Reseaches On The Construction Of Smart Drug Delivery Systems Based On Mesoporous Silica Nanoparticles/Polymeric Micelles And Their Anti-tumor Effects

Malignant tumor is undoubtedly one of the major diseases that threaten human health.Traditional surgery,radiotherapy and chemotherapy were difficult to completely cure tumor especially for malignant tumors due to several issues,such as postoperative recurrence,lacking specificity,multidrug resistance and serious side effects.Due to the enhanced permeability and retention effect（EPR）,nanoparticulate drug carriers play an important role in improving the bioavailability of antitumor drugs,enhancing therapy efficacy and reducing side effects,and have great potential in clinical applications.Currently,a variety of anti-tumor nanomedicine preparations based on liposomes,inorganic nanoparticles and polymer micelles have been developed,and smart drug release system is one of the best nanomedicine preparations.The system can selectively deliver the therapeutic drug to the tumor sites,and then in suit release the chemotherapeutic agents/photosensitizers/siRNA in response to the biological signal.Smart drug delivery system could effectively kill tumors and reduces side effects,which has huge application potential for tumor therapy.Smart drug delivery system are generally constructed using nanoparticles as drug carriers and employing multi-functionalized modification methods,such as integrating stimuli-response release mechanisms and introducing target molecules.Mesoporous silica nanoparticles as well as polymeric micelles have received widespread attention from researchers.Taking advantage of the simple synthesis,large surface areas,adjustable particle size,easy functionality and other characteristics,mesoporous silica nanoparticles are widely used as nanoreservoirs for drug delivery and bioimaging;Polymeric micelles are promising nanocarriers for targeted drug delivery and gene therapy,mainly due to their good biocompatibility,low immunogenicity,high drug loading capacity and biodegradability.However,there are some urgent challenges that should be resolved in practical application:First is the construction of a biological signal-responsive targeted drug delivery system based on mesoporous silica nanoparticle with good biocompatibility and high drug loading capacity,which could specifically deliver chemotherapeutic agents to tumor in response to biological signals just existed in tumor microenvironment and effectively kill tumor cells,as it could significantly improve the bioavailability of chemotherapeutics and reduce side effects on normal tissues;Second is to design a biological signal-responsive targeted drug delivery system based on biodegradable polymeric micelles with good biocompatibility,which could selectively anchor to tumor and enhance endocytosis efficiency of nanocarriers,and subsequently initiated lysosomal escaping and micelles disassembly&therapeutic molecules release in response to intracellular biological signals,leading to the efficient tumor killing in vitro and in vivo.Based on the above discussion,three kinds of mesoporous silica nanoparticles and three kinds of biodegradable polymeric micelles were designed and used to fabricate drug delivery systems,which was achieved by integrating biostimuli-response release/degradation mechanisms and introducing tumor targeting motifs through multi-functional modification methods.Meanwhile,the in vitro and in vivo drug release behaviour,targeted cellular endocytosis,bioimaging,antitumor immune response,tumor suppression and related molecular mechanisms were systematically studied.Above investments laid the theoretical basis for the development of multi-function targeted drug delivery system against tumor therapy.The main contents and conclusions of this work are listed follows:1.Reseaches on the construction of simuli-responsive drug delivery systems based on mesoporous silica nanoparticles and their anti-tumor effects（1）Construction of redox-responsive drug delivery system based on mesoporous silica nanoparticles end-capped with heparin for targeted tumor theapy and its anti-tumor evaluation in vivoMSNs-based redox responsive drug delivery system（MSNs-S-S-HP-LA）were fabricated by employing MSNs as nanoreserviror,disulfide bonds as intermediate linker,heparin as nanocap and lactobionic acid as