Preparation And Characterization Of Stimuli-Responsive Polymer-Camptothecin And Doxorubicin Prodrugs

Camptothecin(CPT)and Doxorubicin(DOX),inhibitors of topoisomerase I and topoisomerase Ⅱ,respectively,have a good effect on inhibiting the proliferation of tumor cells.Unfortunately,CPT and DOX are hydrophobic small-molecular anticancer drugs,leading to the structural instability during the blood circulation and large toxic side effects on the human body,which greatly hinder the applications of CPT in clinic.It is of great significance for the development of the biomedical field to use polymeric prodrug system,which is composed of biocompatible and biodegradable polymer materials and small molecule drugs.Poly(ethylene glycol)(PEG)and polyphosphoesters(PPEs)are biocompatible materials and widely used in drug delivery system,which can effectively improve the water solubility of small molecule drugs,increase the stability of the structure,reduce the side effects on the human body,prolong the time of drugs in the blood circulation.In order to achieve high drug accumulation in tumor cells and effective control of drug release,stimuli-responsive polymer prodrug is a current leading direction by introducing reduction-responsive and acid-sensitive groups into the structure,which are responsed to the difference microenvironments between the normal cells and the tumor cells.Stimuli-responsive prodrug micelles are relatively stable under physiological conditions(pH～7.4,GSH～2.0 to 20 μM)but can be rapidly degraded to control the release of anticancer drugs when reach into tumor tissues responding to the specific microenvironment(pH～4.5 to 6.5,GSH～2 to 10 mM).This article prepared two types of stimuli-responsive prodrugs,including polyphosphoester-camptothecin prodrug and the PEG-doxorubicin prodrug via Michael addition polymerization and CuAAC "click"chemistry reaction,whose chemical structures are verified by different measurements and the applications in biology are studied.The work is mainly contained with two parts:(1)Synthesis and characterization of polyphosphoester-based camptothecin prodrug P(EAEP-PPA)-g-ss-CPT with reduction-responseThe polyphosphoester-based camptothecin prodrug P(EAEP-PPA)-g-ss-CPT was prepared via a combination of Michael addition polymerization and CuAAC "click"chemistry.First,polyphosphoesters(abbreviated as P(EAEP-PPA)),having alkynyl group in the side chain,were prepared by Michael addition polymerization between the unsaturated phosphate monomer(EAEP)and 2-propynylamine(PPA);Second.P(EAEP-PPA)-g-ss-CPT prodrugs were obtained via the CuAAC "click" reaction between azido-functionalized and disulfide-modified drug derivative CPT-ss-N3 and P(EAEP-PPA).The chemical structures were characterized by nuclear magnetic resonance spectroscopy(1H NMR,31P NMR,13C NMR).gel permeation chromatography(GPC),Fourier transform infrared(FT-IR),ultraviolet-visible spectrophotometer(UV-Vis)and high performance liquid chromatograph(HPLC)analyses,respectively.And then,the average particle sizes and morphologies of the prodrug nanoparticles were measured by dynamic light scattering(DLS)and transmission electron microscopy(TEM).The stability of nanoparticles under different medium was also measured by DLS.The study of in vitro drug release behavior indicates that the polyphosphoester-camptothecin prodrug possesses the reduction-response.By MTT assays,it can be known that P(EAEP-PPA)is a favorably biocompatible material and the CPT prodrug nanoparticles can efficiently inhibit the cell proliferation of tumor cells.Moreover,the prodrug nanoparticles could be internalized into cells to release the active CPT by endocytosis in the experiment of in vitro cellular uptake.(2)Synthesis and characterization of PEG-based doxorubicin prodrug DOX-hyd-poly(SS-alt-A)-hyd-DOX)with reduction-and pH-responseThe reduction-and pH-responsive doxorubicin prodrug was synthesized by the highly efficient CuAAC "click" reaction.First of all,acetal-and azido-modified N3-a-PEG-a-N3 and alkynyl-functionalized B-ss-B were obtained.And then,the alkynyl-terminated polymer poly(SS-alt-A)was synthesized by CuAAC "click" polymerization.At the last,N3-hyd-DOX containing hydrazone group was linked onto the both ends of the polymer to get doxorubicin prodrug DOX-hyd-poly(SS-alr-A)-hyd-DOX.The chemical structures were characterized by nuclear magnetic resonance spectroscopy(NMR),gel permeation chromatography(GPC),Fourier transform infrared(FT-IR),ultraviolet-visible spectrophotometer(UV-Vis),respectively.The amphiphilic prodrug can self-assemble into micelles in aqueous solution.The average particle sizes and the size polydispersity indices of the prodrug nanoparticles were measured by dynamic light scattering(DLS).The micellar morphologies were observed by transmission electron microscopy(TEM).DLS measurement was carried out to monitor the size change of nanoparticles at different midiums with the extension of time.The results of in vitro drug release further verify the trigger-controlled release behavior of the prodrug nanoparticles.The anti-proliferation activity of prodrug nanoparticles DOX-hytd-poly(SS-alt-A)-hyd-DOX against tumor cells was investigated using MTT assays and the cellular internalization measurements.