E.coli,TRAIL or its RGD-TRAIL mutant usually formed inclusion body.Their extreme aggregation propensity makes them unstable and easy loss during purification processes,which retards their application in industrial production.To avoid the above problems during RGD-TRAIL production,we employed elastin-like polypeptides(ELPs)for the fusion-expression of recombinant RGD-TRAIL.Recombinant RGD-TRAIL-ELP was expressed in a soluble form and efficiently purified from the clarified cell extracts by three rounds of inverse transition cycling(ITC).SDS-PAGE and Western blotting analyses of purified RGD-TRAIL-ELP indicated that the prepared RGD-TRAIL-ELP was successfully purified and the yield was up to 10 mg/L of bacterial culture.We observed two types RGD-TRAIL-ELP both can self-assemble into nanoparticles at 10?.Apoptosis assay was performed in human colorectal carcinoma cells(COLO-205)and Human breast cancer cell line(MDA-MB-231).Fusion with hydrophobic ELP can effectively enhance RGD-TRAIL's biological activity.RGD-TRAIL-ELP[V5-60]that formed micron particles at physiological conditions are not suitable for in vivo delivery,we redesigned and expression and purification of RGD-TRAIL-ELP[V5-40].The purified RGD-TRAIL-ELP without any chemical conjugation was able to self-assemble into nanoparticle under physiological condition.Flow cytometry and Western blot results confirmed that RGD-TRAIL-ELP exhibited enhanced efficiency in inducing apoptosis.Single intravenous injection of the RGD-TRAIL-ELP nanoparticle induced nearly complete tumor regression in the COLO-205 tumor xenograft model.Histological observation confirmed that RGD-TRAIL-ELP induced significant tumor cell apoptosis without apparent liver toxicity.TRAIL selectively induces apoptosis in tumor cells in vitro and in vivo but not in most normal cells,so its safety has been admired.However,TRAIL exhibit potent resistance for many tumor cell while were administrated repeatedly.Dox and TRAIL complement each other using two distinct pathways that trigger apoptosis for tumor cell.To reduce resistance,many researchers dedicated to develop TRAIL combination therapy with chemotherapy drugs,such as doxorubicin,because the combination of Dox and TRAIL suppresses tumor growth can reduce resistant to treatment with either Dox or TRAIL alone.Dox-associated cumulative and irreversible cardiotoxicity may be induced by overdose.Doxorubicin can insert into the double strand of DNA and preferentially bind to double-stranded 5'-GC-3'or 5'-CG-3'to form a tightly coupled complex without chemical bond links.We express and purified TRAIL-ELP,wherein the ELP motif was positive,and could interact with ployGC-Dox by electrostatic interaction,which contribute to form TRAIL-ELP-Dox nanoparticle.Due to TRAIL motif,TRAIL-ELP-Dox nanoparticle could be internalized into tumor cell by mediating via both TRAIL death receptors(DR4 and DR5).Compared with polyGC-Dox,TRAIL-ELP-Dox can more effectively internalized into tumor cell,achieved better tumor suppression effect and reduce it side effects that caused by the Dox.