targeting agent.The characterizations of TEM,TGA,zeta potential,FTIR,BET and BJH indicated the successful construction of MSNs-S-S-HP-LA step by step.Cumulative release assay demonstrated that the system had pH-sensitive drug release property.Cell viability assays suggested the good compability of MSNs-S-S-HP-LA system.The results of confocal laser scanning microscope（CLSM）,flow cytometry（FCM）,TEM and DNA ladder fragment assays indicated that MSNs-S-S-HP-LA system could be specially recognized and internalized by HepG2 cells,the release of DOX was triggered by intracellular GSH and induced cell apoptosis.In vivo investigations further confirmed that MSNs-S-S-HP-LA@DOX sysem could effective suppressed the growth of tumor and reduced the toxic side effect of DOX.（2）Construction of pH-responsive drug delivery system based on dendrimerlike mesoporous silica nanoparticles for targeted tumor theapy,bioimaging and its anti-tumor evaluation in vivoTo solve the monotonic functionality of MSNs,we constructed a multifunctional drug delivery system（HPSN-Salphdc-FA）based on dendrimerlike mesoporous silica nanoparticles（HPSN）fortumor-targetedtherapyandbioimaging.N,N-phenylenebis-（salicylideneimine）dicarboxylic acid（Salphdc）was used both as the gatekeeper of HPSN via pH-responsive coordination bonds between-COOH of Salphdc and In³⁺ions and as a fluorescence imaging agent.Folic acid was then conjugated to Salphdc as the targeting unit.TEM,SEM,DLS,zeta potential,HNMR and FTIR confirmed the successful fabrication of HPSN-Salphdc-FA system.Cumulative release assay indicated that HPSN-Salphdc-FA system equipped high pH sensitivity for drug release.CLSM,FCM,TEM and endocytosis mechanism assay demonstrated that the HPSN-Salphdc-FA nanoparticles were specially internalized by HepG2 cells and distributed in the cytoplasm via the FA receptor-mediated endocytosis pathway,the sustained release of DOX was triggered by intracellular low pH and locally killed tumor cells in a dose-dependent manner.In vivo investigations demonstrated that the DOX@HPSN-Salphdc-FA system could effective inhibited the growth of tumor with reducing side effects and showed good bioimaging.（3）Construction of pH-responsive drug delivery system based on hollow mesoporous silica nanoparticles end-capped with hyaluronic acid for targeted tumor theapy and its anti-tumor evaluation in vivoTo further improve the drug loading content of MSNs,a pH-responsive drug delivery system（HMSN-PA-HA）based on hollow mesoporous silica nanoparticles（HMSN）was fabricated for targeted tumor therapy by using hydrazone bonds as pH-sensitive linkers and hyaluronic acid molecules as both blocking and targeting agents.TEM,SEM,¹³C NMR,FTIR,TGA and zeta potential indicated the successful construction of HMSN-PA-HA step by step.Cumulative release assay demonstrated that the system had good stability and pH-sensitive drug release.CLSM,FCM,cell viability and TEM demonstrated that HMSN-PA-HA system could effectively enter tumor cells via HA-mediated endocytosis and continuously release of DOX triggered by intracellular low pH and locally killed tumor cells in a dose-dependent manner.In vivo investigations further confirmed that HMSN-PA-HA@DOX system with good biocompatibility could specifically delivered cargo to tumor sites and effective suppressed the growth of tumors with high efficacy.2.Reseaches on the construction of simuli-responsive drug delivery systems based on polymeric micelles and their anti-tumor effects（1）Construction of ROS-sensitive polymeric micelles drug delivery system and its application in photodynamic-chemo combination therapy for liver cancerA ROS-responsive disassemble micellar drug delivery system（PPS-mPEG-Ser-FA）was reported by employing ROS-sensitive poly（propylene sulfide）copolymer（PPS）as the hydrophobic part,PEG as hydrophilic segment,and folic acid as targeting agent.