| Hepatocellular carcinoma(HCC), one of the most common malignant cancer with high rate of recurrence and metastasis, is becoming the most frequent cause of cancer-related death worldwide. Cancer stem cells(CSCs) are considered as the root of tumors initiation, evolution, metastasis and recurrence. But current cancer treatments are mainly acting on bulk tumor cells. It is urgent to seek effective therapies aiming at CSCs to completely cure cancer. The p53 gene, important in cell cycle regulation, is frequently used in gene therapy. And it can induce apoptosis of cell with genetic mutation or damage.Otherwise, p53 mutations associate with a great deal of cancers and CSCs' poorly differentiated characteristics. Doxorubicin, a broad-spectrum chemotherapy drugs, can be inserted into DNA to make it mismatch and hamper replication and transcription of DNA. In addition to causing cytotoxic cells in normal tissue, doxorubicin cannot enter the CSCs to play efficacy because of CSCs' multidrug resistance. However, nanomaterials in virtue of their unique size, charge and surface chemistry can be used as gene and drug carriers for killing CSCs, and it has attracted widespread attention. Nanomaterials as drug carriers can overcome multidrug resistance, while reducing the drug's toxicity to normal tissues. In this connection,the hydroxyapatite being the main component of bones and teeth of vertebrates, is the excellent nanomaterial which surface is positively charged so as to combine with the negatively charged gene by electrostatic attraction. Moreover, the excellent biocompatibility and biodegradability make it possible for hydroxyapatite to be used as a drug and gene carrier. In that regard, HAp loading p53 gene and Dox has enormous potential to improve the situation of cancer treatment.Methods:1. Chemical precipitation was used to prepare two different hydroxyapatite nanoparticules HAp-1 and HAp-2, and modified by PEI. Then FE-SEM, X-ray XRD, FTIR and TGT are used to analyze properties of nano HP particle. such as size, particle morphology and crystallinity. 2. HP's biocompatibility, biodegradability and the efficiencies of p53 gene and doxorubicin loaded were tested. 3. MTT assay, colony formation and hoechst33258 dyeing methods were used to comprehensively assess the inhibitory effect of HP-p53-Dox on hepatic CSCs, which enriched by suspension cultured in serum-free DMEM-F12.Results:1. The main component of HP-1 and HP-2 were hydroxyapatite. HP-1 were short rod-like nanoparticles with length ranged from 20 to 50 nm and width of approximately 20 nm. HP-2 were spherical hydroxyapatite crystals with diameter about 30 nm. 2. Both HAp-1 and HAp-2 have excellent biocompatibility as well as HP-1 and HP-2. They are no significant cytotoxicity to normal liver cells, hepatic cancer cells and breast cancer cells. P53 gene loaded test suggested that HP-1 has a higher load capacity than HP-2. In the HP-1 degradation experiments,HP-1 showed its superior performance that can degrade up to 78%, 60% and 54% respectively at p H 5.6, 6.5, 7.4 on the 90 th days. 3. The survival and colony formation rate of hepatic CSCs treated by HP-p53-Dox was significantly reduced. With Hoechst 33258 staining to CSCs after HP-p53-Dox-treated, the nuclei appeared bright spot, and the cell density is much lower than the control groups. |
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