An NIR-activated Polymeric Nanoplatform With ROS- And Temperature-sensitivity For Combined Photothermal Therapy And Chemotherapy Of Pancreatic Cancer

Background:Pancreatic cancer is the third leading cause of cancer deaths,and the treatment of pancreatic cancer has always been a problem worldwide.In the past 40 years,there has been no significant progress in its treatment.At present,there are many treatments for pancreatic cancer,such as surgical resection,chemotherapy,radiotherapy,immunotherpy,gene therapy,etc.But each single treatment has its own limitations.Surgical treatment is a radical cure for pancreatic cancer,but the onset of pancreatic cancer is hidden,and many opportunities for surgical treatment have been lost when it occurs.Chemotherapy is a traditional treatment method,but for decades,there are still disadvantages of low bioavailability and large side effects.Multi-drug resistance can be induced after prolonged use.Photothermal therapy(PTT)is a minimally invasive tumor treatment technology developed in recent years.It is minimally invasive,highly targeted.efficient and controllable.It can achieve targeted killing,thereby reducing toxic side effects,but PTT is exposed to light Restrictions are not effective on the deep cells in the tumor.Clinical practice and multi-party experimental studies have shown that monotherapy is difficult to completely eliminate tumors.In view of the complexity,diversity and heterogeneity of tumors,the current clinical research trend has gradually shifted from focusing on monotherapy to combination therapy,that is."multi-modal collaborative therapy",that is,several types of monotherapy are integrated into a single nanoplatform.The interaction between them can not only produce significant superadditiveness(ie "1+1>2"),but also can make up for the shortcomings of the single treatment.With the development of nanotechnology,intelligent drug delivery systems for co-administration of two or more therapeutic agents have made combined therapy possible.Among them,polymer micelles are a relatively mature drug-carrying system.The polymer micelles are nanocarriers composed of a hydro phobic core and a hydrophilic shell.It has good thermodynamic stability and the hydrophobic core can be wrapped.Hydrophobic drugs create a microenvironment for lipophilic drugs,thereby increasing the solubility of the drugs and significantly improving the bioavailability,and can be used as a carrier system for many hydrophobic anti-tumor drugs.The commonly used trigger systems for nano-drug delivery systems include endogenous and exogenous stimuli.Exogenous stimuli include light,temperature,magntic field,ultrasound,etc.,while endogenous stimuli use specific stimuli within the tumor's internal environment,including pH value,glucose gradient,hypoxia,cell cytoplasmic GSH concentration and ROS,etc.The normal phase thermosensitive micelle is a micelle with a high critical solution temperature(UCST).When the temperature rises to near the critical temperature,Depolymerization occurs to release the drug quickly,with higher stability and more complete drug release performance.Based on this,this study combined chemotherapy and photothermal therapy to prepare the amphiphilic amphiphilic polymer P(AAM-co-AN)-b-(B-DMAEA)UCST-type normal-phase temperature-sensitive composite micelles,Then the chemotherapeutic drug camptothecin(CPT)is encapsulated in micelles,and then the photosensitizer ICG is adsorbed outside the micelles through cation adsorption to form functional micelles(CPT@PAAB@ICG).with CPT as the core,ICG is the outer layer.The polymer micelles pass through the ERP to the tumor.The excessive active oxygen in the microenvironment inside the tumor reduces the surface potential of the nanopafticles,thereby releasing the absorbed ICG.After the ICG is released,the weakly charged functional micelles become smaller.It can penetrate deeper into the tumor.On the other hand,through the irradiation of 808 nm near-infrared(NIR)laser,ICG will produce a thermal effect,photothermal therapy effectively kills tumor cells,and at the same time,the heat generated by ICG triggers the polymer to change from water-insoluble to water-soluble and release Load drugs and chemotherapy drugs CPT to achieve chemotherapy simultaneously.Synchronously