| ObjectiveIn today's society,cancer has become the biggest killer of human health.Among all kinds of cancers,breast cancer is one of the most common malignant tumors among women.Conventional treatment methods include:radiation therapy,surgical treatment,chemical treatment,and the like.However,traditional methods of treatment all have some disadvantages to a certain extent.Radiotherapy and surgical treatment will cause great harm to the patient's body and mind.Chemotherapy,in addition to having certain side effects on the patient's body,is also resistant to cancer cells.The influence of medicinal properties will reduce the concentration of blood medicine that actually enters the cancer cells,greatly reducing the therapeutic effect.In recent years,with the development of nanomedicine,the nano drug delivery system?NDDS?has become the most promising therapeutic method for cancer treatment,with multi-functional nanomaterials for diagnosis and treatment,and nano-carriersthatstimulateresponsivereleaseof drugs in the tumor microenvironment.The combination of therapeutic methods and nano-systems for cancer treatment can achieve early diagnosis and treatment of cancer and greatly improve the therapeutic effect of cancer.In this study,a polymer nanoparticle containing an iodine atom and a disulfide bond was prepared and used as a CT contrast agent and nano drug carrier with redox stimuli-responsiveness,and then loaded with the antitumor drug doxorubicin?DOX?and the photosensitizer IR780enhances the therapeutic effect of breast cancer through chemotherapy and photothermal therapy.ContentsThe first part isthe material preparation phase:First,the monomer compounds containing iodine atoms were synthesized through chemical reaction.Then iodine-containing nanoparticles with redox responsiveness were prepared by precipitation polymerization.Finally,DOX and IR780 were co-loaded onto the nanoparticles.The second part is the material characterization phase:Transmission electron microscope?TEM?,dynamic laser particle size analyzer?DLS?,inductively coupledplasmamassspectrometry?ICP-MS?,UV-visible-near-infrared spectrophotometer?UV-vis-NIR?,fluorescence spectrophotometers,infrared thermal imagers,CT scanners,etc.,have performed a series of structural and performance characterizations on the nanoparticles.The third part is the in vitro performance of the nanoparticles:Cell uptake,cytotoxicity and photothermic effects of the nanoparticles were detected.MethodsIn the first step,3-amino-2,4,6-triiodobenzoic acid and methacryloyl chloride are used as reactants to generate an iodine-containing monomer compound2-methacryloyl?3-amide-2,4,6-triiodobenzoic acid?.In the second step,The iodine-containing polymer nanoparticles?P?MATIB-co-BAC??were prepareded by precipitation polymerization withMATIB as a monomer,thedisulfide bond-containing compound N,N'-bisacryloylcystamine?BAC?as a cross-linking agent and isobutyronitrile?AIBN?as an initiator.In order to examine the difference in yield,the difference in iodine content,the change in particle size and the degradation under dithiothreitol?DTT?of iodine-containing nanoparticles obtained under different crosslinking degrees,a series of iodine-containing nanoparticles with different cross-linking degrees?15 wt%,20 wt%,30 wt%,40 wt%?were prepared.Finally,the iodine-containing nanoparticle with a cross-linking degree of 30%was used as a carrier to determine its drug loading and release performences.The DOX and IR780were sequentially loaded onto the nanoparticles by physical action to form the DOX andIR780co-loadedP?MATIB-co-BAC?nanoparticles?DOX/IR780P?MATIB-co-BAC??.Then a series of characterizations of the products obtained in each step were performed.The morphology and particle size of the nanoparticles were characterized by TEM.The particle size distribution,surface charge and dispersibility of the obtained nanoparticles were characterized by DLS.The iodine content of the iodine-containingnanoparticleswasdeterminedbyICP-MS.UV-vis-NIR spectroscopy were used to characterize the degradation of iodine-containing nanoparticles in DTT at 10 mM concentration for 24 h period of time,loading capacities of DOX and IR780 on nanoparticles,and the releases of the two drugs from nanoparticles at different pH conditions anddifferent DTT concentrations.DOX and IR780 successfully co-loaded onto the P?MATIB-co-BAC?nanoparticles was determined by their UV absorption characteristic peaks.The changes in the characteristic absorption peaks of the free IR780 and DOX/IR780 P?MATIB-co-BAC?nanoparticles were characterized using a fluorescence spectrophotometer.The photothermal conversion performance of free IR780 and DOX/IR780 P?MATIB-co-BAC?nanoparticles at different concentrations was characterized by an infrared thermal imager.The X-ray absorption properties of iodine-containing nanoparticles at different concentrations of iodine were characterized using a CT scanner.Finally,the in vitro properties of the obtained nanoparticles were investigated.The intracellular distributions of DOX and IR780 after them sequential loading on the nanoparticles were investigated by cell uptake experiments;the iodine-containing nanoparticles,iodine-containing nanoparticles loaded with DOX or co-loaded with