Construction Of Intelligent Nano-Reserveriors Drug Delivery Systems Based On Fe₃O₄ Magnetic And Mesoporous Silica Nanoparticles And Their Biological Evaluations

The convergence of nanotechnology, material science, biology and medicine promotes the rapid development of nano-biomedicine, which plays an important role in relieving the pain of patients and improving the health of human, in particular of cancer therapy. Anticancer drug controlled release system based on nanomaterials could control drug release and keep the drug concentration in blood, in turn to decease the toxic side effects of drugs to patients. Novel intelligent controlled drug release system could not only specifically recognize the targeted tissues but also deliver drugs to lesion sites on demand of time and amount, and then "blast" against abnormal cells to improve drug efficiency and reduce the accumulated drug toxicity. Such intelligent controlled drug release systems have wide application prospect in clinics.During past decades, magnetic nanoparticles （MNPs） and mesoporous silica nanoparticles （MSNs） attracted much attention. MNPs were currently developed as efficient carriers of drugs and molecular probes and applied for magnetic field sentinel treatment （MST） and nuclear magnetic resonance imaging （MRI） in biomedical fields. Owning to their unique mesoporous structures, large surface areas and tunable pore sizes, MSNs were developed to be one kind of novel biocompatible drug nanoreservior, which had potential application in regarding drug and/or gene delivery. MNPs and MSNs based drug delivery systems are facing common problems as follows: firstly, how to construct novel biological signals responsive controlled drug release system that could specifically deliver drugs to malignant cells in targeting, and to further improve the endocytosis efficiency of nanoparticles; secondly, how to assess the mechanism of interactions between nanoparticles and cells so as to provide theoretical evidences for their safe application in vivo.Based on problems as above, the study empoloyed surface functionalized MNPs and MNSs as substrates to construct redox-responsive magnetic molecular nanoreservior drug release system, magnetically triggered reversible controlled drug release microchip, redox-responsive MSNs end-capped with collagen for targeted drug release and multi-functional intracellular pH-responsive anticancer controlled drug release system based on MSNs. Meanwhile, we assessed the targeted cellular uptake and endocytosis mechanism of MSNs composite controlled release systems against hepatoma cancer cell in details, and investigated inflammation responses of macrophages to MSNs/protein composite, so as to lay basis for designing cell-specfic targeting controlled drug release systems. Main contents and conclusions of this study are listed as follows:1. Construction of redox-responsive molecular nanoreservoirs for controlled intracellular anticancer drug delivery based on Fe₃O₄ magnetic nanoparticles and its biological evaluation.We constructed novel redox-responsive molecular nanoreservoirs drug release system （MNPs-S-S-PEI/β-CD） by employingβ-cyclodextrin gfacted poly（ethylene imines） （PEI/β-CD）, which was conjugated onto the surfaces of MNPs via disulfide bonds as intermediate linkers. Scanning electron microscopy （SEM） and transmittance electron microscopy （TEM） observations confirmed that MNPs-S-S-PEI/β-CD displayed uniform, well defined size with average diameters of around 160±32 nm. Fourier transform infrared spectroscopy （FITR）, zeta potential, thermogravimeric analysis （TGA） and fluorescamine tests confirmed the successful fabrication of MNPs-S-S-PEI/β-CD system. TEM observation demonstrated that both MNPs and MNPs-S-S-PEI/β-CD could be endocytosized by HepG2 cells and located in cytoplasm while not penetrating into nuclei of cells. Finally, camptothecin （CAMP）, as the chemotherapeutic anticancer drugs model, was loaded into MNPs-S-S-PEI/β-CD nanoparticles. Cell viability, cell morphology observation and DNA ladder fragment assay was used to investigate the mechanism of cells apoptosis induced by MNPs-S-S-PEI/β-CD/CAMP.2. Fabrication of magnetically triggered reversible controlled drug release microchip based on Fe₃O₄ magnetic nanoparticlesImplantable poly（D,L-lactic acid） （PDLLA） microchip with magnetically triggered reversible controlled drug release property was fabricated by utlizing the magnetic field responseof Fe₃O₄ nanoparticles, micro-arrayed PDLLA as biodegradable substrate and porous polycarbonate （PC） as seal membrane. PDLLA multi-reservoir device with 9×9 micro-array was fabricated by combining slow wire cutting and soft lithography. Uniform Fe₃O₄ nanoparticles with diameters of 196.1±19.8 nm and magnetization value of 76.8 emu/g were synthesized via a solvent thermal reactionh. The imposition of a magnetic field on the device induces the movement of magnetic Fe₃O₄ particles and leads to the pores of the membrane to be filled or emptied depending on the position of the magnetic field in relation to the polycarbonate （PC） membrane. The drug release is thus accordingly switched off or on. SEM and TEM observation demonstrated the successful fabrication of 9×9 micro-array drug microchip. Such polymeric multireservoir device can achieve actively controlled reversible pulsatiled release of drugs according to the measurement of DNA and vitamin B2 release behavior.3. Construction of redox-responsive targeted drug release system based on mesoporous silica nanoparticles end-capped with collagen and its biological evaluation. Redox-responsive nanoreservoirs based on MSNs （LA-Col-Linker-MSN） that are end-capped with collagen were fabricated by using lactobionic acid-conjugated collagen （LA-Col） as cap, disulfide bonds as intermediate linkers and MSNs as nanoreserviors. Synthesized MSNs displayed uniform distribution with diameters of 130 nm, mesopores of 3.8 nm, surface to volume of 889 cm3/g. Lactobionic acid （LA） molecules acted as targeting moiety to achieve the targeted drug delivery. The results of SEM, TEM, Brunauer-Emmett-Teller （BET）, Barett-Joyner-Halenda （BJH）, FTIR and zeta potential measurements confirmed the successful preparation of LA-Col-Linker-MSN step-by-step. Dithiothreitol（DTT）was used as external stimulus to trigger the redox-responsive release of FITC in order to investigate the controlled release behavior of LA-Col-linker-MSN. CLSM images and flow cytometry assay demonstrated that the LA-Col-linker-MSN nanoparticles were endocytosed and located in the cytoplasm of cells. Redox-responsive targeted drug delivery could be achieved within cells.4. Preparation of intracellular pH-responsive anticancer controlled drug release system based on mesoporous silica nanoparticles and its biological evaluation Intracellular pH-responsive anticancer controlled drug release system based on mesoporous silica nanoparticles （LA-BSA-CBA-MSNs） were fabricated by using lactobionic acid-conjugated bovine serum albumin （LA-BSA） as end-caps, 4-carboxyphenylboronic （CBA） as intermediate linkers and MSNs as nanoreserviors. The results of SEM, TEM, FTIR, zeta potential, TGA, BET and BJH confirmed the successful fabrication of LA-BSA-CBA-MSNs step-by-step. Cumulative release profiles of DOX from LA-BSA-CBA-MSNs@DOX particles in response to pH change confirmed property of pH-responsive controlled drug delivery. The results of confocal laser scanning microscope （CLSM） and TEM demonstrated that the internalized nanoparticles distributed in the cytoplasm and accumulated around cells nuclei, while not penetrated into cells nuclei. The endocytosis mechanism analysis of LA-BSA-CBA-MSNs indicated that LA-BSA-CBA-MSNs were likely to be uptook via the receptor-mediated endocytosis pathway. Cell apoptosis or death would be induced by the uptake of LA-BSA-CBA-MSNs@DOX via the observation of cell nuclei morphology and DNA ladder fragment assay.5. Preperation of mesoporous silica nanoparticel （MSNs） and MSN/protein complex and inflammation responses of macrophage to those nanoparticles Gelatin （Gel）, bovine serum albumin （BSA） and lysozyme （Lys） were immobilized onto the surface of MSNs via succinic anhydride as intermediate linker to fabricate MSNs/protein composite systems, respectively. SEM, TEM, FITR and zeta potential instrument were used to monitor the construction processes of SiO₂, MSNs, Gel-MSNs、BSA-MSNs and Lys-MSNs, respectively. The inflammation response of macrophage including cell morphology, cell viability, NO production, ROS and acid phostphtase activity as well as inflammation cytokine expression （TNF-αand IL-1β） were investigated in details in the study. The results suggest that macrophage would be activated by the endocytosis of SiO2 and MSNs, inducing the inflammation response. By contrast, inflammation response of MSNs could be reduced after the modification with gelatin, BSA and lysosome onto the surface of MSNs, respectively.