Preparation Of Amino-Modified Silica Nanoparticles And Its Performance For Gene Transfer Vector

OBJECTIVE: Searching for an applicable gene transfer vector is always a challange in gene therapy and silica nanoparticles (SLNPS) were expected as its favourable biocompatibility, convenience to modification, low toxicity and prospective application. Our previous research confirmed that the suitable SLNPS would not influence the cell proliferation, and its low acute toxicity and reproductive damage were also demonstrated in the animal experiment. This research aims at modifying the SLNPS through animation, establishing an optimal preparation procedure and parameters, improving the surface potential, DNA binding stability and efficiency, and finally, analyzing the transfection efficiency of modified SLNPS by cell transfection experiment.Part I Preparation of amion-modified SLNPS and parameters optimizationMETHODS: The SLNPS was prepared by hydrolysis in a microemulsion system. The surface of prepared SLNPS was coupled with amino groups using silicane coupling agent AEAPS via optmizing the modification conditions. After a process of vacuum freeze-drying, their size and dispersibility were detected by transmission electron microscope. Surface Zeta potential was measured by electrical potential analysator. Changes of the DNA-binding capability among the nanoparticles of the same batch after storing for 6 months at room temperature were determined.RESULTS: The results indicated that SLNPS were prepared under the optimal parameters. The particles were about 65nm, well-distributed in the solution and their average surface Zeta potential was about +31.5mv, leading to an entire DNA binding at 30:1 (SLNPS:DNA, w/w). Compared with the fresh products, the property of SLNPS after storing for 6 monthes did not show any changes except for a minor decrease of the binding abilitily.Part II Cell transfection efficiency analysis of amino-modified SLNPS METHODS: The capability of DNA binding with SLNPS and the subsequent protection of DNA from the degradation in serum were detected by use of gradient agarose gel electrophoresis. Common transfection and the SLNPS-electroporation compound transfection were compared to analyze the differences of the transfection efficiency and stablity in HL7702 cells. The SLNPS toxicity to HL7702 cells and Bel-7402 cells was detected by MTT assay.RESULTS: The results demostrated that SLNPS could completely bind DNA at 20:1(w/w) and protect DNA effectively from degradation in serum. The transfection efficiency of common transfection can stablely be at 20%～30% by detection of flow cytometry. Though the efficiency of SLNPS-electroporation compound transfection is higher than 60%, it is same as those of the electroporation control. Moreover, it was showed that the SLNPS has a lower toxicity to HL7702 cells than the liposomes do, but Bel-7402 cell proliferation was slowed after three days of adding SLNPS.CONCLUSIONS: The prepared amion-modified silica nanoparticles that were about 65nm, well-distributed in the solution and their average surface Zeta potential was about +31.5mv. The particles can carry the human-derived vector stably, indicating a new material and method in gene transfection.