Fabrication And Application Of A PH-responsive And Magnetic Targeting Nanocomposite Greatly Improved Chemotherapy Effect

As one of the most severe threats to humanbeing's health, incidence of malignant tumor has increased in recent years. In clinic, chemotherapy is the most common way to treat those desease. However, chemotherapeutical drugs are usually not specific targeting to the tumor, which makes a low concentration of chemotherapeutical drug in tumor site and a wide range of side effects and damage to normal cells and tissues. These shortcomings severely limit the clinical applicationof chemotherapy and affect the life quality of tumor-bearing patients. Therefore, it is an urgent necessity to design a tumor targeting and conditional responsive system to achieve a high concentration of drugs in the tumor and minimal side effects to the normal cells and tissues.To fabricate the drug delivery system, pH responsive polymer was utilized to load drug and magnetic Fe₃O₄ core is designed for magnetic targeting due to a lower cellular pH value in tumor, Doxorubicin?DOX? was chosen as the modal chemotherapeutical drug?see schematic diagram in appendix?.It is expected that the chemotherapeutic drug is targeted to the tumor site which is located by outlay magnetic field. Then the chemotherapeutic drugs is enriched in the tumor, and released responsive to the cellular pH after endocytosis. By this way, theconcentration of chemotherapeutic drug is elevated in the tumor, with better curative effect and decreased toxicity.In Part ?, pH-responsive polymer PEG-b-PAsp was synthesized through a stepwise process, then Fe₃O₄ magnetic nanoparticles is synthesized by hydrothermal method as the magnetic core of the composite. In order to enhance the stability of Fe₃O₄ magnetic core and reduce its aggregation, SiO₂ layer is coated on the Fe₃O₄ magnetic nanoparticles surface through the St?ber method and sol-gel process,subsequently, pH responsive polymer polyethylene glycol?PEG?-b-poly(aspartic acid?PEG-b-PAsp? was linked onto the surface of the silica layer by amidation reaction. Finally, DOX is loaded as the model drug into thehydrophobic layer of the polymer to form the final product, the p H-responsive and magnetic targeting nanocomposite, Fe₃O₄@SiO₂@PEG-b-PAsp@DOX.The nanoparticle was characterized in various ways: particle size?197.7±1.5? nm and Zeta potential?35.9±0.6? mv by dynamic light scattering?DLS?, DOX drug loading capacity?about 20%? by spectrophotography. Besides, we conducted release test in pH 5.5 and in pH 7.4 buffer solutionrespectively, the resultindicated that the system has a good pH responsive performance.In Part ?, magnetic targeting, biosafety and cytotoxicity of the composite are investigated by fluorescent microscope, biological transmission electron microscopy and CCK-8 kit.Fluorescence microscope and electron microscope images show that composite can effectively internalized by tumor cells, and is enriched in tumor by magnetic effect. In order to analyze the magnetic targeting of the drug carrier, 5?g·mL^-1Fe₃O₄@SiO₂@PEG-b-PAsp@DOX is added to the same cell culture system and culture for 2h. Susquently, the targeting ability of the system in vitro is quantitatively detected by flow cytometry instrument with the help of DOX red fluorescence, flow cytometry datashows the side ofthe cell fluorescence intensity among magnetic field?Xmean = 98.65? is stronger than the side ofthe cell fluorescence intensity out of magnetic field?Xmean = 81.81?, and there was significant difference between the two groups?P < 0.05?, which implied that the composite showed a significant magnetic targeting effect in the magnetic field.In the study of the drug-loading nanoparticle composite carrierkilling tumor cells, we discovered that :The drug composite nanoparticles can effectively kill LLC cells. At 24 h, IC50 of freeDOX and drug carrier is 0.46?g·mL^-1 and 0.89?g·mL^-1, respectively,when at 48 h, IC50 of two is 0.32?g·mL^-1 and 0.46?g·mL^-1, respectively. The results are determined by CCK 8 method, which indicate that the ability of killing tumor cells of the drug carrier is close to the free DOX over time, that may related to the slow release of DOX in nano drug system.The biological safety of the blank carrier is preliminarily investigatedby CCK 8 method and flow cytometry instrument, during the research.When the concentration of the blank carrier is up to 100?g·mL^-1, cellsand blank material are cultured for 48 h, we find that thesurvival rate of three different types are over 80%, and we also find the blank carrier of low concentration has good biological safety,almost has no cytotoxicity.In flow cytometry test, the results also indicates that blank carrier almost does not induce the apoptosis of tumor cells and drug-loaded systemcan effectively induces tumor cell apoptosis, and apoptosis ratio increases with the increase of drug concentration, which is consistent with the free DOX effects on tumor cellsIn Part ?, magnetic targeting and anti-tumor effect in vivo is inspected. LLC tumour-bearing model is established in male C57BL/6N mice; nuclear magnetic resonance imaging is used to evaluate the targeting effect in vivo by comparing the change of signal strength of the controlgroup which is not in the magnetic field. The solution of the composite is intravenously injected in a dose of 4mg·kg^-1, and magnet is fixed in the tumor site for 4 hours, then all the groups are sent to MRI. Data shows the drug delivery compositeis enriched in the magnetic field, that indicated the composite had a good magnetic targeting to the tumor site in the magnetic field.In the experiments on the antitumor research in vivo, tumor-bearing mice are divided into normal saline group?S?, free DOX group?FD?, group out of magnetic field?P?, group in magnetic field?MP?. By recording the change of each mice tumor volume, number of pulmonary metastasis nodule, survival period, body weight, tumor size, and pulmonary metastasis nodule numberto inspect the anti-tumor treatment effect of drugs system, then we find that the survival rates of each group were 40%?S?, 20%?FD? and 80%?P? and 100%?MP?, respectively, after 36 days. Tumor growth curve uncovers that tumor growth faster in normal saline group?S? and group out of magnetic field?P?, and the growth curve of body weight in mice can clearly shows thatonly the average weight of free DOX group?FD? on a downward trend with treatment, combine with the result of survival experiment and tumor growth experiment, shows that free DOX of such concentration administering directly may cause a larger systemic toxicity. Through the observation of the anatomical model mice, we find that the drug-loading composite system inhibits the lung metastasis of the tumor, and the mechanism remains to be further research.In this project, pH-responsive and magnetic targeting nanocompositeis successfully constructed. The composite shows significant magnetic targeting andpH-responsive in vitro and in vivo; and then greatly improve the chemotherapeutic effect and decreases the side-effects of the chemotherapeutic drugs. The composite is expected to be applied in clinic to improve the bioavailability of antitumor drugs, and especially to reduce the side effects of chemotherapeutic drugs.