Nucleolin-Aptamer-Modified Graft Polymeric Micelle With Dual PH/Redox Sensitivity For Preparation And Tumor-targeted Therapy

In this research,two kinds of polymeric micelles with response to specific tumor microenvironment were synethesized which combine multidisciplinary knowledge with a variety of advanced technologies.TLR4 siRNA was firstly introduced into chemotherapeutic drugs for the tumor therapy in order to down-regulate TLR4 protein expression and inhibitie tumor metastasis.Fiestly,we successfully developed a novel drug delivery system with enhanced cellular internalization and efficient drug release based on the stepwise pH/reduction-responsive DA-PLL-LA micelles.The charge-convertible cross-linked copolymer micelles with stable structure and negative surface charge in the blood stream exhibited high tumor-targeting ability via EPR effect.Secondly,In summary,novel polymeric micelles with dual pH and reduction sensitivity were successfully prepared for tumor therapy.These micelles enable the rapid and steady intracellular release of drugs and genes.The present study provided new strategies to design drug-siRNA-loaded micelles with ameliorated efficacy against tumor survival and invasion.The micelles were produced for codelivering small-molecullar chemotherapy drug to inhibit NSCLC growth via enhanced cellular internalization and on-demand intracellular drug release.The polymer micelle was then further modified AS 1411 aptamer to form smart co-delivery system for combine chemotherapy and gene therapy to synergistically inhibit proliferation and metastasis of orthotopic NSCLC,which was expected to provide a promising strategy for precise and effective cancer therapy.Part 1:IntroductionCancer is widely accepted to be characterized by low cure rates,high recurrence rates,and high mortality rates.Substantial effort has been exerted in cancer therapy.Even so,the design and exploitation of novel,safe,and effective codelivery nanosystems for siRNA and chemotherapeutic drugs to cure cancers continue to face tremendous challenges.The process and character focused on the research of characteristic and application of smart nano drug delivery system witch in response to tumor microenvironment in targeted tumor therapy.Besides,the combination of chemotherapy and gene therapy for synergistic antitumor effect was introduced to put forward this study.After that,the idea and proposal of this paper were elaborated.Part 2:Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumorIn this research,a charge-conversional polymer PLL-LA,was prepared by dimethylmaleic anhydride(DA)modification and applied as a carrier with enhanced cell internalization and intracellular pH-and reduction-triggered doxorubicin(Dox)release.The surface charge of dimethylmaleic anhydride-poly-L-lysine-lipoic acid micelles(DA-PLL-LA)was negative at physiological pH and reversed to positive at the extracellular and intracellular pH of cancer cells.The graft ratios of LA and DA were 42.17%and 14.52%,respectively,which were calculated from the 1H NMR spectrum.At tumor extracellular pH of 6.8,the conjugates underwent a rapid charge-reversible process with almost 80%DA cleavage within 2 h,and then endocytosed into the endo/lysosomes more rapidly than at physiological pH of 7.4.The zeta potential of DA-PLL-LA micelles reversed from-16.5 mV to 7.9 mV as the surrounding pH value changed from 7.4 to 6.8.The Dox/DA-PLL-LA micelles(Dox-micelles)demonstrated a sustained drug release in vitro under physiological condition,and rapid Dox release was triggered by both extracellular pH and high-concentration reducing glutathione.In addition,Dox-micelles showed a negative charge under the physiological condition;the EE%and LC%were(93.6±2.26)%and(22.4±2.52)%,respectively.The Dox-micelles also exhibited enhanced internalization at extracellular pH,rapid intracellular drug release,and improved cytotoxicity against A549 cells in vitro.Excellent tumor-penetrating efficacy was also found in A549 tumor spheroids and solid tumor slices.Moreover,the DA-PLL-LA micelles exhibited excellent tumor-targeting ability in tumor tissues and excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing mice.Therefore,the DA-PLL-LA micelles demonstrated great potential for targeted and efficient drug delivery in cancer treatments.Part 3:Nucleolin-targeting AS1411-aptamer-modified graft polymeric micelle with dual pH/redox sensitivity designed to enhance tumor therapy through the codelivery of doxorubicin/TLR4 siRNA and suppression of invasionIn this paper,a novel graft polymeric micelle with targeting function ground on aptamer AS 1411 was synthesized.The micelle was based on chitosan-ss-polyethyleneimine-urocanic acid(CPU)with dual pH/redox sensitivity and targeting effects.This micelle was produced for codelivering Toll like receptor 4 siRNA(TLR4-siRNA)and doxorubicin(Dox).The graft ratios of CSO and UA calculated from the 1H NMR spectrum were 3.33%and 44.4%,respectively.The EE%and LC%of Dox-loaded micelles were 85.81±1.22%and 14.26±1.09%,respectively.Meanwhile,siRNA was compressed into the PEI skeleton via electrostatic interaction.siRNA was efficiently tied to the micelles at an N/P ratio of 6/1 identified.In pH 5.3 PBS or with 10 mM GSH,drugs and siRNA were rapidly released from micelles.Dox-ACPU micelles displayed the strongest cell viability inhibition of A549 cells,followed by Dox-CPU micelles.A greater amount of Dox colocated with the nuclei treated with Dox-ACPU than with the nuclei treated with Dox-CPU after 3 h of incubation.The preponderant cellularuptake was further dynamically confirmed by live cell station.In vitro investigation revealed the sustained gene and drug release from Dox-siRNA-loaded micelles under physiological conditions and this codelivery nanosystem exhibited high dual pH/redox sensitivity,rapid intracellular drug release,and improved cytotoxicity against A549 cells in vitro.Furthermore,the micelles loaded with TLR4-siRNA inhibited the migration and invasion of A549.Excellent tumor penetrating efficacy was also noted in the A549 tumor spheroids and solid tumor slices.In vivo,multiple results demonstrated the excellent tumor-targeting ability of ACPU micelle in tumor tissues.The micelles exhibited excellent antitumor efficacy and low toxicity in the systemic circulation in lung-tumor-bearing BALB/c mice.These results conclusively demonstrated the great potential of the new graft copolymer micelle with targeting function for the targeted and efficient codelivery of chemotherapeutic drugs and genes in cancer treatment.