Hyperbranched Polycaprolactone / Poly Glycidyl Ether Copolymers And Gene Delivery Carrier Application

Biodegradable materials based on poly(ε-caprolactone) were widely used for drug delivery and gene transfection due to their good biocompatibility and long-term degradability. Polyglycidol is a soluble and biocompatible polymer which has hyperbranched architecture with numerous hydroxyl end groups.In present study,a novel di-block copolymer of poly(ε-caprolactone)-b-hyperbranched polyglycidol(PCL-b-HPG) was synthesized.Compared to PCL homopolymer,the PCL-b-HPG has improved solubility and modifiability owing to the introduced hydroxyl end groups.The PCL-b-HPG copolymer was used to prepare nanomicelles with reporter GFP gene and cytotoxicity and gene transfection efficiency of the gene nanomicelle were evaluated.The PCL-b-HPG-based nanomicelles were further chemically coupled with RGD4C peptide,which we thought to lay a preliminary foundation for developing new targeted drug delivery system.First,a hydroxyl-terminated PCL was prepared by ring-opening polymerization using 1-dodecanol as initiator and stannous octanoate as catalyst.The obtained PCL was treated with naphthalene potassium to get a PCL-based macro-initiator,which was used to initiate glycidol polymerization to form the amphiphilic copolymer of PCL-b-HPG.Chemical structure of the PCL-b-HPG was characterized by ~1H NMR,GPC and IR.Optimal parameters for the copolymerization procedure were established by series of experiments,i.e.1mL/hour of glycidol adding rate and 5 hours of total reaction time.A novel living copolymerization was designed for better control of the copolymer configuration.The living poly(ε-carprolatone) was synthesized by an ionic-coordination catalyst,named bimetallicμ-oxoalkoxides.The living system of PCL further initiated the polymerization of glycidol to form the block copolymer of PCL-b-HPG.In comparison with anion polymerization,PCL-b-HPG obtained from living copolymerization has lower polydispersity with Mw/Mn of 1.3.Within this system,optimal production conditions were set as follows:reaction time for 10 hours,glycidol adding rate at 2mL/hour and CL/glycidol ratio of 1:2.Chemical Structure of the resultant PCL-b-HPG was characterized by ~1H NMR, GPC,DSC and IR.Nanomicelles of the amphiphilic poly(epsilon-caprolactone)-b-hyperbranched polyglycidol(PCL-b-HPG) were prepared for further evaluation as targeted drug delivery vehicles.Size and morphology of the nanomicelles were characterized by TEM,SEM and DLS,respectively.The nanomicelles were spherical in uniform shape with diameter ranging from 170±10.0nm.Cytotoxicity of the PCL-b-HPG nanomicelles was evaluated by means of MTT assay.Subsequently,a reportr gene of pEGFP-C1 was loaded into the PCL-b-HPG nanomicelles of 225 nm in size.The loading efficiency is measured over 85%and the ratio of pEGFP-C1 to nanomicelles is about 1:50.In gel electrophoresis,obvious smears appeared when plasmid DNA was packaged into nanomicelles,indicating PCL-b-HPG-based nanomicelles have potential to serve as an efficient gene carrier.The yields of transfection efficiency were found to be 15%when the polyplexes were transfected to EA.hy926.These results demonstrated that the PCL-b-HPG/DNA nanomicelles are effective gene vector in vitro.RGD4C peptide was conjugated on the surface of PCL-b-HPG nanomicelle by a series of chemical reactions,such as direct coupling using N-glycine maleic anhydride as crosslinker, or modifying the hydroxyl end groups of the PCL-b-HPG molecules with carboxyl groups followed by coupling using acylamide chemical bond.The conjugation of RGD4C was confirmed by IR or XPS spectrum.It was hypothesized that the RGD4C-conjugated PCL-b-HPG nanomicelle be capable of specifically recognizing tumor neovascular endothelium,therefore may offer great opportunities for developing novel tumor-targeting drug delivery system.