¹H NMR,GPC,MS and TEM indicated the successful construction of PPS-mPEG-Ser-FA copolymer step by step,and drug loaded micelles displayed uniform and monodispersed spherical morphology with average diameters of approximately 80 nm,low CMC value and good structural stability in serum.The results of TEM,DLS,¹H NMR and cumulative release assays demonstrated that the system had ROS-sensitive micelles disassembly and drug release.CLSM,FCM,cell viability and western-blotting assays demonstrated that PPS-mPEG-Ser-FA system could be specially internalized by HepG2cells via recepotor mediated endocytosis pathway,and the endocytosed micelles was disassembled and released DOX in response to inherent intracellular ROS,in turn leading to severe cell apoptosis/death.Furthermore,PPS-mPEG-Ser-FA@DOX@ZNPC micelles could efficiently generate ROS upon laser irradiation and accelerate the disassembly of micelles&drug release as well as cell apoptosis.In vivo investigations confirmed that PPS-mPEG-Ser-FA@DOX@ZNPC system combining with laser irradiation could effectively targeted tumor tissue to suppress tumor growth with minimal toxic side effect and prolong the survival time of tumor-bearing mice.（2）Construction of cascade-responsive disassemble prodrug micellar drug delivery system with dual-targeting potential and its application in photodynamic-chemo combination therapy for cervical cancerAcaspase-responsivedisassemblemicellardrugdeliverysystem（FA-PEG-PDBO-BPT）were constructed by using disulfide bond linked CPT prodrug as the hydrophobic core,pH-responsive segment of poly（2-（diethylamino）ethyl methacrylate）（PDEA）as a middle layer,PEG as the outer hydrophilic layer and FA as thetargetingmotif.Anewcationicporphyrinderivative5-（3-hydroxy-p-（4-trimethylammonium）butoxyphenyl）-10,15,20-triphenylporphyrin chlorine（MTPP）was synthesized as a mitochondria-targeting photosensitizer and loaded into FA-PEG-PDBO-BPT system.¹H NMR,MS,GPC,HPLC and TEM confirmed the successful synthesis of FA-PEG-PDBO-BPT copolymer and MTPP molecule step by step,and the FA-PEG-PDBO-BPT copolymer could self-assembled into an suitable size of micelles with low CMC value and good stability.The FA-PEG-PDBO-BPT@MTPPsystemdisplayedpH-responsiveprotonation,redox-responsive CPT release,caspase-responsive micelles disassembly and drug release detected by TEM,HNMR and drug release assays.The results of CLSM,FCM and apoptosis assays demonstrated that FA-PEG-PDBO-BPT system could be specially endocytosed by HeLa cells via receptor mediated endocytosis pathway,the lysosomal escaping mechanism and disassembly of micelles as well as drug release were quickly activated in response to pH and redox stimuli.The released MTPP could be specifically accumulated in mitochondria,and the ROS produced by MTPP locally caused mitochondrial damage and activated the mitochondria apoptotic pathway under laser irradiation.PDT combined with CPT effectively induced tumor cells apoptosis with high efficiency.In vivo investigations confirmed that the FA-PEG-PDBO-BPT@MTPP system combined with laser irradiation effectively delivered CPT and MTPP into tumors through dual-targeting strategy,it significantly improved the bioavailability of CPT&dramatically suppressed tumor growth with minimal toxic side effect and prolonged the survival time of tumor-bearing mice.（3）Construction of size/charge changeable pH-responsive micelleplex and its application in photodynamic-PD-L1 combination therapy for melanomaA size/charge changeable pH-responsive micelleplex were fabricated for photodynamic immunotherapy,in which PD-L1-targeting siRNA（siPD-L1）and mitochondria-targeting photosensitizer MTPP were both incorporated for simultaneous PD-1/PD-L1 interaction suppression and antitumor immune response activation.The micelleplex（PCPP）was prepared from two block copolymers comprising PEG-CDM-PDEA and PEI-PDEA at a mass ratio of 1.5:1.¹H NMR,GPC and TEM demonstrated the successful synthesis of the two copolymers,and the prepared PCPP micelles displayed suitable size,low CMC value and good stability.TEM,¹H NMR and drug release assays demonstrated that PCPP system had pH-sensitive size reduction&charge increase&micelle disassembly and drug release.CLSM,FCM and apoptosis assays demonstrated that the micelleplexes not only improved tumor penetration and cellular endocytosis,but were also capable of activating lysosomal escaping mechanism and inducing micelle disassembly and drug release in response to pH stimuli,in turn leading to effective PD-L1 silencing and severe cell apoptosis.The curative effect in vivo showed that PD-L1 blockade photodynamic immunotherapy effciently activated immune response and inhibited immune resistance mediated by the checkpoint gene PD-L1.Consequently,tumor growth and metastasis were effectively inhibited by triggering systemic antitumor immune responses.