Study On The Effect Of Electroacupuncture Combined With MOF Drug Loaded Microneedles On The Inhibition Of Melanoma By TME

ObjectiveMelanoma is a skin tumor with a high degree of malignancy.Its formation,growth and metastasis are closely related to the tumor microenvironment(TME).The composition of TME is complex,but its acidity and chronic inflammation play a very important role in promoting tumor growth and metastasis.Research has confirmed that targeting TME is an effective treatment strategy for improving efficacy and preventing metastasis.Traditional Chinese medicine believes that the pathogenesis of melanoma is complex,with deficiency in origin and excess in appearance,and the treatment needs to eliminate pathogens and strengthen the body.The role of acupuncture and moxibustion in regulating inflammation has been confirmed by many experiments.Acupuncture at Zusanli(ST 36)can not only strengthen the body and strengthen the deficiency,but also inhibit the occurrence of inflammation.The new metal organic framework(MOF)drug delivery drug targeting TME for tumor treatment,combined with the clinical anti-tumor strategy of comprehensive therapy,this experiment intends to treat melanoma by electroacupuncture at Zusanli combined with MOF drug loaded microneedle to regulate the tumor microenvironment,observe its anti-tumor effect,and explore whether electroacupuncture at Zusanli can improve NF in TME-κB inflammatory signal pathway to alleviate the inflammatory reaction in TME,and verify the anti-tumor mechanism of MOF drug loaded iron death,jointly target TME to treat tumors and inhibit the growth of melanoma.MethodsThis experiment is divided into two chapters.Experiment 1:Preparation and anticancer efficacy of MOF drug loaded microneedlesMOF materials are a type of zeolite material with a repetitive network structure formed by self-assembly of organic ligands and metal ions.According to the literature method,the nano MOF material Cu BTC was prepared by the liquid solid solution method with related materials,and the MOF drug carrier was synthesized by using the attraction of chemical coordination bond to carry the drug diethyldithiocarbamate(DDTC)on Cu BTC with the aid of magnetic stirrer and ultrasound Cu-BTC@DDTC。Observation of Cu BTC and CuBTC@DDTC The morphological characteristics of Cu BTC and Cu BTC were detected by Fourier transform infrared spectroscopy Cu-BTC@DDTC Chemical composition and structure;Determination by UV visible spectrophotometry Cu-BTC@DDTC The drug loading capacity.Then the mouse melanoma B16F10 cells cultured in vitro Cu-BTC@DDTC To investigate the in vitro anticancer ability of.Firstly,the CCK-8 method was used to study Cu-BTC@DDTC The inhibitory effect on B16F10 cells was calculated Cu-BTC@DDTC IC50 was divided into normal group,DDTC group,and Cu BTC group for cell experiments,Cu-BTC@DDTC 2(μG/mL group Cu-BTC@DDTC 4(μG/mL group,DDP 20(μG/mL)group;Then detected through EDU cell proliferation experiment and scratch experiment CuBTC@DDTC The effect on the proliferation and migration ability of B16F10 cells was observed through immunofluorescence detection of iron ions and lipid peroxidation(LPO)Cu-BTC@DDTC The effect of iron ion accumulation and lipid peroxidation on B16F10 cells.Prepare soluble MOF drug loaded microneedles using microneedle molds.After observing its external characteristics,the physical and chemical properties of the microneedle are tested.Specifically,the mechanical strength of the drug loaded microneedle is tested to determine whether it can penetrate the skin surface.The solubility of the microneedle is tested to roughly understand the time when all the drugs inside the microneedle enter the body.The skin damage after microneedle treatment is observed,and the skin irritation of the drug loaded microneedle is roughly evaluated;Finally,the antitumor effects of drug loaded microneedles and direct injection drugs on melanoma model mice were evaluated.Specifically,18 mice were randomly divided into a model group,a MOF drug loaded microneedle group,and a MOF drug loaded injection group.After modeling,treatment began when the tumor volume of the mice reached 50-100mm 3,and continued for 10 days.Observe and record the changes of the tumor volume of the three groups of mice every day,measure the tumor weight after treatment,observe the histopathology changes of the tumor through HE staining,and evaluate the anti-tumor efficacy.Detect the changes in liver and kidney function of three groups of mice using low light/spectrophotometry to evaluate the toxic side effects of MOF drug loading.Experiment 2:Study on the in vivo anticancer efficacy and mechanism of electroacupuncture at Zusanli combined with MOF drug loaded microneedlesThe melanoma model mice were treated with electroacupuncture at Zusanli combined with MOF drug loaded microneedles to evaluate its anti-tumor efficacy and mechanism.Specifically,30 mice were randomly divided into 5 groups,namely,normal group,model group,MOF drug loaded microneedles group,electroacupuncture at Zusanli combined with MOF drug loaded microneedles group,and positive drug group.After modeling,the mice were treated when the tumor volume reached 50-100 mm3 for 10 consecutive days.During the treatment,observe and record the changes of tumor volume of the five groups of mice every day.After the treatment,measure the tumor weight,observe the histopathology