Construction Of Intelligent Controlled Drug Release Systems Based On Magnetic Nano-carriers And Their Biological Evaluations In Vitro And In Vivo

The advancements of nanotechnology and molecular biology promote theapplication of nanoparticles with special functions in the diagnosis and treatment ofdiseases, in particular of tumor therapy. Through surface modification of nanoparticleswith functional molecules, the functionalized nanoparticles would be endowed withproperties, including real-time diagnosis and drug delivery, which could efficientlyimprove the treatment efficiency and reduce the side effects of drugs.Magnetic nanoparticles (MNPs) attracted much attention due to their inherentmagnetic property. MNPs were widely applied in the fields of magnetic separation,biosensors, medical imaging in vivo, drug delivery and tissue repair, mainly due to theirsimple and diverse synthesis, good biocompatibility and convenience for surfacemodification. As for tumor therapy, MNPs are facing problems as follows: firstly, howto construct stimuli-responsive intelligent nano-carriers for controlled drug releasesystem based magnetic Fe3O4nanoparticles that could achieve on-demand drug deliveryat maglicant tumor tissue; secondly, how to evaluate the biological performancesmagnetic nano-carrier of Fe3O4nanoparticles in vivo of animal, in turn providingscientific evidences for the its safe application in clinic.Based on problems as above, the study employed biomacromolecules to surfacemodify or encapsulate MNPs, thus constructing temperature-responsive magneticliposomes drug delivery system, redox-responsive self-assembled magnetic drugdelivery system and hydrazide bearing poly (methyl methacrylate)(PMMA) decoratedmagnetic pH-responsive drug delivery system. A series of materials characterizations,controlled drug release and biological evaluations in vitro were performed for thosesystems.In addition, the tumor inhibition evaluation in vivo was performed forpH-responsive PMMA decorated magnetic drug delivery system and investigated itsside effects to different organs of nude mice. The study provides scientific evidence forthe potential clinic application of MNPs. Main contents of this study are listed asfollows:1. The synthesis of small scale Fe3O4nanoparticles.The Fe3O4nanoparticles with dimensions below100nm were synthesized bychemical sol-gel method, chemical co-precipitation method and thermal decomposition method. Transmittance electron microscopy (TEM) observations confirmed that MNPsprepared by sol-gel method displayed round morphologies with bad dispersity anddimensions of90.4±4.7nm; Oleic acid-MNPs prepared by chemical co-precipitationmethod showed round morphologies with good dispersity and dimensions of14.3±3.9nm; MNPs prepared by thermal decomposition method displayed differentmorphologies depending on the decomposition temperature, i. e., round features for320°C and cubic features for380°C. Both nanoparticles demonstrated good dispersitywith16.5±2.1nm and30±4.1nm respectively. Fourier transform infraredspectroscopy (FTIR) confirmed the successful synthesis of Fe3O4nanoparticles withcharacteristic peaks at590cm-1. Magnetic hysteresis loops (VSM) results showed thatthe magnetization values for four kinds MNPs were27.113emu/g，57.759emu/g，38.599emu/g and26.998emu/g respectively.2. Construction of temperature responsive magnetic liposomes based intelligentcontrolled drug release system and its biological evaluation.To construct temperature responsive controlled drug release system, hydrophobicFe3O4nanoparticles with dimensions of14.3±3.9nm were mixed thermosensitiveliposomes. The pure thermosensitive magnetic liposomes were obtained throughmagnetic separation. TEM and scanning electron microscopy (SEM) observationsconfirmed that thermosensitive magnetic liposomes displayed good dispersity withaverage diameters of105.3±13.0nm. FTIR, VSM and thermogravimeric analysis(TGA) confirmed the successful fabrication of thermosensitive magnetic liposomes.After loading hydrophilic model drug of5-(and-6)-carboxylfluorescein (CF) andhydrophobic anticancer drug of doxorubicin (DOX), the drug release behaviors ofthermosensitive magnetic liposomes at different temperatures was characterized byfluorescence spectrophotometer. The results showed the controlled drug release systemcould be triggered by external temperature. Confocal laser scanning microscopy (CLSM)and TEM observations demonstrated thermosensitive magnetic liposomes could beendocytosed by breast cancer cells and distributed in the cytoplasm of cells. Cellviability assay and cell morphology observation revealed that the endocytosedthermosensitive magnetic liposomes within cells could release drugs, thus leading tocell apoptosis or death.3. Construction of redox-responsive amphiphilic copolymer/Fe3O4nanoparticlesself-assembled controlled drug release system and biological evaluation.Amphiphilic copolymer was synthesized by crosslinking hydrophilic hyaluronic acid and hydrophobic poly(lactic acid) via disulfide bonds as intermediate linkers. Theredox responsive magnetic controlled drug release system with hydrophilic shell wasconstructed through the self-assembly of amphiphilic polymer, leading to theencapsulation of hydrophobic anticancer drug of paclitaxel (PTX) and MNPs into thehydrophobic inner cavity of the formed micelles. TEM observation showed that thedimension of the system was405.4±28.0nm. FTIR, VSM and TGA analysisconfirmed amphiphilic copolymer and magnetic drug system were successfullyfabricated. A burst release of PTX was observed after addition of10mM dithiothreitol(DTT), which was measured with a UV spectrophotometer. TEM observationdemonstrated the cell uptaken amount of the drug delivery system was much higherthan that of hydrophobic MNPs. Cell viability assay and CLSM observationsdemonstrated the fabricated drug delivery system was biocompatible and PTX could beintracellularly released, leading to Hela cell apoptosis.4. Fabrication of pH-responsive PMMA-functionalized magnetic nanotubes forremotely targeted cancer therapy in vitro and in vivoThe controlled drug release system was fabricated by employing poly (methylmethacrylate)(PMMA) to cover magnetic Fe3O4cubes, and then covalently couplinghydrazide with PMMA. The coupled hydrazine was used to react with DOX to formpH-responsive hydrazone bonds. Thus, drug loading was achieved simultaneously. TEMobservations suggested that PMMA was successfully coated onto the surfaces of Fe3O4nanocubes. The PMMA coated Fe3O4nanocubes displayed good dispersity with edgelengths of32±3nm. FTIR, VSM and TGA analysis confirmed that the designedcontrolled drug release system was successfully fabricated. The real-time release profileof DOX was measured by a UV spectrophotometer. TEM and CLSM observationsshowed that the endocytosis amount of the system was much higher than that of nativeFe3O4cubes. The endocytosed Fe3O4nanoparticles were distributed at cytoplasm ofcells. Cell viability assay and cell nucleus morphology observations suggested that theendocytosed drug delivery system could intracellularly release DOX, leading to cellapoptosis. More importantly, the carriers could be guided to the tumor tissue with anexternal magnetic field (targeting). The weak acidic condition within tumormicroenvironment led to the breakage of hydrozone bonds, resulting in drug releasefrom the Fe3O4cubes carriers. Tumor model of nude mice in vivo suggested that thetumor growth was obviously inhibited by magnetic field guided therapy. Meanwhile,TUNEL staining and H&E staining assays showed that magnetic field guided therapy led to the necrosis of tumor. Besides, H&E staining assay of main organs of nude micesuggested that the fabricated system could drastically reduce the side effects to themajor organs of nude mice when applying an external magnetic field.