| Objective: To develop cancer vaccines is costly and tumor cells can escaped from immunesystems, this experiment based on polyclonal antibody preparation technology and ligand binding theory, the introduction of new immunogens(DOX-BSA) to stimulate the body to produce antibodies(anti-DOX), and targetedto tumor cells bya small molecule(FA-DOX) like bridge, to detection and diagnoses of tumor by enhance tumor immunogenicity. Immunotherapy is less toxic or side effectsand moderate long-lasting, but the period of immunotherapy is longer, to prevent tumor development over to contain, so useof smaller particle delivery system(niosomes) to inhibiting tumor’ early development process to ensure that the effect of immunotherapy.Methods:(1) F127-Chol synthesis by acetal vesicle preparation and its purification and identification.(2) Preparation of Fu niosomes by solvent injection method, encapsulation efficiency was determined by ultrafiltration centrifugation and dialysis.To particle size and entrapment efficiency evaluation, optimize formulation and preparation process of niosome by using single factor method, the particle size, potential, encapsulation efficiency prepared by TEM and NLS, fited the release curve of Fu obtain fitting equation.(3) In order to investigate the in vitro antitumor activity, murine 4T1 breast cancer cells was choosed as tumor cell model to study the cytotoxicity by cytotoxicity assay(MTT method), and further through the flow cell cytometry to detect drug’s dose totally uptake by cells.(4) 4T1 as cell model, 6-7 week old female balb/c mice as an animal model for multiple tumor volume growth and the inhibition rate index, the effects niosomes actual anti-tumor effect.(5) In order to obtain the desired immunogen, glutaraldehyde cross-linked hapten synthesis of exogenous DOX-BSA, by UV and FTIR spectrophotometry to identify them, purified immunogen through dialysis.(6) for evaluation wherein the anti-doxorubicin antibody titer and specificity, 5-6 week old female balb/c mice as a model animal, polyclonal antibodies, inhibition from free DOX competition and draw the corresponding standard curve.(7) synthetic molecules bridge FA-DOX, through the LC-MS and NMR.(8) To confirm the presence of immune complex macromolecules in cells 4T1 cells as a model, including anti-Doxorubicin antibodies in serum antibodies in mice, measured absorbance value of its cellular enzyme-linked immunosorbent assay method. Treatment and in vitro study of molecular targeting function of the bridge, were carried out flow’s cell uptake test, fluorescence microscopy and MTT test.(9) In order to investigate the anti-tumor effect of immunotherapy combined with chemotherapy to 4T1 cells model, 5-6 week old female balb / c mice as an animal model for multiple tumor volume and tumor inhibition rate of growth indicators, study the actual combination therapies antitumor effect; in tissue sections map, based on the effects of different dosing groups killing tumor cells as well as their toxicity to other organs.Results:(1) FTIR and NMR results demonstrate the synthetic material F127-Chol structure.(2) obtained for the more regular spherical vesicle, the average particle size of(240.4?10.1) nm, Zeta potential-68.37 m V, FALT1.83 nm; encapsulation efficiency of 32.76%; in vitro release in line with Weibull release model in simulated body fluid normal environment significantly slow(t1/2 for free Fu of 3.47 times).(3) cytotoxicity test(MTT method) showed cytotoxicity 4T1 niosomes presentation time and concentrationdependent manner, 24 h when each group IC50(μg.ml-1) were: niosomes: 38.02±1.93; control niosomes: 71.78±2.17; blank blister + free drugs: 193.57±2.29; free drug: 207.068±3.58; blank niosomes: 927.14±5.93. Flow’ cell cytometry showed that cellular uptake of niosomes above the blank niosomes and free drug dosing.(4) In the DOXinistration of the 23 days, the niosomes group and the control group niosomes, Fu and saline groups multiple tumor volume growth rate were 64.44,81.16,105.87,150.28; inhibition rate in each group were 52.49%, 33.90% and 20.85%, niosomes group were significantly higher than the control group and Fu group(p<0.05).(5) UV and FTIR show that pured DOX-BSA immunogen synthesis success.(6) to obtain polyclonal antibodies can obviously be free doxorubicin competitive inhibition and IC50 value 75.77±2.81 ng.ml-1, fully achieve the required sensitivity of the experiment.(7) LC-MS and NMR proved the synthesis of FA-DOX.(8) The absorbance values + serogroup intramolecular bridge is much higher than the rest of the group, and the intramolecular bridge + significant difference between the negative sera group; either free doxorubicin- serogroup or intramolecular bridge-negative serogroup the absorbance values were slightly higher than the PBS group; absorbance value of the minimum molecular bridge + PBS group. Flow’s results show that the uptake of Doxorubicin when DOXinistered 2h for each group were: FA-DOX> FA + FA-DOX>DOX; FA+FA-DOX, FA-DOX group were higher than free DOX group; and free folic acid is added to a restricted molecular bridge intake trend. Fluorescence intensity in all group were enhanced with time, indicating the progress of the increase over time of drug intake. 2h, the uptake of each group were less, so the difference between the two groups did not significantly reflected; 4h, intake of FA-DOX group not only significantly higher than the free DOX group and significantly higher than the added folic acid molecule free bridge group; 6h of results similar to 4h, have proved FA-DOX in tumor cell surface with good targeting. IC50 of MTT test FA-DOX at different time(μg.ml-1) were: 24h: 0.99 ± 0.02; 48h: 0.28±0.07; 72h: 0.26±0.04, each group of 4T1 cell growth inhibition rates were : FA-DOX> FA+FA-DOX>DOX.(9) In the DOXinistration of the first 27 days, multiple tumor volume growth rate :saline was 36.16, immune+FA-DOX+niosomes was 8.99, immune + FA-DOX + control niosomes was 12.75, immune + FA-DOX + Fu was 15.08, immune+ FA-DOX+ saline was 17.68, immune +FA+DOX+ niosomes was 18.98, immune + FA + DOX + control niosomes was 22.40 and FA-DOX + niosomes was 13.04; inhibitory percentages were 0, 75.15, 64.73, 58.29, 47.48,48.89,38.06 and63.93. H&E staining showed that:the damge rate of tumor is: immune+FA-DOX+nios> immune + FA-DOX +Fu > immune + FA-DOX +saline≈ FA-DOX+nios > immune + FA+ DOX + nios > saline。Conclusion: The prepared niosomes better physical and chemical properties, facilitates the transport of drugs to cancerous tissue release, cellular assays, animal experiments show that a significant effect. Synthetic immunogen resulting in a better immune effect may be generated when used in combination with the corresponding immune complex macromolecules intramolecular bridge, help to improve the body’s ability to identify tumor cells, in combination with chemotherapy and immunotherapy may improve both treatments inherent flaws, get a good anti-tumor capabilities. |
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