changes of the tumor through HE staining,and evaluate the anti-tumor efficacy.At the same time,observe the pathological changes of the heart,liver,spleen,lung,kidney,and liver and kidney functions of the five groups of mice through HE staining and micro light/spectrophotometry,and evaluate the safety of various treatment methods.Next,we will discuss the research of acupuncture and moxibustion and drug anti-tumor mechanism respectively.On the one hand,we will detect interleukin-6(IL-6)and tumor necrosis factor(TNF)in serum by enzyme-linked immunosorbent assay(ELISA)-α(Tumor necrosis factorα,TNF-α)、The content of interleukin-10(IL-10)was used to observe the changes of the overall immune status of the body,and then the IL-6 and TNF in the melanoma tissue of mice were detected by immunofluorescence-α、IL-10 fluorescence intensity,detection of inflammatory pathway nuclear factors by immunoblotting κ B(nuclear factor kappa-B,NFκB)Expression of related proteins p65/P-p65,IKK/P-IKK,IKB/P-IKB,and explore whether electroacupuncture at Zusanli can regulate inflammation in TME and inhibit the growth of melanoma.On the other hand,the content of iron ion,malondialdehyde(MDA)and glutathione(GSH)in mouse melanoma was detected by low light level/spectrophotometry,to observe whether MOF drug loaded microneedles can promote lipid peroxidation in melanoma tissue,and the expression of iron death pathway related proteins GPX4 and SLC7A11 was detected by Western blot to observe whether iron death occurred in melanoma tissue.ResultsExperiment 1:Preparation and anticancer efficacy of MOF drug loaded microneedlesThis experiment successfully prepared nano MOF material Cu BTC,and loaded DDTC onto Cu BTC to synthesize a MOF drug carrier with high anticancer activity CuBTC@DDTC o The transmission electron microscopy results show that Cu BTC and CuBTC@DDTC The size of the drug is approximately in the range of 150-200 nm,belonging to the nanoscale drug,which can be well absorbed by cells and will not penetrate the cell membrane randomly;Fourier transform infrared spectroscopy results display CuBTC@DDTC Having both CuBTC and DDTC characteristic peaks,DDTC was successfully loaded onto Cu-BTC;Expression of results by UV visible spectrophotometry CuBTC@DDTC With a high drug loading capacity,1 gram of Cu BTC can carry 1.84 grams of DDTC.The results of the CCK-8 experiment show that,Cu-BTC@DDTC It has significant inhibitory effect on B16F10 cells and is dose-dependent,with IC50 approximately equal to 3.479 μ Cu BTC and DDTC showed no significant cytotoxicity to B16F10 cells at the same concentration of g/mL.The results of EDU cell proliferation experiment and scratch test show that Cu-BTC@DDTC It can inhibit the proliferation and migration of B16F10 cells,and Cu-BTC@DDTC As the concentration increases,the number of cell proliferation gradually decreases,and the migration ability gradually weakens CuBTC@DDTC 4(μ The proliferating cells in the g/mL group had the lowest number and shortest migration distance.Iron ion and Lipid ROS immunofluorescence results display CuBTC@DDTC It can cause the accumulation of iron ions in B16F10 cells,produce lipid peroxidation,and Cu-BTC@DDTC The effect of increasing concentration is enhanced.The mechanical performance analysis of the microneedle shows that the MOF drug loaded microneedle has sufficient strength to penetrate the skin;The results of the microneedle solubility experiment showed that MOF drug loaded microneedles had good dissolution performance.After 10 minutes of penetration into the skin,a large amount of dissolution was achieved,and after about 30 minutes,complete dissolution was achieved;The skin irritation test results showed that after stripping the MOF loaded microneedle for 0 minutes,there were small pinholes on the surface of the skin,no obvious damage or bleeding was observed.After 30 minutes,there was slight redness and swelling in the local area,and after 3 hours,it returned to normal,with less irritation to the mouse skin.The experimental results showed that the tumor volume and weight of the MOF loaded microneedle group were smaller than those of the model group compared to the injection group.There was no difference compared to the MOF loaded injection group.The H&E staining results of the tumor body showed local necrosis and increased vacuoles in the tumor tissue of both the MOF loaded microneedle group and the MOF loaded injection group,proving that the drug loaded microneedle has similar anti-tumor efficacy as injection.In summary,the drug loaded microneedle can achieve similar therapeutic effects as direct injection of drugs,but has advantages such as minimal trauma,low risk of infection,and painlessness,which can replace injection as the drug delivery tool in this experiment.Experiment 2:Study on the in vivo anticancer efficacy and mechanism of electroacupuncture combined with MOF drug loaded microneedlesThe results of the anti-tumor efficacy evaluation experiment showed that the combination of electroacupuncture at Zusanli and MOF drug loaded microneedle group,MOF drug loaded microneedle group,and positive drug group can effectively reduce tumor volume.The volume of the electroacupuncture at Zusanli combined with MOF drug loaded microneedle group was significantly smaller than that of the model group on the third day(P<0.05),and the volume of the positive drug group was significantly