| Colorectal cancer(CRC)is a commonly diagnosed cancer of the digestive system,which seriously endangers human health and life quality.In recent years,morbidity and mortality of people that suffered from CRC have rised at an alarming rate worldwide.And it has become one of the most serious public health concerns for China and most countries around the world.Currently,clinical outcome of CRC treated via conventional therapies such as surgery,radiotherapy and chemotherapy is unsatisfactory due to various limitations.Development of safe and effective colon cancer therapy has therefore become a crucial and urgent need.Anti-angiogenesis therapy has always been one of most well-researched anti-tumor approach against solid tumors.Anti-angiogenesis therapy inhibits tumor growth mainly by blocking the VEGF/VEGFR signaling pathway to cut off blood supply surrounding the tumors.Breakthrough progress of anti-angiogenesis therapy against certain types of cancer including CRC has been achieved.In addition,it was also reported that suppressing tumor angiogenesis could synergize and enhance efficacy of tumor immunotherapy.The latest research showed that using biomaterials as in vivo carriers could deliver anti-angiogenesis drugs,immunotherapy drugs or tumor antigens safely and efficiently.Moreover,biomaterial-based drug delivery system also promotes synergy between different molecules by simultaneous delivery.Alginate is one of the most well-established polyssachoride biomaterial used for drug delivery.In this study,we report an alginate-based hydrogel as a sustained release system for in vivo delivery of an anti-angiogenic drug recombinant human endostatin(commercially named Endostar),and tumor cell lysate loaded(TCL)polydopamine nanoparticles(TCLN)against CRC.We evaluated its effect on tumor microenvironment and anti-tumor immune response in CRC tumor-bearing mice.Moreover,we also investigated the potential of synergistic cooperation of in vivo delivery and prolonged release of Endostar as an anti-angiogenesis therapy with TCLN as an active immunotherapy.This thesis includes two parts as the following:The first part describes optimization of conditions for preparation of alginate hydrogel(AH).The hydrogel was prepared by ionic cross-linking between sodium alginate and calcium ions.We observed that duration of in vitro drug release has been prolonged to 21 days and it is up to 26 days in vivo.The drug encapsulation efficiency(EE)and the Endostar content(EC)of Endostar loaded hydrogel(EH)varied with the concentration of EH.The EE of 2.75%EH reached 91.8%,and only 17.26%of the release rate was observed on the first day,thus the 2.75%EH was selected in the subsequent experiments.MC-38 colon cancer tumor-bearing mouse model was established to detect the expression of tumor angiogenesis-related factors and cytokines by ELISA assay.The results showed that,treatment of EH with 42 mg/kg Endostar(EH42)significantly inhibited angiogenesis,accompanied by declined level of pro-angiogenic factors and altered expression of 5 cytokines in the tumor tissues.Significantly increased proportion of CD4+and CD8+ T cells and delayed tumor growth were also observed.The above results suggested that the controllable sustained release of Endostar by using Endostar hydrogel formulation improved the bioavailability of Endostar,and subsequently enhanced its efficacy on inhibiting angiogenesis and tumor immunosuppression.EH enabled sustained-release of Endostar treatment could be a potential candidate for combinational therapy to enhance the effect of tumor immunotherapy.The second part focused on the investigation of the combination therapy of EH and tumor cell lysate nanoparticle vaccine(TCLN).In vitro cytotoxicity tests showed that AH,AH mixed with polydopamine nanoparticles(AH/PDA),and EH combined with TCLN(EH/TCLN)were non-toxic to mouse embryonic fibroblasts NIH3T3 cells.ELISA results exhibited that treatment of EH42/TCLN led to dramatic changes in intratumoral levels of cytokines,indicating that it caused changes in the tumor immunosuppressive microenvironment in mice.The proportions of T cell subsets and dendritic cells(DCs)in the spleen,lymph nodes and tumors were detected by flow cytometry.It was found that the combination treatment of EH42/TCLN significantly increased the proportion of CD4+ and CD8+ T cells,facilitated DC maturation,and effectively promoted antigen presentation,improved lysis activity of cytotoxic T Lymphocytes(CTL)in spleen on MC-38 cells(76.5%).The data observed by confocal microscopy showed that the microvascular density(MVD)of the tumor in the EH42/TCLN group was the lowest(0.57),and the number of apoptotic cells in tumors was the highest(74.2%).In the survival experiment of tumor-bearing mice,EH42/TCLN treatment led to complete tumor regress in most of tumor-bearing mice,and the survival rate of mice after 62 days of tumor inoculation was 62.5%,while the other groups were 0%.Mice that survived from the EH42/TCLN treatment remained tumor-free following re-challenge with the CRC tumor cells.All our results showed that the combination therapy not only significantly inhibited tumor angiogenesis significantly,but also elicited dramatic anti-tumor immune response,showing a high degree of synergy effect,implicating a possible relationship between active immunotherapy and anti-angiogenesis therapy.In conclusion,we report that EH42/TCLN could be a potential therapeutic strategy for colon cancer. |
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