Design、Synthesis And In Vitro Evaluations Of Organic/Inorganic Hybrid Nanocarriers For Drug Delivery

Organic/inorganic hybrid nanoparticles that integrate the virtues of organic segments such as intelligence,biocompatibility and biodegradability and the enhanced stability of inorganic moieties have drawn considerable attention for drug delivery applications.Of all the reported hybrid systems,silica-based hybrid nanoparticles probably represent the most investigated ones due to the obvious advantages of silica-based in-situ cross-linking catalyzed by acid or base without the addition of any small molecules as a cross-linker.However,the degradation of the formed silica network after cross-linking remains a long-term issue due to its high density and low dissociation constant,which apparently hampers the clinical translations of silica-based hybrid systems.Great progresses have been devoted to develop biocleavable silica nanoparticles,however,the approaches developed so far generally suffered from tedious multistep modifications after sol-gel process.Therefore there is considerable scope for the development of facile yet effective strategies toward bioreducible silica-based hybrid nanoparticles.For this purpose,in this thesis a novel reducible silica monomers were designed and synthesized.To demonstrate the potential of these monomers for the construction of biodegradable silica-based hybrid nanoparticles,amphiphilic block copolymers were synthesized by a combination of ring-opening（ROP）polymerization and reversible addition-fragmentation chain transfer（RAFT）polymerization.The structure and composition of the synthesized polymers was characterizaed by nuclear magntic resonance（¹H NMR）spectrometer as well as size exclusion chromatography and multi-angle laser light scattering（SEC-MALLS）analyses.The size and the morphology of the formed self-assemblies were examined by dynamic light scattering（DLS）and the transmission electron microscopy（TEM）.The cytotoxicity of the drug-loadedmicelleconstructswasevaluatedinvitrousingthe3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium（MTS,Promega）assay.The cellular uptake efficiency of the drug-loaded micelles was further quantified using flow cytometry（FCM）.The detailed contents and the primary results of this thesis are summarized as follows,1.In the second chapter,a reducible silica monomer based on 3-aminopropyl triethoxysilane,(2-（（2-（methacryloyloxy）ethyl）dithioalkyl）ethyl（3-（triethoxymethylsilyl）propyl）carbamate（TESSPMA）that integrates a polymerizable methacrylate structure and in-situ cross-linking sites of silica precursors via a reduction-sensitive disulfide bridge,was designed and successfully synthesized.Further polymerization of this monomer was carried out using ATRP and RAFT techniques.The structure of the synthesized polymers was characterizaed by¹H NMR and SEC-MALLS analyses.Then the amphiphilic block polymer of POEGMA-b-PTESSPMA was constructed based on the new reducible silica monomer.The crosslinked organic/iorganic hybrid nanocarriers were then prepared with the addition of a catalytic amount of triethylamine（TEA）.DMF that is a good solvent for all the building blocks was added in the micelle solution to examine whether the cross-linking is sufficient.The significantly increased size rather than destroyed for the SCL micelles were observed after dilution with 10-fold amount of DMF strongly supports the preservation of micelle structure even after transfer into an organic solvent.In order to investigate whether disulfide bond can be destabilized at the lesion site,the size change of the particle were monitored by DLS in PBS buffer solution with 10 mM GSH mimicking the intracellular reducing environment of tumor tissue.The overall results confirmed that the novel reducible silica monomer can not only improves the stability of hybrid micelles,but also promotes its destabilization at the localized disease tissue toward enhanced therapeutic efficency.2.In the third chapter,to further increase the drug loading capacity of crosslinked hybrid micelles,a core-shell-corona（CSC）hybrid micelle was fabricated based on an amphiphilic triblock copolymer of PCL-b-PTESSPMA-b-POEGMA.The degree of polymerization（DP）of the central PTESSPMA block was optimized to realize the elegant tradeoff between the sufficient stabilization of micelle structure and then amphiphilic copolymer of PCL₂₅-b-PTESSPMA₆-b-POEGMA₇₇ was used for loading DOX to investigate the drug loading,drug release behaviors and the cytotoxicity in HeLa cells.The results confirmed that the destabilization of the organic/inorganic hybrid nanocarriers is accelerated by disulfide bond cracking with10 mM DTT.In vitro drug release experiments showed that the cumulative drug release of nanocarriers was increased after cross-linking under reductive environment.Therefore,the reducible silica monomer developed herein offers a highly straightforward and robust strategy toward biocleavable silica-based hybrid nanoparticles for controlled drug release.3.In the fourth chapter,to realize complete destabilization of the nanocarriers at the tumor tissue toward greater therapeutic efficency,a series of amphiphilic triblock copolymers with disulfide bonds in both the main and side chains were prepared using a reducible double-head agent.The polymers were synthesized with an almost identical polymer composition and structure to the polymer constructs prepared in the third chapter and was used to investigate the effect of additional disulfide bond in the polymer main chain on the in vitro properties and performance of the hybrid nanocarriers for anticancer drug delivery.A detailed comparison study revealed that the introduction of disulfide bonds in the main chain can promote the full decomposition of the hybrid nanocarriers toward greater intracellular drug release and therapeutic efficency.4.In the fifth chapter,the structure of TESSPMA was modified to further increase the drug loading capacity of the resulting organic/iorganic hybrid nanocarriers without compromised micelle stability.A new reduction-sensitive silica monomer 2-（（2-methacryloxy）ethyl）dithioalkyl)ethyl（3-diethoxymethylsilyl）propyl carbamate（DESSPMA）was developed.¹H NMR and SEC-MALLS were then used to investigate the controllability of RAFT polymerization of this silica monomer.The results demonstrated that DESSPMA with less crosslinkable functions than the previously used TESSPMA provided the resulting SCL micelles with lower cross-linking density without compromised micelle stability,leading to simultaneously greater drug loading capacity and therapeutic efficacy of DESSPMA-based constructs.Therefore the“less is more”silica-based cross-linking strategy developed herein provides a facile and robust route to engineer organic/inorganic hybrid nanocarriers with enhanced therapeutic efficacy.