realize the combination of hyperthermia and chemotherapy,kill the cells inside and outside the tumor,avoid the release of drugs during the delivery process,and reduce toxic side effects.Object:We aim to gain NIR-activated polymeric nanoplatform with ROS-and temperature-sensitivity for combined photothermal therapy and chemotherapy of pancreatic cancer,and discover the potential of CPT @ PAAB @ ICG and in vitro anti-pancreatic cancer.And the ability to penetrate the deep penetration of pancreatic cancer-Methods:1.the amphiphilic polymer PAAB was prepared by the high temperature thermal decomposition method and the reversible addition method(RAFT)respectively.and the CPT@PAAB temperature-sensitive micelles were prepared by the thin film dispersion method,and the ICG was adsorbed by the charge adsorption method To the outer layer of micelles,CPT@PAAB@ICG polymer micelles are formed,and a series of related characterizations of their properties are carried out through DLS,electron microscopy,etc.2.The construction of CPT@,PAAB@ICG drug delivery system and the evaluation of in vitro photothermal effects.CPT@PAAB@ICG solutions of different concentrations were placed in a 50 ? Celsius water bath,and the value of CPT released by UV was measured.3.Using BXPC-3 pancreatic cancer cells as a model,reactive oxygen species and cytotoxicity experiments were performed to evaluate the effect of NIR-triggered CPT@PAAB@ICG drug delivery system against BxPC-3 pancreatic cancer cells in vitro.4.Using tumor-bearing mice as a model,the effect of CPT@PAAB@ICG in vivo photothermal-chemotherapy combined treatment was evaluated.First,in order to evaluate the distribution of nanoparticles in mice,in vivo fluorescence imaging experiments were performed.Check the in vivo biodistribution of BXPC-3 tumor-bearing mouse model after tail vein injection.Secondly,the nude mice with BXPC-3 tumor cells were randomLy divided into 5 groups(PBS.CPT@PAAB.PAAB@ICG + NIR.CPT@PAAB@ICG and CPT@PAAB@ICG + NIR),and regular follow-up measurements the tumor size and weight of each groupResults:The results showed that the two-block copolymer P(AAM-co-AN)-b-(B-DMAEA)was successfully prepared,including temperature-responsive block P(AAm-co-AN)and active oxygen-responsive block P(B-DMAEA).The blank micelle PAAB has a particle size of about 110 nm,and the polymer micelle CPT@PAAB@JCG is temperature sensitive,with a uniform shape and a spherical shape,with a particle size of about 110 nm.and a phase transition temperature of about 50?.BXPC-3 pancreatic cancer cells were selected as a model to investigate the toxicity of blank micelles PAAB and CPT@PAAB.The results showed that the blank vector had no significant toxicity.In vitro release experiments show that CPT can be released quickly when the UCST temperature is reached.CPT@PAAB@ICG has been found to have an excellent photothermal effect.It can absorb NIR light energy and convert it into heat energy to achieve rapid temperature rise.The effect of reactive oxygen species in vitro was evaluated.After H₂O₂ treatment,DLS and potential were measured.It was found that the potential gradually decreased and the size of micelles became smaller,indicating that it has a response to reactive oxygen species and can form a potential reversal to release ICG.Reactive oxygen experiments show that in the tumor microenvironment,the concentration of CPT@PAAB@ICG is significantly increased by near-infrared light irradiation.In addition to the contribution of ICG.CPT may also have a certain contribution.The anti-tumor effect of the CPT@PAAB@ICG drug delivery system was evaluated by the MTT method.The results showed that even if the concentration was as high as 30 ?gmL-1,there was no obvious cytotoxicity in the PAAB group,and the cell survival rate was still higher than 90%.Therefore,PAAB micelles have low toxicity and are suitable as drug carriers.The CPT@PAAB@ICG or CPT@PAAB group also did not make BXPC-3 cells not significantly inhibited,which indicates that monotherapy(photothermal therapy or chemotherapy)has limited cancer treatment effects.However,when PAAB@ICG is irradiated and NIR laser is used,the cell survival rate is significantly reduced,less than 50%.After loading CPT,CPT@PAAB@ICG nanoparticles have high cvtotoxicity,and