Construction Of Tumor Homologous Exosome Nano-sonosensitizer EXO-DVDMS And Its Experimental Study On Sonodynamic Killing Of Breast Cancer

Background and Objective:Cancer is a serious public health concern worldwide.According to the report of"Cancer Statistics,2019",breast cancer has become the leading malignancy among women in the worldwide.Currently,treatment such as surgery,chemotherapy,radiotherapy,endocrine therapy,targeted therapy and traditional Chinese medicine have made great achievements in clinic controlling,but accompanied by severe side effects,high tumor metastasis and recurrence rate.Therefore,research on the noninvasive breast cancer treatments with high targeting efficiency and low toxicity has always been a major issue of biomedicine research.Sonodynamic therapy(SDT)is noninvasive strategy for tumor treatment developed in recent years.Ultrasound(US)with appropriate tissue attenuation coefficient for penetrating allows energy focus into small volumes and activates sonosensitizer which specific enriched in tumor tissue,and exerts anti-tumor effect through multiple mechanisms including ultrasonic cavitation,production of free radicals,reducing drug resistance,inducing cell apoptosis and immune regulation.SDT combines the selective enrichment of its low-toxicity/non-toxicity sensitizer to the lesion tissue and the precise focus of ultrasound,thereby makes it possible to damage the pathological site but not peripheral healthy tissues.Therefore,SDT can be an effective alternative for patients who are difficult to undergo surgery or with advanced cancer.Sonosensitizers are critical factors in SDT.The ideal sonosensitizers to achieve effective and safe SDT should be highly sensitive to ultrasound irradiation and exhibit high tumor specificity.However,most traditional sonosensitizers come with their own limitations such as low bioavailability,poor tumor accumulation,poor stability in physiological environment.Thus,exploring novel high-efficiency sonosensitizers,overcoming the defects of current sonosensitizers as well as improving the drug delivery efficiency are urgent in SDT field.Compared with small-molecule drugs,nano-scale sensitizers are more easily to be accumulated in tumor tissues through passive targeting via the enhanced permeability and retention effect(EPR)of solid tumors and thus reduce their accumulation in non-tumor tissues.Nanoparticles act as the drug-delivery carrier or just play the part of sonosensitizers to simultaneously improve the physical and chemical properties of sonosensitizers and enhance the SDT outcome.However,on the one hand,the clinical translation of synthetic nanoparticles still stymied due to the potential immunogenicity,cytotoxicity of supporter molecules and the degradation products and may rapidly be removed by the reticuloendothelial system.Besides,tumor heterogeneity and the exist of physiological and pathological barriers limited the actual effect of EPR.To overcome the limitations of conventional delivery systems,increasing efforts have been focused on developing carriers derived from endogenous cells or subcellular structures.Exosomes are nanoscale(30-150 nm)cell-derived membrane vesicles that carry macromolecules from their parental cells,and mediate cell-to-cell communication.Exosomes have been developed for use as diagnostic markers in various malignancies,and as delivery vehicles for anti-cancer therapy.Their endogenous origin imparts excellent biocompatibility,intrinsic targeting property and the ability to evade uptake by the reticuloendothelial system(RES),which protects the encapsulated drug till delivery.Among various attempts to promote EPR,ultrasound targeted microbubble destruction(UTMD)technology displayed great potential in assisting drug delivery and permeation.Based on the above background,we designed a functionalized smart nanoparticle system by loading sinoporphyrin sodium(DVDMS),an excellent porphyrin sonosensitizer with potential therapeutic and imaging applications,onto homotypic tumor cell-derived exosomes.By using the tumor exosome camouflage strategy,the resulting EXO-DVDMS was harnessed as biological fluorescence-guided "spy drones" to maintain drug stability in the tumor microenvironment,ensuring targeted delivery of DVDMS to both primary tumor and metastatic lesions.A guided-ultrasound(US1)was first introduced to promote local deposition of EXO-DVDMS to all regions of tumor,followed by the therapeutic-ultrasound(US2).We systematically investigated the drug protection effect,ultrasound-mediated drug release profile,intracellular transport characteristics and homologous tumor targeting ability of EXO-DVDMS;Mouse breast cancer 4T1 cells were used as experimental models to study the combined enhanced anti-tumor effect of US1 guided EXO-DVDMS-SDT.The ultrasound-manipulated EXO-DVDMS platform provides insights into the application of cell-derived vesicles as spatio-temporally controlled