Construction Of Multiple Interactions-tuned Supramolecular Nanoamplifier And Their Use In The Treatment Of Lung Metastases

ObjectivePrimary lung cancer is the most common cancer in our country.As the first cause of death of cancer patients,it is still an important public health problem in our country.The latest cancer statistics in China show that the number of new cases of lung cancer in China is about 828,000/4,064,000.In addition to primary lung cancer,the lung is also one of the most common sites of tumor metastasis,and pulmonary metastasis is very common in clinical practice.At present,surgical resection is still used for the treatment of lung cancer,and it has been reported that chemotherapy and radiotherapy can improve the prognosis of postoperative patients,but its efficacy is still unsatisfactory and the adverse effects are large.Therefore,it is of great significance to seek new treatment strategies for lung cancer.Photodynamic therapy(PDT)has attracted wide attention because of its advantages of minimally invasive,controllable,efficient and low adverse reactions,and is a treatment method that uses radical reactive oxygen species(ROS)toxic substances such as singlet oxygen and free radicals produced by photosensitizer excitation to produce cytotoxicity,resulting in damage and even death of tumor cells.However,intratumor redox homeostasis is a serious challenge for ROS-mediated antitumor therapy.Disrupting the balance between ROS and antioxidants in tumors can sensitize ROS-mediated tumor treatment.Herein,we proposed a supramolecular nanoamplifier PαLA@TAPP-Mn O2,which co-assembled 5,10,15,20-tetrakis(4-aminophenyl)porphyrin photosensitizer(TAPP)and manganese dioxide nanoenzyme(Mn O2)with a reductionsensitive anticancer drug carrier(poly α-lipoic acid,PαLA)by electrostatic adsorption and metal coordination.Supramolecular nanoamplifiers could be efficiently endocytosed by tumor cells and released payloads in response to high concentrations of glutathione(GSH)within cells.Mn O2 had the ability to eliminate GSH and generate hydroxyl radicals(·OH),working with TAPPmediated PDT to amplify redox homeostatic imbalance within tumor cells.On this basis,the therapeutic effect of Pα LA@TAPP-Mn O2 was evaluated on ectopic colon cancer tumor models and colon cancer lung metastasis mouse models.Methods1.We first prepared the carrier poly α lipoic acid(PαLA)with GSH reduction responsiveness through a simple heating process.Next,a supramolecular nanoamplifier(PαLA@TAPP-Mn O2)with PαLA polymer,TAPP,and Mn O2 co-nano-assembled was prepared by the "one-step nanoprecipitation method".The nanoparticles were then characterized using particle size analyzers and transmission electron microscopy(TEM).Computer molecular simulations were used to study the assembly mechanism of the nanoamplifiers.The in vitro release behavior of Pα LA@TAPP-Mn O2 was investigated using PBS(p H 7.4)containing 20%(v/v)DMF as the release medium.The in vitro stability and GSH responsiveness of Pα LA@TAPP-Mn O2 were analysed by simulating physiological environment mediators and simulating tumor high GSH environmental mediators,respectively.2.The cell uptake efficiency of Pα LA@TAPP-Mn O2 was evaluated using colon cancer cells(CT26)as a model,and the cytotoxicity of PαLA@TAPPMn O2 was investigated by MTT assay and live dead cell staining.Annexin VFITC/PI staining detected the ability of PαLA@TAPP-Mn O2 to induce apoptosis.DCFH-DA was used as the ROS probe to study the level of ROS produced by PαLA@TAPP-Mn O2 induced cells.3.Using CT26-tumor-bearing mice as a model and labeling PαLA@TAPPMn O2 with Di R,the tumor accumulation capacity of PαLA@TAPP-Mn O2 and the distribution of organ tissues in vivo were investigated by small animal in vivo imaging technology.4.The anti-tumor effects of each group of preparations were evaluated by subcutaneous ectopic tumor(CT26 cells)mice and lung metastasis model mice.ResultsThe results showed that PαLA@TAPP-Mn O2 was successfully prepared by TEM and particle size analyzer,with a particle size of 226±2.4 nm,an approximate spherical structure,and uniform size distribution.The results of molecular docking experiments showed that electrostatic adsorption and metal coordination drive the co-assembly of TAPP,Mn O2 and PαLA.In addition,in the simulated physiological environment medium,the particle size of the nanoparticles fluctuated little within 12 h,but was cleaved in a high GSH environment.In vitro release results also showed that PαLA@TAPP-Mn O2 had a higher cumulative release in high GSH environmental media,reaching 75%release after 24 h.The results of cell uptake showed that PαLA@TAPP-Mn O2 had better cell uptake efficiency and was the most toxic to CT26 cells.From the staining results of live dead cells,it was more intuitive to verify that PαLA@TAPP-Mn O2 caused the most significant cell death effect.The results of apoptosis experiments showed that the apoptosis ratio of PαLA@TAPP-Mn O2 group was 90.3%,which was higher than that of PαLA@TAPP(70.1%).PαLA@TAPP-Mn O2 induced CT26 cells to produce more ROS by confocal microscopy and flow cytometry.Fluorescence imaging was used to find that PαLA@TAPP-Mn O2 was efficiently accumulated in tumor tissues.We evaluated the antitumor effects of Mn O2 suspension,TAPP solution,PαLA@Mn O2,PαLA@TAPP and PαLA@TAPP-Mn O2 in subcutaneous ectopic tumor-bearing mice,and the results showed that PαLA@TAPP-Mn O2 had the best effect of inhibiting tumor growth and good biosafety in vivo.We further verified that PαLA@TAPP-Mn O2 had the best anti-metastasis efficacy in mouse lung metastases models,which further confirms the advantages of PαLA@TAPP-Mn O2 in tumor treatment.ConclusionsWe successfully prepared PαLA@TAPP-Mn O2 drug-loaded nanoparticles,whose good colloidal stability enhanced cellular uptake,and TAPP-mediated PDT combined with Mn O2 with the ability to consume GSH and generate ·OH,synergistically increasing the production of ROS in tumor cells.In vitro,PαLA@TAPP-Mn O2 had the strongest cytotoxicity and ability to induce apoptosis.In vivo,intravenous injection of PαLA@TAPP-Mn O2 effectively increases the enrichment of drugs in tumors,so it showed a strong anticancer therapeutic effect in subcutaneous tumors and lung metastases.