the cell survival rate is less than 10%,indicating that the combination therapy has excellent anti-cancer properties.In addition,we also conducted a penetration test,using the 3D model of multicellular tumor spheres(MTS).observed by CLSM,and evaluated the penetration rate of CPT@PAAB@ICG.The results showed that CPT@PAAB@ICG can penetrate the 3D cell model up to 70 ?m instead.The free CPT will penetrate into the 3D unit model less than 40 ?m.This indicates that this delivery system can deliver CPT in polymer micelles to the deep layer of tumor tissue to kill cells inside the tumor.Cell experiments show that the CPT@PAAB@ICG platform can deliver CPT to the deep layer of tumor tissue to kill cells inside the tumor,and the combination therapy has an excellent anti-tumor effect.First,in order to evaluate the distribution of nanoparticles in mice,in vivo fluorescence imaging experiments were performed.Check the in vivo biodistribution of BXPC-3 tumor-bearing mouse model after tail vein injection.The results showed that,compared with other organs,strong fluorescence was observed in tumor tissues,indicating that CPT@PAAB@ICG is highly enriched in tumor tissues,and high tumor enrichment is the basis for efficient tumor treatment.Secondly,the nude mice with BXPC-3 tumor cells were randomLy divided into 5 groups(PBS.CPT@,PAAB,PAAB@ICG+NIR,CPT@PAAB@ICG and CPT@PAAB@ICG + NIR),and regular follow-up measurements the tumor size and weight of each group.The results showed that compared with the PBS group of the control group,the NIR treatment group and CPT@PAAB@ICG,CPT@PAAB group had no obvious anti-tumor effect,indicating that CPT@PAAB vector and CPT@PAAB@ The ICG carrier does not inhibit the growth of tumors.Compared with other treatment groups.CPT@PAAB@ ICG + NIR has the smallest tumor volume,indicating that it has a good anti-tumor effect.This is because light promotes the release of CPT,photothermal therapy and the combination of chemotherapy kills tumor cells by mutually enhancing the treatment.Therefore,theCPT@PAAB@ICG laser group has the best therapeutic effect in the body.In addition,weight loss in mice is an indicator of the safety of nanomedicine.Our follow-up results show that no significant weight loss was observed in the treatment group under any circumstances,which means that this combination therapy has no systemic toxicity.In addition,after the mice were sacrificed,the tumor tissues of each group of mice were analyzed histologically by H&E staining.The results showed that the CPT@PAAB@ICG tumor tissue section study group of the irradiation group showed that the tumor cells were severely destroyed,indicating that it had the strongest inhibitory effect on tumor cell proliferation,while the morphology of other tumor groups remained normal.It is important that the other There were no obvious inflammatory lesions or injuries in the heart,liver,kidney,and spleen,indicating that the carrier has low organ cytotoxicity.The anti-tumor effect in vivo shows that the combined treatment effect of CPT@PAA @ICG + NIR is significantly better than that of pure hyperthermia and chemotherapy.Conclusion:we have designed a polymer micelle with a dual response of temperature and reactive oxygen species.It is an intelligent nano-platform that can effectively synergize chemotherapy and photothermal therapy for the comprehensive treatment of pancreatic cancer.Under the action of active oxygen,the conversion of amino copolymer is triggered to form carboxyl copolymer,and the loaded ICG is released to reduce the size of nanoparticles.The reduced nanoparticles with weakly positive charges have a strong penetrating function.On the other hand,in Under the irradiation of 808 nm near-infrared light,the heat of ICG produces a photothermal effect to kill tumor cells.At the same time,it triggers the dissociation of nanoparticles to release CPT and quickly transport CPT to the deeper tissues of the tumor to effectively kill deep tumor cells.In vitro and in vivo experiments show that this functional nanoparticle has good anti-tumor activity,which is produced by synergistically enhanced combination therapy.This functional platform shows great potential in anti-tumor therapy and provides a new perspective for the design of intelligent drug delivery systems.