Calcium Carbonate Microsperes For p53Gene Deliverpy

Author :HanHuaFeng
Tutor:KongXiangDong
School :Zhejiang University of Technology
CLC :R730.5
TYPE :Masters thesis
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Year:2014
Abstract:
Gene therapy for tumor is significantly important since tumor occurs mostly with geneticmutations.
The safety and efficacy of gene carrier to achieve gene therapy is one of the keyfactors. Currently,
developing an efficient and safe gene vector is attracting much more attention.In this study, three
different properties of calcium carbonate microspheres were prepared by thechemical precipitation
method. The as-prepared calcium carbonate microspheres modified by thepolyethyleneimine (PEI)
was used to load p53gene. The possibility, safty or the antitumoreffects of the modified calcium
carbonate microparticles as gene delivery carrier in vivo and invitro were studied.Methods(1) Three
kinds of spherical CaCO3microparticles were synthesized by the chemicalprecipitation method. These
samples were characterized by field emission scanning electronmicroscopy (FE-SEM), X-ray
diffraction (XRD), fourier infrared spectrum (FTIR) and dynamiclight scattering (DLS) on the surface
morphology, particle size and crystallinity of the particles.A stable process route to synthesize calcium
carbonate microparticles could be obtained.(2) The PEI was used as the surface modification agent to
modify the CaCO3microparticles.The p53gene was loaded to the modified particles to form the
carrier-gene complexes, and thenmultiple cells (such as Hep3B, H1299, Hela, QSG-7701and293a)
were transfected by thecomplexes. GFP-P53protein expression was observed under a fluorescent
microscope.(3) The MTT assay was conducted to study the biosafty of the calcium
carbonatemicroparticles and the antitumor effects of the prepared CaCO3-PEI-pEGFP-C1p53complexes.And the mechanism of their antitumor effects was studied via Hoechst33342staining.
(4) The Hep3B xenograft tumor-bearing of nude mice model was constructed. Byintratumoral
injection of the carrier-gene complexes, the antitumor effects in vivo of the modifiedcalcium
carbonate particles were studied. HE staining and TUNEL histopathologic analysis oftumor sections
were completed to study the tumor-suppressor mechanism.Results:(1) By adding different control
agents, three kinds of spherical, rough CaCO3microparticles were prepared successfully, with an
average particle size of960nm,976nm, and510nm,respectively.(2) By studying the degradation of
calcium carbonate microparticles under different pHconditions, it shows that calcium carbonate
particles degraded rapidly under the acid condition.Comparing with the other microparticles, SCa was
depended more on the acidic pH. Thedegradation tests indicate that the particles are able to respond to
pH value. The toxicity test ofcalcium carbonate microparticles with different concentrations implies
that all the particlespresented good biocompatibility, especially in ACa group.(3) In vitro experiments
confirm that the modified CaCO3microparticles could deliver p53gene to the cells effectively and
express the GFP-P53protein, which results in suppressing theproliferation of cancer cells.(4) In vivo
animal experiments show that the SCa-PEI-pEGFP-C1-p53inhibited tumorgrowth effectively.