Experimental Study Of Exosomes And Exosome Mimetics As Drug Carriers For Tumor Therapy

Objective:Tumor-derived exosomes(TEX)have shown great potential for drug delivery and tumor targeting.Here,we developed a novel multi-drug loaded exosomes nanoprobe for combined antitumor chemotherapy and photodynamic therapy,and monitoring the drug delivery capabilities with pre-targeting technique.Methods:TEX of human colorectal cancer HCT116 was prepared,and Doxorubicin and the photodynamic therapy agent 5-aminolevulinic acid(ALA)were loaded and named as TEX@DOX@ALA.Tumor uptake was first examined using fluorescence imaging of the fluorescent dye Cy5(TEX@DOX@ALA@Cy5).Visualization of exosome aggregation in tumor were realized by positron-emission tomography/computed tomography(PET/CT)with pre-targeting technique.Tumor-bearing mice were first injected with TEX@DOX@ALA labeled with azide(N3)(TEX@DOX@ALA@N3),and then 68Ga-(2,2’-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)hexan-2-yl)azanediyl)diacetic acid-dibenzocyclooctyne(68Ga-L-NETA-DBCO)was injected after 24 hours for PET/CT imaging via in vivo click chemistry.For the antitumor therapy with photodynamic and/or chemotherapy,seven groups of tumor-bearing mice with different therapy were monitored,and the tumor size,animal weight and the survival time were recorded.Furthermore,the samples of blood and interested tissues were harvested for hematological analysis and H&E staining.Results:The drug loading process did not influence the structure or the function of the HCT116 TEX membranes.In a fluorescence imaging experiment,higher fluorescence could be seen in tumor after TEX@DOX@ALA@Cy5 injected,and reached the highest signal at 24 hours.From PET/CT images with subcutaneous and orthotopic colon tumor-bearing mice,clear radioactivity could be seen in tumors,which suggested the successes of TEX accumulation in tumors.TEX@DOX@ALA(+)group with photodynamic therapy and chemotherapy had the best tumor inhibition effect compared with the other groups,with the longest survival time(36 days,37.5%).No significant damage was found on histological observation and the blood biochemical analysis,which suggested the safety of the multi-drug loaded exosomes.Conclusions:We successfully engineered an exosome-based nanoprobe integrating PET imaging components and therapeutic drugs.This drug-loaded exosome system may effectively target tumors and enable synergistic chemotherapeutic and photodynamic antitumor effects.Background:Photodynamic therapy(PDT)is a promising antitumor strategy,with improved efficacy and less toxicity than conventional therapy.In this study,we designed an aminolevulinic acid(ALA)-loaded exosome mimetic(EM)(EM@ALA)and labeled it with radionuclide 131I to overcome the challenge of PDT application in deep tumors in vivo.The systemic side effects of CR-PDT can be reduced using ALA,which is mainly transformed into the photosensitizer protoporphyrin IX(PpIX)in tumor cells.Methods:EM@ALA was prepared using the lipid extrusion method.EM@ALA was labeled with 131I using the chloramine T method to form 131I-EM@ALA.Tumor uptake was examined by confocal microscopy,flow cytometry,and radionuclide uptake.An in-vivo imaging system(IVIS)was used to record the CR signals from 131I sources under different conditions.The in-vivo circulation characteristics of EM@ALA were explored by fluorescence imaging in small animals.For antitumor therapy in vivo,five groups of tumor-bearing mice treated with different therapies were monitored,and the tumor size,animal weight,and survival time were recorded.Blood and tissue samples of interest(heart,lung,liver,kidney,spleen,muscle,and thyroid)were collected for hematoxylin and eosin staining.In addition,blood and liver tissue samples were collected at 7,14,and 21 days after the injection of 131I-EM@ALA or 131I-EM@PpIX to assess differences in systemic toxicity between the 131I-EM@ALA and 131I-EM@PpIX groups.Finally,protein expression in tumor cells was examined by Western blotting to analyze the primary mechanism of tumor cell death by 131I-EM@ALA.Results:EM@ALA was spherical in morphology,with an average particle size of 154.8±2.8 nm.Western blotting showed that EM@ALA inherited 4T1 cell membrane components,but it did not carry genetic material in the tumor nucleus or cytoplasm.The labeling rate of 131I-EM@ALA was 96.65%,and the radiochemical stability in phosphate-buffered saline over 48 h was 80.5%±1.3%.Confocal microscopy,flow cytometry,and radionuclide uptake experiments showed that EM@ALA or 131I-EM@ALA could effectively target 4T1 tumor cells,and the binding rate increased with time.The CR imaging results showed that the CR signal emitted by 131I decay was collected by the IVIS,and the greater the concentration of 131I,the stronger the CR signal.When the 131I dose and the total volume of solution were consistent and the PpIX solution was added to the 131I solution,the higher the concentration of PpIX,the weaker the CR signal measured by the IVIS.Fluorescent imaging revealed that EM@ALA had a long circulation time in vivo and accumulated at the tumor site approximately 24 h after injection,and EM@ALA was mainly metabolized by the liver.At 19 days after treatment,the 131I-EM@ALA group demonstrated greater tumor inhibition than the other groups.The tumor volume in the 131I-EM@ALA group(580.0±146.4 mm3)was significantly smaller than the tumor volume in the 131I-EM group(1081.5±273.5 mm3),suggesting that combination therapy had greater antitumor efficacy than single 131I radiotherapy.Blood biochemical analysis and histological observation suggested excellent biocompatibility of 131I-EM@ALA.When comparing systemic toxicity between 131I-EM@ALA and 131I-EM@PpIX,the data demonstrated varying degrees of damage to hemocytes and hepatocytes in the 131I-EM@PpIX group.Finally,Western blotting showed that the apoptosis-related protein caspase 3 was highly expressed in tumor cells and lowly expressed in hepatocytes after 131I-EM@ALA treatment,suggesting that the molecular mechanism leading to tumor cell death involves the caspase 3 pathway,which is induced by reactive oxygen species.Conclusion:In this study,drug-loaded nanoparticles 131I-EM@ALA were successfully prepared to perform CR-PDT independent of an external light source.131I internal irradiation combined with CR-PDT achieved an excellent antitumor effect.By comparing systemic toxicity differences between the 131I-EM@ALA and 131I-EM@PpIX groups,we confirmed that use of the photosensitizer precursor ALA reduced systemic side effects.This study provides new insights into the synergistic antitumor effects of multiple therapies and provides a new vision for the optimization of CR-PDT independent of an external light source.