| This study focused on anti-tumor effect of p53gene loaded hydroxyapatite (HAp) nanoparticles and the detection of biodistribution, biodegradation and tumor targeting of HAp nanoparticles in vivo. The antitumor effects of pEGFP-Cl-p53loaded HAp nanoparticles with different shapes and sizes were detected. DiR was used to label the HAp nanoparticles. The biodistribution, biodegradation and tumor targeting of HAp nanoparticles in vivo were observed by in vivo imaging system (IVIS). The results in this work will provide some theoretical basis for HAp nanoparticles used as gene or drug vectors in vivo. In addition, We built a new technique to investigate the biodegradation and biodistribution of nanoparticles in vivo. Methods:1) Different shapes and sizes HAp nanoparticles were synthesised through changing preparation conditions, such as pH, T (℃)and so on. Field Emission scanning electron microscopy (FE-SEM), transmission electron microscope (TEM), fourier Infrared spectrum (FTIR), X-ray diffraction (XRD) and dynamic light scattering (DLS) were used to analyse the physicochemical properties of HAp nanoparticles.2) Different doses of pDNA were added into the HAp suspension and1%agarose gel electrophoresis was used to detected the affinity of HAp and pDNA. The influence of affinity was investigated with different treated temperature (4℃and37℃).3) The transfection efficiency of the four kind of HAp nanoparticles with or without PEI modification (named HAp and HAp-PEI) was tested by fluorescence microscope and flow cytometry (FCM), respectively.4) QSG-7701cells were used to investigated the biocompatibility of four kind of HAp nanoparticles to find the proper doses for cell proliferation. The killing effects on pEGFP-Cl-p53loaded HAp and HAp-PEI nanoparticles (HAp-p53, HAp-PEI-p53for short) to cancer cells were examined by MTT assay. Hocehst33348staining tests and Western blot were used to study the mechanism of the four kind of HAp-p53and HAp-PEI-p53.5) Injecting four kind of HAp-PEI-p53intratumorlly to4T1/luc xenograft tumor-bearing BalB/C mice to observe the differences of antitumor capacity of S-HAp-PEI-p53, N-HAp-PEI-p53, R-HAp-PEI-p53and SR-HAp-PEI-p53in vivo. IVIS were used to study the tumor metastasis in vivo. Hematoxylin and eosin staining (HE), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and P53immunohistochemical (P53IHC) and transmission electron microscope (TEM) of tumor sections were completed to analyse the mechanism of antitumor effect of HAp-p53and HAp-PEI-p53.6) DiR was used to label HAp nanoparticles and the tagged HAp was injected into ICR mice via tail vein. The biodistribution, biodegradation and tumor targeting were detected by IVIS and TEM.Results:1) Four kind of HAp nanoparticles were prepared. The shapes and size are as follows:spherical HAp nanoparticles (S-HAp, diameter:50nm), needle-like HAp nanoparticles (N-HAp, average lenth:110nm and width:30nm), rod-like HAp nanoparticles (R-HAp, average lenth:100nm and width:30nm) and short-rod-like HAp nanoparticles (SR-HAp, average length:40nm and width:30nm).2) Compared with R-HAp and SR-HAp, S-HAp and N-HAp can load more amounts of pDNA, the pDNA can be absorbed on HAp nanoparticles firmly after4℃treatment.3) The transfection efficiency of S-HAp, N-HAp, R-HAp and SR-HAp are1.76%,1.03%,0.76%and1.13%. After modified by PEI, the transfection efficiency of the S-HAp-PEI, N-HAp-PEI, R-HAp-PEI and SR-HAp-PEI were13.00%,14.60%,9.62%and16.60%, respectively.4) Four kind of HAp-p53have no effects on cancer cells because of their low transfection efficiency. However, HAp-PEI-p53shows good killing effect toward a variety of cancer cells especially for Hep-3B. Hoechst33348staining and Western blot confirmed that the cell apotosis was induced by P53expression.5) Four kind of HAp-PEI-p53could inhibit the tumor growth in vivo and HE staining, TUNEL, P53IHC and TEM of the tumor sections shows that the mechanism of tumor growth inhibition. In addition, we found that tumor metastasis could be suppressed significantly by the N-HAp-PEI-p53treatment by IV1S.6) DiR to HAp with the ratio of1:50was chosen to tag HAp. HAp nanoparticles with size of40-200nm were only distribute in cytoplasm of liver cell.100μg S-HAp nanoparticles degradated completely in3weeks. S-HAp nanoparticles with the average size of100nm can target solid tumor in vivo. |
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