The Construction Of Hydrogel Carrier System By Traditional Chinese Medicine Ecklonia Kurome For The Treatment Of Cancer

As a novel treatment modality of tumors,hypothermal hyperthermia employed relatively lower temperature?<45°C?to damage cancer cells with mild toxicity to normal tissues.However,beyond that inducible heat resistance of tumor cells,the discounted therapeutic effect of low temperature hyperthermia was also ascribed to poor penetration of exogenous light stimulation and low accumulation of photothermal agents in tumor sites.Herein,we constructed a multifunctional in situ hydrogel of sodium alginate?ALG?which was the active ingredient of traditional Chinese medicine ecklonia kurome via Ca²⁺coordinated with ALG to encapsulate the photothermal agent of Ink and azo initiator of2,2?-azobis[2-?2-imidazolin-2-yl?propane]dihydrochloride?AIPH?.Local hypothermia stimulation induced by 1064 nm near-infrared-II irradiation triggered the generation of alkyl radicals and enhanced the hypothermia effective tumor treatment.Objective:Using ALG and Ca²⁺coordination cross-linking mechanism to initially construct the hydrogel carrier system,and utilize in vitro experiments to investigate the photothermal effect and free radical generation capacity of hydrogel after Ink and AIPH were encapsulated.Multifunctional hydrogel was applied to colon cancer model to evaluate its synergistic photothermal treatment effect on tumor cells,and to explore the mechanism of multifunctional hydrogel's anti-tumor effect.Methods:1.Prepare AIPH+Ink+ALG multifunctional hydrogel,and evaluate the morphology and characterization of the gel by scanning electron microscope,DLS ditector and infrared spectrum scanning.The gelatinization characteristics of ALG at different concentrations were studied and the sustained release effect of the encapsulated drug was evaluated by UV-vis scanning experiment.The photothermal effect and photothermal stability of multifunctional hydrogels after laser irradiation were monitored by infrared thermal imaging experiment.The free radical production ability of multifunctional hydrogels was evaluated by ABTS detection agent and methylene blue decolorization experiment.DCFH-DA fluorescent probe was used to detect the formation of free radicals after the gel was applied to HCT-116 cells.2.In vitro,HCT-116 and 293T cells were selected to determine the biosafety of AIPH+Ink+ALG multifunctional hydrogel without laser irradiation by CCK-8 method.CCK-8 test was used to evaluate the antitumor activity of multifunctional hydrogels containing different concentrations of AIPH and Ink after combined photothermal treatment.Confocal microscopy with Calcein-AM/PI staining,flow cytometry quantitative analysis with Annexin V-FITC/PI staining,and mitochondrial membrane potential assay were also used to evaluate the effect of multifunctional hydrogel on apoptosis of colon cancer cells after laser irradiation.The anti-tumor mechanism of multifunctional hydrogels was analyzed using the lipid omics LC-MS platform.3.Establishment of colon cancer nude mouse model,specimen collection and determination of related indexes.High resolution photoacoustic imaging system was used to monitor the gel formation and fixation after intratumor injection.HCT-116 tumor-bearing nude mice were randomly divided into 6 groups.The therapeutic effect of functional gel combined with hypothermal hyperthermia in vivo was monitored by the changes of body weight and tumor volume.Pathological changes of tumor tissues were studied by hematoxylin/eosin?H&E?staining and TUNEL immunofluorescence staining.Frozen sections of tumor tissue were prepared for DCFH-DA/DAPI staining to study the changes of free radical content in tumor.Immunohistochemical experiments were conducted to investigate the pathological changes of major organs after treatment,and to evaluate the toxicity of multifunctional hydrogels in vivo.Results:1.?A mass of Ink nanoparticles were observed to accumulate on the gel surface through the material characterization experiment,and the infrared absorption spectrum of AIPH+Ink+ALG at 360 nm had a strong absorption peak similar to that of AIPH,and the remaining bands were almost the same as that of Ink,indicating that AIPH and Ink could be effectively sealed into the hydrogel through the cross-linking between ALG and Ca²⁺.?The gelatinization experiment showed that when the ALG concentration exceeded 5mg/m L,Ca²⁺solution could be rapidly gelatinized,so this concentration was adopted for the follow-up experiment.?After constant temperature oscillation of AIPH+Ink+ALG for 24 h,the released AIPH and Ink have almost no US-Vis absorption,thus ALG hydrogel can be used as a drug sustained-release carrier.?In addition,in the evaluation of the photothermal effect of functional gels,the temperature of the multifunctional hydrogel encapsulated with 25?g/m L Ink can rise to 44?after 1064 