Prepration Of Co-delivery System With Sequential Release Of Gene/chemotherapeutic Behavior And Anti-tumor Effect

Objective:Cancer has become the second leading cause of human health. Chemotherapy is an important auxiliary program after cancer treatment, but traditional chemotherapy drugs lack of tumor-selective due to strong side effects on cancer patients normal cells, and long-term use can cause resistance to chemotherapy, seriously affecting the treatment of chemotherapy effect. Gene therapy can effectively enhance the efficacy of concurrent chemotherapy, further study found that gene therapy combined with chemotherapy, gene sequence with chemotherapy drugs administration, dosing intervals have a great impact on the effect of tumor suppression. For the MDA-MB-231 breast cancer cells, the maximum inhibition rate appeared earlier mi R-21 inhibitor is administered four hours then the administration of doxorubicin, while the administration or to administration of doxorubicin has shown antagonism, and combination therapy inhibition rate of tumor cells is lower than medication alone. The findings suggest that gene therapy combined with chemotherapy and medication administration time must pay attention to sequence a problem that timing issues of combination therapy, the drug carrier which has been raised higher requirements: First, we must realize gene / chemotherapy co-load, making gene with the chemotherapy drug delivery to the same tumor cells play a pharmacodynamics; second, to control gene and the timing of the release of chemotherapy drugs to achieve sequential combination therapy, but in the present study, the carrier does not meet the timing requirements. We built a hollow sphere with gold-based delivery system timing control application which plasmon resonance intensity of near-infrared light absorption characteristics, to achieve a precise release of mi RNA and DOX, and through the vivo and vitro experiments confirmed that tumor inhibitory effect, providing a theoretical basis for clinical application.Methods: 1、Preparation and Characterization of Materials1)Silver ions as the template for the preparation of the gold ball carrier replacement reaction HGPAD. 2)We modified gold balls with PAMAM dendrimer to get HGNPs,which can implement the connection path between the micro RNA with the carrier. 3)The HGNPs through electrostatic adsorption carry chemotherapy drugs doxorubicin hydrochloride become HGPAD. 4)We used MDA-MB-231 cells to confirmed the different inhibition concentration under different timing intervals. 5)After a good sample preparation we used TEM, UV absorption, NMR, mass spectrometry and other methods to characterize nanomaterials. 2、Cellular level verification killing effect on tumor cells and its mechanism of nanomaterials 1)We used the IC50 and MTT experiments to test gene and drug timing therapy with respect to changes of free drug on tumor cells of 50% inhibition concentration. 2) We confirmed the mi RNA inhibition of tumor cells by RT-PCR technique at the genetic level verification. 3) The inhibitory mechanism of nanomaterials on tumor verified was identified by immunofluorescence and western blot experiments 3、The validation drug inhibition of tumor was found by nude subcutaneous tumor model 1) The 4-6-week-old nude mice were inoculated with tumor cells to establish tumor-bearing mouse model. 2) Periodically injection of nano-medicine in nude mice by intravenous with infrared light to stimulate nano carriers achieved timing therapy the release of drug and gene. 3) Regular amount of tumor volume and mice weight assessed the nano medicine. 4) Small animal imaging technology orientation distribution of nanomaterials in mice determined the tumor targeting of HGNPs. 5) Mice were sacrificed and taked the major organs and tumor tissue to be tested by HE staining analysising nano drug toxicity.Results: 1)Replaced by silver ions reacting hollow gold ball,, using gene-modified gold ball carrier with PAMAM to get HGPA, which stable carrier particle size is 86 nm, that through positive and negative charge adsorption with the chemotherapy drug doxorubicin combination get HGPAD.It can release as-mi R-21 stablly in the simulated vitro environment, and in response to the near-infrared light to stimulate achieve controlled release of doxorubicin. 2)Gene and drug timing combination therapy reduced the breast cancer cells MDA-MB-231 IC50 nine times, while confocal microscopy shows the timing of the treatment group significantly increased the amount of drug accumulation within cells by flow cytometry and immunofluorescence experiments confirm the timing of treatment can induce cytochrome C release from mitochondria to cytosol, inducted of apoptosis. 3)Western blot confirmed the timing of the treatment group, both mechanisms may induce apoptosis, including cell apoptosis pathway Fas L-Fas and caspase protein family activation of the mitochondrial apoptotic cytochrom C. 4)Free DOX can not inhibit breast cancer in nude mice transplanted tumor growth.However,the timing combined treatment group tumors almost had no growth during the three weeks,which indicat that the gene and drug timing combination therapy has a good antitumor effect. 5)4 hours later the subcutaneously intravenous into nude mice by injection HGNPs can be observed almost all HGNPs accumulation at the tumor site,and after 24 hours there was still a very strong fluorescence signal at there that confirmed HGNPs has good tumor retention. HE staining showed that in addition to the tumor site is toxic but the major organs were all normal, indicating HGNPs is safe, non-toxic and effective drug delivery systems.Conclusion: We constructed the hollow gold sphere as a gene and drug timing combination therapy nano carrier delivery system, by plasmon resonance effect to achieve geneand drug release timing has a better inhibition effect on tumor growth, and it has higher biological safety, which is a good prospect in the clinical aspects.