Construction And Evaluation Of Chemotherapeutics And Nucleic Acids Coloaded Polymeric Carriers

Cancer is a leading cause of death,and the treatment for cancer now is some combination of surgery,radiation therapy and/or drug.The mechanisms of anticancer drugs for cancer therapy include:(1)using cytotoxic agents(chemotherapeutics)to kill cancer cells for cancer therapy,(2)blocking the nutrition supply by inhibiting tumor angiogenesis for cancer therapy,(3)regulating the tumor microenvironment to inhibit tumor growth,(4)enhancing the cancer cells recognization by innate immune system for cancer therapy.Due to the heterogeneity and complexity of cancer,combination therapies using anticancer drugs with different mechanisms are the clinical approach for cancer therapy.Of which,the combination therapy involving both gene therapy and chemotherapy can efficiently inhibit the proliferation of cancer by targeting different cancer cell signaling.The co-delivery of chemotherapeutics and nucleic acids using single carrier with rationally designed doses and release profiles can maximize the benefit of combination therapies.However,the loading of chemotherapeutics and nucleic acids into one carrier is a challenging task due to their different properties,such as hydrophobicity,molecular weight and metabolic stability.Polymers are emerging as promising vectors for the simultaneous co-delivery of chemotherapeutics and nucleic acids due to their controllable physicochemical properties and desirable biological features.Construction of multifunctional polymeric carriers for co-delivery of chemotherapeutics and nucleic acids by simple methods is beneficial for industrial manufacture.The Aza-Michael reaction is a versatile synthetic methodology for the efficient coupling of electron poor olefins with a vast array of nitrogen-donor nucleophiles.The versatility of the Aza-Michael reaction in terms of monomer selection,solvent environment,and reaction temperature permits the synthesis of sophisticated macromolecular structures under conditions where other reaction processes will not operate.In order to solve the problems mentioned above,three novel polymer-based co-delivery carriers were designed and synthesized based on Aza-Michael reaction.1.Poly[platinum(IV)-alt-PEI]/Akt1 shRNA complexes for enhanced anticancer therapy.The co-delivery of platinum(IV)prodrugs and nucleic acids has emerged as a new modality for cancer therapy.However,as a result of the different molecular properties of nucleic acids and platinum(IV)prodrugs,the co-delivery of platinum(IV)prodrugs and nucleic acids encounters challenges(e.g.,insufficient drug loading capacity and efficiency,less definable ratios of the two agents).To address these issues,a novel polymeric platinum(IV)prodrug,poly[platinum(IV)-alt-PEI](DP),was synthesized by one-step Michael-addition reaction of platinum(IV)diacrylate with low molecular weight PEI(PEI 800).Platinum(IV)prodrug content in the polymer was 35.946 wt%,and the loading efficiency of the platinum(IV)prodrug was nearly 100 %,for cases where the platinum(IV)prodrug content in the polymer could be precisely regulated by simply changing the molar ratios of the reactants.The poly[platinum(IV)-alt-PEI]/Akt1 shRNA complex was constructed by the electrostatic condensation of negatively charged Akt1 shRNA(a RNAi sequence for serine-threonine kinase AKT pathway)with positively charged poly[platinum(IV)-alt-PEI].Cellular assays involving A549,MCF-7,and PC-3 cancer cells indicated that poly[platinum(IV)-alt-PEI]/Akt1 shRNA complexes for platinum(IV)prodrug and Akt1 shRNA co-delivery offered combinational anticancer efficacy.In conclusion,we proposed a convenient carrier assembly,which could achieve the co-delivery of small molecular chemotherapeutics and macromolecular nucleic acids.2.Polymeric demethylcantharate prodrug/Akt1 shRNA complexes for enhanced cancer therapy.Due to the carboxylic acid groups,demethylcantharate carries negative charges when in a physiological environment.Therefore,it's hard for demethylcantharate to enter into the cancer cells.This report demonstrated a one-step assembly for co-delivering chemotherapeutics and therapeutic nucleic acids,constructed by integrating drug molecules into a nucleic acid condensing polymeric prodrug through degradable linkages.Demethylcantharate was selected as the model drug and pre-modified by esterifying its two carboxylic groups with 2-hydroxyethyl acrylate.The synthesized demethylcantharate diacrylate was then used to polymerize with linear polyethyleneimine(PEI 423)through a one-step Aza-Michael reaction.The obtained cationic polymeric demethylcantharate prodrug was used to pack Akt1 shRNA into complexes through electrostatic interaction.The formed complexes could release the parent drug demethylcantharate and Akt1 shRNA through the hydrolysis of ester bonds.Cellular assays involving cell uptake,cytotoxicity,and cell migration indicated that demethylcantharate and Akt1 shRNA co-delivered in the present form significantly and synergistically suppressed the growth and metastasis of three human cancer cells.This work suggests that incorporating drug molecules into a nucleic acid-packing cationic polymer as a polymeric prodrug in a degradable form is a highly convenient and efficient way to co-deliver drugs and nucleic acids for cancer therapy.3.Construction of multinuclear polymeric nanoparticles to co-deliver of nucleic acids and hydrophobic chemotherapeutics for enhanced cancer therapy.A rational designed polymer-based co-delivery system,which was constructed by Aza-Michael reaction,was reported for the first time.Firstly,the cationic GSH-responsive poly(amidoamine)s were synthesized by the Aza-Michael reaction of N,N-bis(acryloyl)cystamine and agmatine at a molar ratio of 1.2.To synthesize poly(amidoamine)s with the terminal primary groups,ethanediamine was reacted with the terminal acrylamide groups of the synthesized poly(amidoamine)s through Aza-Michael reaction.The cationic block copolymer,which was prepared by conjugating GSH-responsive poly(amidoamine)s with PLGA through amide linkages,was able to form polymeric nanoparticles and load with doxorubicin(DOX)nucleic acids(siBcl-2).The cleavage of disulfide linkages at intracellular glutathione-rich reduction environment significantly decreased the cytotoxicity,and promoted more efficient drug release and gene transfection.The post-adsorbing lipid-BSA complexes could stabilize the cationic core and prevent the leakage and degradation of nucleic acids.The co-delivery system entered into the cancer cells through the caveolae pathway and could successfully escape from endosomes.Most importantly,the co-delivery of DOX and B-cell lymphoma 2(Bcl-2)small interfering RNA(siRNA)exerted a combinational effect against tumor growth in three different cancer cells(A549,SMMC-7721 and MCF-7 cells)in vitro,which was much more effective than either DOX or Bcl-2 siRNA-based monotherapy.This multinuclear polymer-based co-delivery system may constitute a promising stimuli-responsive system for efficacious co-delivery of nucleic acids and hydrophobic chemotherapeutics.