nm laser irradiation for 10 min,and the photothermal stability is good under 5 times of repeated irradiation,so as to achieve the purpose of low-temperature hyperthermia.?The ABTS detection agent was used to evaluate the ability of AIPH decomposition to generate free radicals.It was found that the absorbance of ABTS+·at 44?was significantly higher than that at37?,indicating that AIPH decomposition rate was faster at a higher temperature.?After 10 min of laser irradiation,the ABTS+·generated by AIPH+Ink+ALG functional gel incubated with ABTS reached the peak,and the fading rate also reached 47.2%after incubation with MB,indicating that the multifunctional hydrogel could decompose to produce a large number of free radicals after laser irradiation.?ROS expression in HCT-116 cells was detected by staining.It was found that the groups AIPH+Ink+laser and AIPH+Ink+ALG+laser showed strong green fluorescence signals,suggesting that the functional gel had a good inhibitory effect on tumor cells.2.?CCK-8 essays showed that the components of the multifunctional hydrogel had no obvious cytotoxicity to both HCT-116 and 293T cells.?To evaluate the in vitro photothermal synergistic therapeutic effect of multifunctional gel in HCT-116 cells,the survival rate of cells in hypothermic treatment group after 1064 nm laser irradiation was73.6%,while that of the functional gel group was 19.8%.?Confocal qualitative evaluation of apoptosis showed that AIPH+Ink+laser group and AIPH+Ink+ALG+laser group performed strong red light.?The flow quantitative results showed that the apoptosis rates of two groups were 44.38%and 45.0%,respectively.?The red fluorescence was observed to turn green in JC-1 staining experiment,indicating the decrease of mitochondrial membrane potential,which was significantly different from other groups.The above results further confirmed that low temperature photothermal effect of Ink induced by NIR-II laser irradiation triggered a mass of free radicals generated by AIPH and effectively induced the apoptosis of cancer cells.?LC-MS lipidomics platform was used to explore the changes of cellular lipid level after involvement of free radicals.A total of 14 lipid molecules were detected and 99 lipids were screened for hierarchical clustering analysis.Compared with Ink+laser group,the content of polyunsaturated fatty acids in AIPH+Ink+laser group significantly decreased,and the trend of lipid metabolites was similar.It is concluded that the mechanism of cytotoxicity of AIPH under heat is related to free radical mediated lipid damage.3.?With Ink as a contrast agent,it was found in the photoacoustic imaging experiment that the PA signal strength was still 0.907 after ALG hydrogel was injected into mice for 24 hours,showing strong fixation ability.?In the subcutaneous tumor model,it can also be intuitively seen that ALG effectively reduces drug leakage after gel formation in the tumor.?The AIPH+Ink+ALG+laser group rapidly heated up to 44.9?five minutes after the injection into the tumor-bearing mice with the infrared thermal imager,showing a good photothermal effect in vivo.?In the intratumoral free radical detection experiment,it was found that tumor sections of the AIPH+Ink+ALG+laser group showed strong DCF fluorescence signal,which proved that free radicals generated by the thermal decomposition of AIPH could significantly enhance the oxidative stress in tumor.?Pharmacodynamic evaluation of HCT-116 subcutaneous tumor model showed that the tumor inhibition rate of AIPH+Ink+ALG+laser group reached 88.24%,and the multifunctional hydrogel group was observed to have the most obvious tumor cell apoptosis in immunohistochemical and immunofluorescence experiments,showing superior in vivo photothermal synergistic treatment effect.?There was no significant difference in body weight during treatment in nude mice,and no significant pathological changes in major organs,indicating that the functional gel had good biocompatibility.Conclusions:1.A multifunctional injection gel which use the effective component of traditional Chinese medicine ecklonia sodium alginate as the carrier was constructed,encapsulating photothermal material of Ink and thermal-degradable azo initiator.Under the hypothermal hyperthermia,lots of free radicals were generated along with the thermal degradation of AIPH to synergistically kill tumors.2.We confirmed the efficacy of synergistic therapy by inducing lipid oxidation of cancer cells in vitro and eradicating tumors in vivo.3.Simultaneously,the strong immobilization capacity of hydrogel was attested both in vitro and in vivo,which effectively prolongs the duration of drug action.4.Furthermore,the formulation could also exert strong photoacoustic signals,which were utilized to monitor the stability of the composite hydrogel.5.Systematic toxicity evaluation also proved that the strategy had high biological safety and biocompatibility.