smaller than that of the model group on the fifth day(P<0.05)Cu-BTC@DDTC On the sixth day,the tumor volume of the group was significantly smaller than that of the model group(P<0.05).After 10 days of treatment,the tumor volume of the electroacupuncture Zusanli combined with MOF drug loaded microneedle group,MOF drug loaded microneedle group,and positive drug group were significantly smaller than that of the model group(P<0.01).The tumor volume of the electroacupuncture Zusanli combined with MOF drug loaded microneedle group was significantly smaller than that of the MOF drug loaded microneedle group(P<0.05).The tumor weight results showed that the electroacupuncture Zusanli combined with MOF drug loaded microneedle group The tumor weight of the MOF drug loaded microneedle group and the positive drug group was significantly reduced compared to the model group(P<0.01).The tumor weight of the electroacupuncture Zusanli combined with MOF drug loaded microneedle group was also significantly lower than that of the MOF drug loaded microneedle group(P<0.05).The H&E staining results of the tumor body showed that the electroacupuncture Zusanli combined with MOF drug loaded microneedle group,MOF drug loaded microneedle group,and positive drug group all showed local necrosis and increased vacuoles,indicating that the above three treatment methods can inhibit tumor growth,The combination of electroacupuncture at Zusanli and MOF drug loaded microneedle group has the best effect;The results of liver and kidney function showed that there was no significant difference between the liver and kidney functions of each group.The HE staining results of organ tissue sections showed that the results of spleen histopathology in the Zusanli electroacupuncture combined with MOF drug loaded microneedle group were better,and there was no significant difference between the other groups,indicating that the treatment methods of each group were not significantly toxic to the body.The combination of electroacupuncture at Zusanli and MOF drug loaded microneedle group showed better pathological results in the spleen,with clear boundaries between red and white marrow and an increase in white marrow,indicating that electroacupuncture at Zusanli may have improved the body’s immunity.Enzyme linked immunosorbent assay for detecting IL-6 and TNF in serum-α、The results of the IL-10 experiment showed that the DDP group had lower levels of IL-6 and higher levels of IL-10 in serum compared to the model group,which weakened the overall immune anti-tumor function of the mouse body.There was no significant difference between the other groups.Immunofluorescence detection of IL-6 and TNF in mouse melanoma-α、The IL-10 fluorescence intensity experiment results showed that the combination of electroacupuncture at Zusanli and MOF drug loaded microneedle group TNF-α The fluorescence intensity of IL-6 was significantly reduced compared to the model group and MOF microneedle drug loading group(P<0.01),indicating that electroacupuncture at Zusanli acupoint inhibited TNF in TME-α Expression of IL-6,inhibition of inflammatory response in TME,immunoblotting detection of inflammatory pathway NF-κ The experimental results of B-related proteins Pp65/p65,P-IKK/IKK,P-IKB/IKB showed that electroacupuncture at Zusanli acupoint can reduce the ratio of P-p65/p65,P-IKK/IKK,P-IKB/IKB,and inhibit NF-κ The activation of B signaling pathway can reduce the inflammatory reaction in TME and inhibit the growth of melanoma.The content of iron ions,MDA,and GSH in the tumor body was detected by low light/spectrophotometry.The experimental results showed that the combination of electroacupuncture at Zusanli and MOF drug loaded microneedle group and MOF drug loaded mcroneedle group both increased the content of iron ions and MDA(P<0.01),decreased the content of GSH(P<0.01),and increased the content of Fe2+compared to the model group,The results of Western blotting test of iron death pathway related proteins GPX4 and SLC7A11 showed that both the Zusanli electroacupuncture combined with MOF drug loaded microneedle group and the MOF drug loaded microneedle group could significantly reduce the relative protein expression of GPX4 and SLC7A11 compared with the model group,indicating that MOF drug loaded can promote lipid peroxidation in melanoma tissue,induce iron death,and inhibit the growth of melanoma.Conclusion1.MOF drug loading Cu-BTC@DDTC It is a new type of nanomaterial drug with stronger killing ability on B16F10 cells compared to Cu BTC and DDTC alone.It can inhibit cell proliferation and migration.2.Drug loaded microneedles are a new type of drug delivery tool that has similar therapeutic effects to injection drug delivery,but is non-invasive,painless,and has minimal side effects.It is a novel drug delivery method worth studying.3.Electroacupuncture Zusanli Combination Cu-BTC@DDTC Drugs and Simplicity CuBTC@DDTC Drugs can inhibit the growth of melanoma,electroacupuncture combined CuBTC@DDTC The drug has a better effect.4.Electroacupuncture at Zusanli can inhibit TNF in tumor microenvironment-α Expression of IL-6 and inhibition of NF-κ The activation of the B signaling pathway reduces inflammation in TME and inhibits tumor growth.5.Cu-BTC@DDTC It can target the tumor microenvironment,enable melanoma to accumulate iron ions and lipid peroxidation,induce iron death,and inhibit tumor growth.