The Preparation Of Redox-responsive Polymer Hollow Microspheres For Delivery Of Anti-cancer Drug And Gene And Tumor Targeted Superparamagnetic MRI Contrast Agents

Objective:Much attention should be paid on the diagnosis and treatment of cancer as a main disease threatening to human in a few past decades.Since the antitumor drugs have no selectivity between somatic cells and cancer cells and chemotherapy has a poor therapeutic effect,it is very necessary to design and develop the targeting drug carriers.At the same time,gene therapy has been a significant subject,in which the gene transfer plays an important role.According to the diagnosis,contrast agent has made a progress in recent years,especially in NMR-imaging.Based on these,the research project in this thesis is focused on the design and development of a targeting drug and gene carrier as well as a contrast agent.Methods:The functional monomer methacrylic acid and N,N-bis(acryloyl)cystamine were distillation precipitation polymerized to get the drug and gene carrier,the core-shell PMAA/P(MAA-co-BAC).Then the linear core of PMAA were removed selectively by ethanol from the core-shell PMAA/P(MAA-co-BAC).The hollow P(MAA-co-BAC)microspheres were modified with PEI to get the final carrier.The particle size,size distribution and polydispersity index of the hollow P(MAA-co-BAC)microspheres can be calculated through the TEM picture and the hollow structure can also be confirmed.The size and charge changing can prove that the PEI are successfully modified onto the microspheres and elemental analyzer is used to confirm the content of PEI.The turbidity change of hollow P(MAA-co-BAC)/PEI microspheres monitored by a UV-visible spectroscopy at the wavelengh of 630 nm and MS were used to characterize the degradation and molecular weight,respectively.Dox was chosen for the investigation of the drug loading and controlled release behavior of the hollow P(MAA-co-BAC)/PEI microspheres.The in vitro gene transfection was evaluated by Hela cells with the transfection of plasmid DNA(pDNA)encoded with green fluorescent portein(GFP)and the results were determined by confocal fluorescence microscopy.Furthermore,the cytotoxicities of hollow(P(MAA-co-BAC)/PEI)microspheres before and after loading of Dox were assessed via WST-1 assay.Superparamagnetic Fe3O4 nanoparticles were prepared by chemical co-precipitation method with subsequent modification of oleic acid.Then the Fe3O4 aggregates were prepared by emulsion evaporation method.Silica(SiO2)was deposited on the surface of the Fe3O4 aggregates via hydrolysis of tetraethoxysilane.Finally,FA were conjugated to the surface of Fe3O4-SiO2 composites with the aid of 3-aminopropyltriethoxysilane(APS)to prepare superparamagnetic MRI contrast agent with tumor-targeting.The morphologies and structures of Fe3O4-SiO2-FA composites were characterized by TEM,FTIR,XPS and UV spectra.The saturation magnetic intensity,relaxation rate and contrast effect in solution of the Fe3O4-SiO2-FA composites were measured by magnetometers and a clinical MRI instrument.Results and Conclusion:The microspheres with redox-response used as drug and gene dilivery were successfully synthesized.The size of the hollow P(MAA-co-BAC)microspheres was 339 nm,the shell thickness being 26.5 nm and polydispersity index being 1.00?1.05.The zeta-potentials of hollow P(MAA-co-BAC)/PEI microspheres were +31.09 mV after surface modification in the contrast with-29.22 mV of hollow P(MAA-co-BAC)microspheres.The white color of the microspheres solution were quickly changed to shallow and disappeared within 20 min when 10 mM GSH,as a reducing agent,were added in.Upon the addition of the PBS buffer without GSH,the microspheres began to aggregate slightly and degraded gradually for about 2 days.Dox cumulative release from hollow P(MAA-co-BAC)/PEI microspheres was about 12 wt%at pH 7.4 after 25 h in the absence of GSH and in the presence of 10 mM GSH,the cumulate release of Dox increased to 48 wt%in a physiologic pH at the same time scale.In the absence of GSH,Dox released from the hollow P(MAA-co-BAC)/PEI microspheres in pH 5.0 was about 17 wt%in 25 h which was more than the Dox release under the neutral condition at the same period.These results show that the carriers have a response to the pH change and the redox trigger.The remarkable difference of fluorescence level in all cases was observed.Almost no green luminescence can be observed in the absence of hollow P(MAA-co-BAC)/PEI microspheres.While hollow P(MAA-co-BAC)/PEI microspheres were demonstrated good transfection efficiency above a N and P ratio of 168:1.From the result of WST assay,the hollow P(MAA-co-BAC)/PEI microspheres didn't show the obvious toxicity even at high concentration of 1000 ?g L-1,while PEI,a synthetic cationic polymer used as gene carrier,showed 81%cell lehtality even at the concentration of 100 ?g mL-1.The IC50 of free Dox was 17.27?g/ml,contrasting with the IC50 of Dox loaded hollow P(MAA-co-BAC)/PEI microspheres being 72.91 ?g/ml,and the higher IC50 of Dox loaded hollow P(MAA-co-BAC)/PEI microspheres was on the count of partial releasing of Dox.The Fe3O4 aggregates have the size of below 80 nanometers and about 100?120 nm after coating with SiO2.Fe3O4-SiO2 composite particles were spherical and nearly monodispersed.TEM,FTIR,XPS and UV spectra suggest that tumor targeting molecule FA is modified on Fe3O4-SiO2 composite particles.The saturate magnetization Ms of the Fe3O4-SiO2-FA composite particles is 29.75 emu·g-land the T2 relaxivity is obtained as 0.101 × 106 mol-1 s-1.The saturate magnetization Ms and the T2 relaxivity of the Fe3O4-SiO2-FA composite particles showed that the Fe3O4-SiO2-FA composite particles had a potential application for MRI contrast agents because of their saturate magnetization Ms and the T2 relaxivity.