nanosensitizers and drug delivery platforms,in order to achieve safe,accurate,effective and personalized anti-cancer therapy and finally allow us to lay a theoretical and experimental foundation for such a promising therapy in clinical cancer treatment.Methods and Results:I.Preparation and characterization of EXO-DVDMS:The reported PEG precipitation method was improved by screening the molecular weight and concentration of PEG as well as adding pre-extraction and post-extraction treatment steps.The improved method(PEG-8K method)was compared with ultracentrifugation method and commercial sedimentation kit to assess the applicability of the modified PEG-8K protocol.Fluorescence spectrophotometer was used to determine the encapsulation efficiency and drug loading efficiency of EXO-DVDMS.The EXO-DVDMS particles were characterized by TEM.The particle size,zeta potential,and PDI of various exosomal formulations were measured by NTA and DLS.Western blot was used to detect exosomal markers.FCM and CLSM were used to verify the purity of the prepared EXO-DVDMS to eliminate the interference of unloaded exosomes.? The amount of exosomes separated by PEG-8K modified method is more than 10 times that of ultracentrifugation method.The exosomes separated by PEG-8K modified method process good stability with particle size about 110 nm.And this improved protocol can be applied for isolation of various cells derived exosomes and blood exosomes.?We finally prepare the optimal EXO-DVDMS by co-incubation of DVDMS and 4T1 derived exosomes at a mass ratio of 1:15.5(5.46 ?g DVDMS loading per 100 ?g exosomal protein)for 30 min at 25?,which resulted in a comparatively higher drug loading capacity(EE%=84.75%,DL%=5.18%),low polydispersity(0.18 ±0.05)and suitable size distribution(mean size at 126.71±3.86),and were thus used in subsequent experiments.Classical exosomal makers tetraspanin CD9 and CD63,directly involving in the sorting of extracellular vesicle contents,were abundant in the obtained exosomes.?The result of flow cytometry shows that the ratio of drug loaded exosomes(with the high fluorescence signal of DVDMS)is above 98.5%.Therefore,the obtained high purity EXO-DVDMS could be considered as a uniformly dispersed system.2.Exosomal encapsulation maintained DVDMS stability and enhanced singlet oxygen yield:The time-dependent colloidal stability of Blank-exosome and EXO-DVDMS in conditioned DMEM containing 10%,20%and 50%FBS at 4? or 37?were evaluated after 0,2,4,6,12 and 24 h of incubation by DLS.The absorption spectra and emission fluorescence spectra of free-DVDMS and EXO-DVDMS were measured in PBS or 50%serum(pH 7.4 or 5.0)to simulate physiological conditions,using a fluorescence spectrophotometer.The levels of ROS generated from the EXO-DVDMS and free-DVDMS in neutral and low pH conditions were measured in terms of the fluorescence intensity of SOSG at 525 nm.?Both blank-EXO and EXO-DVDMS exhibited excellent colloidal stability,with no visible precipitation in either complete DMEM or PBS containing 10%,20%or 50%serum at 4? or 37? even after 24 h,indicating good biocompatibility of the particles.?Free-DVDMS in PBS exhibited a strong Soret absorption peak at-370 nm,and Q-hands between 500 and 700 nm.The UV-vis spectrum of EXO-DVDMS shows a superimposition of DVDMS peaks and an enhanced absorbance on Q-hands,and showed a new peak at 430 nm.The fluorescence spectra of free-DVDMS showed fluorescence emission peaks at?640 nm,which did not change upon loading onto exosome,indicating that the fluorescence spectral properties of DVDMS are not significantly affected by the carrier.The fluorescence intensity of the loaded DVDMS was significantly enhanced which is highly promising for real-time visualization of in vivo DVDMS delivery and molecular imaging-guided SDT.The pH and serum concentration of the buffer significantly affected the spectra of DVDMS but not that of EXO-DVDMS.?The singlet oxygen yield of EXO-DVDMS at pH 7.4 and pH 5.0 was 4.74 and 14.4 times higher than that of free-DVDMS at 24 h time point,indicating exosomal coating protected the serious dysfunction of naked DVDMS in tumor micro-environment,and EXO-DVDMS would be more beneficial to ultrasound activation and ROS production.3.Ultrasound-triggered DVDMS release from EXO-DVDMS:The release of DVDMS from EXO-DVDMS in simulated physiological environment following ultrasound stimulation(LP:1-3W,irradiation time:30-90s)was evaluated using the dialysis method.Acoustic cavitation was evaluated using TA method.The effect of ultrasonic treatment on the morphology of EXO-DVDMS was observed by TEM.The intracellular dissociation of DVDMS from composite EXO-DVDMS under ultrasonic stimulation in 4T1 cells was tracked using LSCM.?DVDMS release from EXO-DVDMS can be triggered by low pH condition and ultrasound irradiation.?The·OH content in the EXO-DVDMS suspension increased in a DVDMS-dose dependent manner after ultrasonic exposure,and showed