| Chitosan, the chemical name is2-Amino-2-deoxy-(1,4)-β-D-glucopyranan,it is obtained by deacetylation of chitin. As chemotherapy drug carriers, chitosan has been extensively in attention since its excellent biological properties. It is non-immunogenicity, non-toxicity, non-hemolytic reaction, non-irritating, no heat source reaction, and it offer a high degree of biocompatibility, biodegradability, bio-adhesivity, anti-tumor effects etc. chitosan is considered as novel drug carriers with brilliant prospects.5-fluorouracil (5-FU) is a broad-spectrum chemotherapy drug, and it can be used to treat digestive neoplasms, breast cancer, skin cancer, lung cancer, cervical cancer, bladder cancer, ovarian cancer, etc. In recent years, many researchers have studied on the antitumor effect of chitosan and its derivative, and the feasibility of chitosan as drug carriers. In this study, we used chitosan as drug carrier, preparated5-FU-CN controlled release formulation, measured its characteristics, and tested its anti-ovarian cancer cell effect.Aims1. Exploring experimental conditions to prepare5-fluorouracil-chitosan nanoparticles (5-FU-CN), to prepare and characterize5-FU-CN.2. To study cell proliferation effects induced by5-FU-CN on ovarian cancer A2780cell in vitro.3. To study cell apoptosis effects induced by5-FU-CN on ovarian cancer A2780cell in vitro.Methods1. Preparing5-FU-CN by ionotropic gelation method,discovering the influences of the different mass ratio of CTS to TPP, CTS solution mass concentration and mass ratio of CTS to5-FU on the reaction solution system. Observing the form of5-FU-CN by transmission electron microscopy. Compared the release characteristics of5-FU with5-FU-CN in vitro by dialysis method.2. Human ovarian cancer cell A2780cells were treated with5-FU-CN,the morphological change of cell was observed by inversion microscopy, the growth inhibitory effect was tested by MTT assay, the effect of proliferating cell nuclear antigen (PCNA) protein expressions was detected with immunocytochemistry, the change of cell cycle was detected by flow cytometry.3. Human ovarian cancer cell A2780cells were treated with5-FU-CN, the effect of cell apoptosis was detected by flow cytometer and TUNEL method, the change of apoptosis-related genes (caspase-3,caspase-8,bax,bcl-2) were detected by Real Time PCR technique. Results1. Fixed the CTS solution mass concentration as1mg/mL, as the mass ratio of CTS to TPP increasing, the reaction solution system turn turbid gradually; similarly, Fixed the mass ratio of CTS to TPP as5to1, as the CTS solution mass concentration increasing, the reaction solution system also turn turbid. According to the exploration for experimental conditions, when we prepared CN and5-FU-CN,we chose the mass ratio of CTS to TPP as5to l,the CTS solution mass concentration as1mg/mL and the mass ratio of CTS to5-FU as1to1.Transmission electron microscopy studies shown that CN is regular spherical, smooth-surfaced, approximately30-60nm in diameter. The nanoparticles in the liquid had ideal suspension stability and uniformly dispersion. After encapsulated5-FU, the diameter of nanoparticles increased with increasing the mass proportion of5-FU,and the particle size distribution turn wider. Dialysis experiment in vitro indicated that free5-FU didn’t have controlled-release characteristics. But5-FU-CN had controlled-release characteristics, and the release-time could be sustained about120hours.2. After given length of time, the A2780cells treated with5-FU-CN showed growth slowly,decrease in the population and significant morphological changes including volume decreased, cell shrinkage, and appeared small vesicles. These changes were more obvious as extending treatment time. The MTT assay result showed that5-FU-CN had certain inhibitory action on the growth of A2780cells, and such an inhibition was concentration-time dependent. Compared with free5-FU,before24hours, the anti-ovarian cancer effect of5-FU-CN was poorer than free5-FU, but after24hours,5-FU-CN’s antitumor effect was better than free5-FU, and the difference between both groups was statistically significant (P<0.05),this result indirectly proved5-FU-CN had the property of medicine controlled-release.5-FU-CN could inhibit the expression of cell’s PCNA (P<0.05). At the same time, Compared with free5-FU,5-FU-CN made the proportion of cell on S phase down, and made the proportion of cell on G1phase up. As the extension of medication time, the inhibition effect of cell cycle on G1phase of5-FU-CN was more obvious.3. Flow cytometer and TUNEL method proved that5-FU-CN counld induce apoptosis of ovarian cancer cell A2780cells. Compared with free5-FU, after24cultures, the apoptosis-inducing effect of5-FU-CN was poorer than free5-FU, but after48h cultures, the apoptosis-inducing effect of5-FU-CN was better than5-FU, and the difference between both groups was statistically significant (P<0.05).5-FU-CN could increase the mRNA expression level of caspase-3, caspase-8, bax and decrease the the mRNA expression level of bcl-2to induce cell apoptosis, Although the gene regulatory speed of5-FU-CN was slower than free5-FU,the effect of gene regulatory of5-FU-CN was much more potent than5-FU,and the actuation duration is was more longer.Conclusion1. CN and5-FU-CN can be prepared by ionotropic gelation method. When the mass ratio of CTS to TPP is5to l,the CTS solution mass concentration is1mg/mL and the mass ratio of CTS to5-FU is1to1,we can make CN and5-FU-CN have good stability,good dispersion, similar size,and their diameter distribution is narrow.2. Release study of5-FU-CN in-vitro indicated that it had a controlled release feature. The release process included two parts:burst release and stagnant release,and the release process could last for about120h。3.5-FU-CN has certain growth inhibitory effect on ovarian cancer cell A2780cells in vitro, the effect appeared concentration-time dependent.,and the growth inhibitory effect on A2780cells of5-FU-CN was was much more potent than5-FU and CN. Compared the anti-ovarian cancer effect of5-FU with5-FU-CN in vitro,5-FU-CN exhibited disadvantaged antitumor effect in the earlier, but superior in the later, this result indirectly proved5-FU-CN had the property of medicine controlled-release.4.5-FU-CN could inhibit the cell cycle in G1phase,at the same time inhibit the expression of cell’s PCNA,thereby5-FU-CN counld inhibited A2780cells’ proliferation. 5.5-FU-CN could induce ovarian cancer cell A2780cells apoptosis, and the effect significantly enhanced as extending the action time.It could be the result of medicine controlled-release.6.5-FU-CN could induce A2780cells apoptosis by regulating apoptosis-related gene. The up-regulating of mRNA expression were caspase-8, caspase-3, bax, The down-regulating of mRNA expression was bcl-2. |
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