DOX and IR780 with or without laser irradiation were examined by MTT assays.The effect of photothermal therapy combined with chemotherapy on breast cancer cells was further examined by staining of live dead cells.ResultsTEM showed that the iodine-containing P?MATIB-co-BAC?nanoparticles obtained with differentcross-linking degrees are uniformly spherical and showed good dispersibility.As the cross-linking degree of the obtained P?MATIB-co-BAC?nanoparticles increased,the particle size increased.The results of DLS showed that the particle size distributions of the P?MATIB-co-BAC?nanoparticles with different cross-linking degrees were narrow,and the polydispersity coefficient?PDI?were small,which further verified that the P?MATIB-co-BAC?nanoparticles had good dispersibility.The surface charge of the P?MATIB-co-BAC?nanoparticles exhibited negative charge.After the DOX and IR780 were loaded,the particle size of the P?MATIB-co-BAC?nanoparticles slightly increased,and the surface potential was still negative.ICP-MS was used to determine the iodine content of the P?MATIB-co-BAC?nanoparticles.It was found that the iodine content decreased with thecross-linking degree increased.The iodine content of the P?MATIB-co-BAC?nanoparticles with cross-linking degree of 15 wt%was 59.3%,the iodine content of the P?MATIB-co-BAC?nanoparticles with cross-linking degree of 40 wt%was only18.3%.The degradation results of the nanoparticles in the presence of DTT showed that the degradation rate and final degradation degree of P?MATIB-co-BAC?nanoparticles increased with the increase of the cross-linking degree of the nanoparticles.The P?MATIB-co-BAC?nanoparticles with cross-linking degree of 40wt%showed nearly 90%degradationafter 24 h,while the P?MATIB-co-BAC?nanoparticles with cross-linking degree of 15 wt%showed only about 50%degradation.The results of CT detection showed that the CT value increased with the increase of iodine concentration of the P?MATIB-co-BAC?nanoparticles with different cross-linking degrees,suggesting their good X-ray attenuation performance.Considering their yield,size and size distribution,iodine content and the degradation in the presence of reducing agent,the P?MATIB-co-BAC?nanoparticle with a cross-linking degree of 30 wt%was chosen as the carrier for DOX and IR780.Drug-loading experiments showed that the drug loading of DOX and IR780 increased with the initial concentration of the drug.At a certain initial concentration,the drug loading reached saturation.The final DOX and IR780 loadings on the P?MATIB-co-BAC?nanoparticles were 28.3%and 11.3%respectively.The drug release showed pH and redox sensitivity.With the decrease of pH value,the DOX and IR780 release rates have increased to some extent.In the presence of DTT,the release of DOX and IR780 increased significantly.In the UV-vis-NIR absorption spectrum of DOX/IR780 P?MATIB-co-BAC?nanoparticles,characteristic absorption peaks of DOX and IR780 appeared at 490 nm and 800 nm,respectively,indicating the successful loading of DOX and IR780.The fluorescence spectra of free IR780 and DOX/IR780 P?MATIB-co-BAC?nanoparticles show that the absorption peak of IR780 in the P?MATIB-co-BAC?nanoparticles has a certain degree of red shift compared with that of free IR780.The photothermal property of DOX/IR780 P?MATIB-co-BAC?nanoparticles shows that the temperature their suspension rose to 52°C at IR780 concentration of 100?g mL-1after 10 min laser irradiation,indicating that the DOX/IR780 P?MATIB-co-BAC?nanoparticle has good photothermal conversion effect.Cellular uptake experiments showed that both DOX and IR780 are located in different locations in the cell.DOX is mainly distributed in the nucleus while IR780is distributed in the cytoplasm.MTT assays showed that the P?MATIB-co-BAC?nanoparticles without DOX and IR780 still had high cell activity at high concentrations(1000?g mL-1),suggesting their good biocompatibility.The DOX and DOX/IR780loadedP?MATIB-co-BAC?nanoparticlesshowedthesimilar cytotoxicity as free DOX After laser irradiation,the DOX/IR780 P?MATIB-co-BAC?nanoparticles showed significant cytotoxicity.The live dead cell staining experiment further demonstrated that the DOX/IR780 P?MATIB-co-BAC?nanoparticles have more obvious cytotoxicity under near-infrared light irradiation compared to the control groups.ConclusionIn this study,DOX and IR780 co-loaded iodine-containing nanoparticles with redox responsivity were successfully prepared for CT imaging and combined photothermal-chemotherapy of breast cancer.The iodine-containing nanoparticles have good dispersibility,stability and X-ray attenuation properties.The drug loadings for DOX and IR780 were 28.3%and 11.3%,respectively.The in vitro release results showed that the drug release had pH and redox responsiveness.The results of cell experiments showed that the nanoparticles co-loaded with DOX and IR780 could enter tumor cells,and have good photothermic-chemotherapy combined effects under near-infrared laser irradiation.At the same time,the nanoparticle showed good biocompatibility.This makes the nanoparticles have potential application prospect in the integration of cancer diagnosis and treatment and combined treatment of tumors. |
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