Study On The Reverse Of Photothermal Tolerance Of Tumor By Melanin Composite Nano System

At present,tumor is still one of the most serious diseases threatening human health.Traditional tumor treatment methods include tumor resection,chemotherapy and radiotherapy,but these traditional methods have some limitations,and the treatment effect is often not ideal.Photo thermal therapy(PTT),as a new type of tumor treatment technology,the principle is to kill tumor cells by using high heat generated by materials with high photothermal conversion efficiency,Because of its minimally invasive,high efficiency,low adverse reactions and can inhibit tumor metastasis,it has attracted great attention in tumor treatment.But there is a general phenomenon of heat tolerance in tumor photothermal therapy,which is a key factor affecting photothermal therapy.Heat shock protein(HSPs)is one of the main factors leading to tumor tolerance.HSPs can improve the stress ability of cells,especially heat tolerance,it can form complex with a variety of tumor antigens,which can enhance its resistance to external stimulation,thus producing tolerance,reducing the effect of photothermal therapy,leading to treatment failure.Therefore,in order to overcome the problem of heat tolerance in photothermal therapy,it is essential to reduce the HSPs.The synthesis of HSPs needs to consume a lot of ATP,which are mainly attained from the tricarboxylic acid cycle in the glycoaerobic oxidation pathway.Therefore,this paper proposes to overcome the problem of heat tolerance in photothermal therapy by blocking the ATP supply of HSPs.Because the metal organic frame work zeolites ZIF-8(MOF)can release Zn2+,which can inhibit the oxidase of Cytochrome C in the tricarboxylic acid cycle.It should block the cycle and restrict the production of ATP.So as to reduce HSPs,inhibit heat tolerance,reverse tumor and increase the effect of hyperthermia.In this paper,MOF was firstly prepared by a solvent method,and then the melanin nanoparticles(MNP)were synthesized.MNP was successfully wrapped in MOF to construct the melanin nano delivery system(MNP@MOF).In tumor lysosomes,The carrier MOF can disintegrate and release MNP and Zn2+encapsulated in melanin nanoparticles.Furthermore,the antitumor activity of melanin nano delivery system in vitro was studied with breast cancer cell 4T1 of mouse as the cell model,and the imaging and pharmacodynamics of MNP@MOF in vivo were studied with female BALB/c mice as the experimental animals.The details are as follows:Part 1:The preparation and characterization of melanin nano delivery system.Firstly,MOF and MNP were synthesized,and then,MNP was loaded into MOF to construct MNP@MOF.The maximum absorption peak of MNP at 580 nm was detected by UV spectrophotometer.The average particle sizes of MOF,MNP and MNP@MOF were about 60 ± 6.8 nm,5.4±1.2 nm and 67.6±5.4 nm,respectively,The average potentials were about 10.12±0.48 mV,-22.3±0.8 mV,-9.8 ±2.1 mV,respectively;The results of TEM are consistent with those of particle size analyzer.At 4?,for one month,the particle size potential,encapsulation efficiency and drug loading efficiency of the nanoparticles had no significant changes,indicating that the stability was good.Ultrafiltration centrifugation and UV-vis spectrophotometry were used to determine the encapsulation efficiency of the nanoparticles,which was 92%and the drug loading rate was 10%.The experiment of photothermal conversion performance showed that the temperature of MNP@MOF increased rapidly to more than 42? in three minutes under the exposure of 808 nm laser transmitter,which indicated that it had a high photothermal conversion efficiency.The results of release in vitro showed that the release rate of MNP in MNP@MOF was over 70%at pH 5.0,but only about 10%at pH 7.4.The concentration of Zn2+released from MNP@MOF was 1.4 mg/L at pH 5.0,and 0.055 mg/L at pH 7.4.Therefore,the release of MNP and Zn2+ at pH 5.0 was much higher than that at pH 7.4 release under conditions.Part 2:Antitumor activity of melanin nano delivery system in vitroIn this paper,4T1 mouse derived breast cancer cells were used as cell model,The biocompatibility of blank MOF and the effect of the nano delivery system of melanin on the survival rate of 4T1 cells were studied by MTT;According to the results of cell uptake,the uptake capacity of 4T1 cells to the final preparation was significantly higher than that of free FITC,The results showed that nano preparation was helpful to cell uptake;The expression of ATP in 4T1 cells was detected by ATP kit;Western blot was used to investigate the effects of various preparations on the expression of HSP70/90 in 4T1 cells and evaluate the photothermal tolerance reversion ability of nano delivery system;The photothermal effects of different preparations on 4T1 cells were studied by thermography;AM/PI kit was used to investigate the effect of the preparation on the number of living and dead cells;Flow cytometry was used to analyze the effect of each group on cell apoptosis.The results showed that:MTT test showed that MNP@MOF group had the strongest inhibitory effect on tumor cell activity compared to other groups under 808 nm laser irradiation.The results of uptake experiments showed that the cancer cells could uptake more MNP@MOF,which enhanced preparation concentration in cells.ATP test results showed blank carrier MOF and MNP@MOF+NIR could significantly reduce ATP production,indicating that the carrier MOF could block the production of ATP in tumor cells.The results of Western blot showed that MNP@MOF+NIR had a significant inhibitory effect on the expression of HSP70/90 protein.It was confirmed that the nano preparation could lower HSP70/90 protein;The experimental results of photothermal efficiency show that the temperature of MNP@MOF group could rise to 42? in 3 minutes under 808 nm laser irradiation,and the light energy could be transformed into heat energy successfully.In the experiment of cell viability,compared to other preparation groups,there was the largest number of dead cells in the group treated by MNP@MOF which showed the strongest tumor inhibition activity under 808 nm laser irradiation.In the MNP@MOF group,more cells were induced apoptosis under 808 nm laser irradiation.All results showed that MNP@MOF can significantly inhibit the reversal of tumor photothermal tolerance.Part 3:In vivo imaging and pharmacodynamics of melanin nano delivery system.We selected female BALB/c mice as experimental animals and established the animal model with mouse breast cancer cell 4T1.We investigated the imaging and pharmacodynamics of MNP@MOF in vivo.The pharmacodynamics of the nano system was studied by the changes of body weight,tumor volume.HE staining and TUNEL experiment before and after administration.The results showed that MNP@MOF+NIR group had the strongest antitumor activity,which indicated that MNP@MOF+NIR group could inhibit heat tolerance and enhance antitumor effect.HE staining pathological section showed that MNP@MOF+NIR group had no significant toxic and side effects on all tissues and organs of mice,which indicated that the final preparation could effectively release in the tumor and reduce the toxic and side effects of the preparation.The distribution of MNP@MOF in mice was investigated by in vivo imaging experiment.The results showed that the fluorescence signal intensity of MNP@MOF in tumor site was significantly higher than that of other tissues,indicating that the nano system had good antitumor effect.Based on the above results,it can be seen that the nano delivery system studied in this project has good biosafety,it will lay a good foundation for solving the photothermal tolerance of tumor.