Studies On Combining Capacity Between Mannose-conjugated Chitosan Nanoparticles (mCNPs) And Tumor Tissues

1. Observation of expression of macrophage (TAMS) receptor related to tumor in pathological specimens of liver cancer, breast cancer, gastric cancer and colorectal cancer and tumor in normal tissue.2. Searching a simple and economical mannose-conjugated chitosan particle preparation process and method. Using the principle that mannose can specifically bind with mannose receptors expressed in TAMS Cells of tumor tissues, observing combining capacity between mannose-conjugated chitosan nanoparticles (mCNPs) and tumor tissues in the above tumor tissues and normal tissues so as to explore a method of targeted therapy for tumors. Methods:50 cases of tissue sections of colon cancer, breast cancer, gastric cancer and liver cancer were separately collected,including tumor and normal tissues, staining was carried out on specific antibody CD68 and CD 163 of tumor-associated macrophages (TAMS) by using immunohistochemical methods,the cinnamon-dark brown positive stained cells were counted under the view of high magnification light microscope.Proportions of TAMS in various types of tumors and normal tissues were evaluated. Chitosan nanoparticles with different particle sizes were constructed with ion crosslinking method, Take 19mg chitosans and prepare a solution with concentration of 4mg/ml, and then add 1% glacial acetic acid to the solution and stir it for 4 hours at room temperature until becoming clear. Prepare 0.5mol/L sodium hydroxide solution, adjust the pH value of chitosan solution to 6.23 and then add 5mg mannose to the chitosan solution; after the end of reaction, continue to dropwise add 0.5mol/L sodium hydroxide solution to the synthetic solution; when there is a great deal of precipitation, transfer the synthetic solution to the test tube for being centrifuged at the rate of 3000rpm/min., and after removing the supernatant, put the distilled water, methanol and ethyl alcohol in the test tube for washing for two times successively, and finally get the solid sediment. After using distilled water to completely dissolve the obtained solid sediment, put the solid sediment in the dialysis bag (molecular weight cutoff:800) and use clear water to dialyze for 24 hours, then pour the remaining solution to the culture dish for freeze drying for 2 days. The resulting product is the mannosylated chitosan. Take 100mg mannosylated chitosan powder, dissolve it in distilled water and prepare the solution with concentration of 1mg/ml, and then use 0.5mol/L sodium hydroxide solution to adjust the pH value of said solution to 7.48; prepare 4mg/ml ZOL solution under the water bath condition at 30℃ , add ZOL at the mass ratio of 1:3 to the mannosylated chitosan solution and stir it for 30 minutes at room temperature, and put the resulting synthetic products in the dialysis bag (molecular weight cutoff:800) respectively and use clear water to dialyze for 24 hours, and then freeze dry the dialyzed solution for 2 days. The resulting product is the mannosylated chitosan nanoparticle. The corresponding substituted thiourea was obtained through reaction between isothiocyano group in FITC structure of fluorescent dye and primary amine group in the nanoparticles in the chitosan, subsequently the fluorescent secondary wavelength and the maximum absorption wavelength was measured with fluorescence scan. Using the principle that mannose carried by the nanometer granules can specifically bind with mannose receptors expressed in TAMS Cells of tumor tissues, after dewaxing paraffin, antigen retrieval was carried out on the paraffin tumor tissue section,chitosan nanoparticles PBS suspension of different particle size was added with an appropriate proportion, overnight incubation was carried out at 37 "C.The solution was discarded, then it was rinsed with PBS solution and freeze drying was carried out. The binding of tumor & normal tissues and nanoparticles was observed under a fluorescence microscope, distribution of fluorescence stained cells was observed to determine its binding site andcombining capacity between the two elements can be reflected through counting of positive stained cells.Finally, t test statistical analysis was carried out. Results:Positive strength of immunohistochemical expression was judged based on number of positive stained cells under high power microscope,CD68 receptors showed strong positive expression in nests of human liver cancer, breast cancer, gastric cancer & colorectal cancer and on cell membrane of part of the peritumoral cells of the above cancers,but they showed negative expression in normal control group. CD 163 receptors showed positive expression on peritumoral cell membrane and cytoplasm in the observation group of the above cases, but they show negative expression in normal tissues. Ion crosslinking chitosan nanoparticles with diameter of 0.05um-lum were successfully prepared with ion crosslinking method. Its physical and chemical properties are stable. Chitosan nanoparticles were separately observed for histopathologic sections and normal sections after staining, cancer nests and tissues around the tumor were carried out 5HP/per piece,in addition, immunofluorescence stained cells were counted, stained cells in peritumoral tissues were significantly more than those in cancer nest tissues. CD63, CD 163 positive stained cells are significantly different between the observation group and the control group and the difference was statistically significant. CD63 and CD 163 expression was significantly higher than that in observation group. Number of positive stained cells in nanometer granule immunofluorescence staining is significantly different between the observation group and the control group and the difference was statistically significant. Number of positive stained cells in nanometer granule immunofluorescence staining of the observation groupis significantly more than that in the control group. Conclusion:Tumor-correlated macrophages gather in cancer tissues including hepatocarcinoma, breast cancer, gastric cancer and colorectal cancer. Expression of these macrophages is rarely seen in the normal tissues of breast, gastric, colorectal that adjacent to the above tumor positions. Chitosan nanoparticles with particle size 0.05 um-1um has the specific combining ability with the mannose receptor expressed in tumor-correlated macrophages, The nanoparticles can concentrate beside cancer and in cancer nests,but they are rarely combined with normal tissues adjacent to the tumors, The above indicates that chitosan nanoparticles have a certain tumor-targeting property, it will provide a new way of thinking for tumor treatment regarding the therapeutic target selection and development of targeting drugs.