Construction Of The Functional Dendrimer-based Celastrol Delivery System For The Cancer-targeted Treatment

Cancer has become a major "killer" that threatens human health in recent years,and chemotherapy has become one of the most important means for the treatment of cancer.However,due to the non-specific distribution of chemotherapeutic drugs,their significant toxic side effects and lack of efficacy have become the main reasons for the failure of clinical trials.Nanotechnology-based targeted drug delivery system has been playing an important role in cancer treatment in recent years,because it can break through the biological barriers and overcome the disadvantages of pharmacokinetics of traditional chemical drugs.Especially,dendrimers as a good carrier of drug delivery have gained great attention by researchers due to their small size effect and the abundant surface functional groups.Celastrol has shown favorable anti-cancer efficacy in many cancers.Nevertheless,its clinical application is limited for the obvious toxic and side effects.Our study aimed at solving the problems of celastrol in safety and efficacy for colon cancer treatment,and constructed a new site-specific delivery system by using polyamidoamine dendrimers as nanocarriers to achieve the excellent cancer treatment with high efficiency and low toxicity.In this study,we took the EDC catalytic methods to prepare the functional dendrimers-celastrol bioconjugates with the surface modification of PEG and EpCAM aptamers.UV/fluorescence spectroscopy and urea gel electrophoresis were used to identify whether the nanoconjugates were successfully synthesized.Dynamic light scattering and scanning electron microscopy were used to characterize the particle morphology and size of nanoconjugates.The conjugation efficiency and the release rate under the pH environment of celastrol were,respectively,calculated and evaluated based on the unique UV absorption characteristics of celastrol.The biological stability of the nanoconjugates was then studied according to the measurements of change in the hydrodynamic size and zeta potential.The selective binding abilities of G5-PEG-EpCAM to human colon cancer SW620 cells and human embryonic kidney AD293 cells were investigated by using confocal microscopic imaging in vitro.The cell proliferation-inhibited and apoptosis-induced effects of nanoconjugates on SW620 and AD293 cells were then evaluated by MTT and Western Blot analyses.The antitumor capability in vivo and the toxicity-reduced effects in vivo were further evaluated in the xenograft nude mice models and the mouse acute toxicity assays.The biosafety in vivo was further evaluated by the zebrafish model.Our results showed that the water-soluble dendrimer-celastrol nanoconjugates were successfully prepared,with a hydrated particle size of about 120 nm and a potential of-3.49 mV,which could maintain the stability in physiological environment and exhibit biphasic release in different pH environments that ensured the nanodrugs has sufficient time to enrich at tumor sites when releasing celastrol.The nano drug carrier could selectively target colon cancer SW620 cells not AD293 cells and realize the time-dependent accumulation and internalization in cancer cells.MTT results showed that the IC50 values of celastrol-G5-PEG-EpCAM on SW620 cells were 0.70 and 0.64 times that of celastrol at 7 h and 24 h,and 1.32 times and 1.62 times on AD293 cells,respectively.Therefore,the nanodrugs had the better anticancer activity to S W620 cancer cells and more significantly reduced the toxicity to normal AD293 cells in vitro than free celastrol.The acute toxicity test and tumor inhibition experiment in vivo further confirmed the good antitumor effect of nanomedicine and weakened the toxicity of celastrol on liver and kidney.The evaluation results of the zebrafish model also confirmed that the nanomedicine has higher biosafety than free celastrol.In a word,we designed and constructed the functional dendrimer-celastrol nanoconjugates,thereby greatly improving the anti-tumor efficiency and reducing the toxic side effects for colon cancer treatment both in vitro and in vivo.Our study will provide the research reference for the cancer treatment of celastrol in pre-clinical application.