significantly higher HTA fluorescence compared with that of the free-DVDMS,indicating that exosomal coating and ultrasonic stimulation increase cavitation which enhances drug release and SDT.?The TEM images show that the morphology of Blank-EXO did not change much before and after US irradiation.However,the morphological impairment was observed in EXO-DVDMS(US+)group,indicating EXO-DVDMS increased the sonosensitive response to US exposure.?Exogenous ultrasonic achieved spatio-temporal manipulation of EXO-DVDMS.4.In vitro cellular uptake and homologous targeting of EXO-DVDMS:The FCM and CLSM were used to detect the intracellular uptake of EXO-DVDMS.?EXO-DVDMS were dispersed in the cytoplasm after 3 h,and gradually increased after 6,12 and 24 h.?The fluorescence intensity of the exosome delivered DVDMS was significantly higher than free-DVDMS(1.62 fold)and Lipo-DVDMS(1.44 fold).?4T1 cells exhibited the highest uptake efficiency,with 2.35,2.05,2.08 and 1.81-fold higher fluorescence intensities compared to that of the NIH/3T3,CT26,MDA-MB-231 and MCF-7 cells,respectively.Thus,the EXO-DVDMS possessed intrinsically superior selectivity to its homologous tumor cells in vitro.?Ultrasound stimulation increased the cellular uptake of DVDMS,with significantly higher uptake of the exosomal-encapsulated compared to the free-DVDMS.The proportion of 4T1 cells with high FD500 fluorescence intensity increased by ultrasound stimulation,with higher frequencies seen in cells incubated with EXO-DVDMS(61.65%)compared to free-DVDMS(38.40%,p<0.01),indicating that the enhanced cell membrane permeability induced by sonoporation increased EXO-DVDMS uptake.5.In vitro cytotoxicity of EXO-DVDMS:The in vitro SDT effect was detected using MTT assays and calcein-AM/PI staining.DCF and DHE was used to assess intracellular ROS level and superoxide anion(O2·-)production.Subcellular re-localization of exsomal DVDMS under ultrasonic stimulation was observed by CLSM.?The cell-damaging effect was enhanced with the increase of US intensity and DVDMS dose.EXO-DVDMS exhibited much stronger sonodynamic cytotoxicity compared with free-DVDMS under the same parameters.?EXO-DVDMS resulted in higher DCF and DHE fluorescence than free-DVDMS when combined with ultrasound.?In EXO-DVDMS group,DVDMS was localized in the endo/lysosomes,indicating cellular uptake via endocytosis.EXO-DVDMS partly shifted from the lysosomes to the mitochondria after ultrasonic stimulation.6.In vivo homotypic targeting and ultrasound guided EXO-DVDMS accumulation and permeation in exposed tumors:The DVDMS content in plasma extraction was measured by fluorescence photometer to detect the long circulation ability of EXO-DVDMS.The in vivo biodistributions and homotypic targeting potential of EXO-DVDMS were investigated by IVIS Spectrum Imaging System.A dual-xenograft model was established and used with IVIS Spectrum Imaging System to determine the ability of ultrasound to guide exosome accumulation in the tumor tissues.The fluorescence distribution of DiO/DVDMS in the different depths of the tissues was observed under a stereo fluorescence microscope.?Exosomal loading significantly elongated the circulation of DVDMS.?The average radiant efficiency of EXO-DVDMS in 4T1 tumor were about 2.17-fold higher than that in CT26 tumor.4T1-EXO-DVDMS displayed much higher EXO-DVDMS fluorescence(2.04-fold)in homotypic tumors compared with that of B-EXO-DVDMS.?The DVDMS fluorescence signals in the tumor with US1 were significantly higher(2.05-fold)than that of the tumor without US1,indicating that selective irradiation by focused ultrasound could promote nanoporphyrin accumulation at the specific site.Meanwhile,ultrasound promoted the penetration and intra-tumoral diffusion of EXO-DVDMS.?EXO-DVDMS greatly improved the in vivo imaging ability,bio-distribution and tumor accumulation of DVDMS.The average radiant efficiency of tumor in EXO-DVDMS group was about 3.22-fold greater than that of Free-DVDMS group.7.In vivo anti-tumor effects of US1 augmented EXO-DVDMS-SDT:Mouse breast cancer 4T1 cells were used as experimental models to study the combined enhanced anti-tumor effect of US1 guided EXO-DVDMS-SDT.?US1+US2 along with free-DVDMS and EXO-DVDMS resulted in maximum tumor growth inhibition indicating the crucial role of the guiding ultrasound which markedly strengthened the antitumor effect by increasing accumulation and permeation of the sensitizers.In addition,the tumor size in EXO-DVDMS-SDT group was much smaller than that of the Free-DVDMS-SDT group.Histological examination indicated more extensive tumor tissue damage in the US1+US2 plus EXO-DVDMS treated group compared to US2 plus EXO-DVDMS and US1+US2 plus free-DVDMS groups.PCNA levels declined significantly and TUNEL staining increased markedly after US1+US2 plus EXO-DVDMS treatment compared to the other treatment modalitie,indicating inhibition of tumor cell proliferation and apoptosis induction following EXO-DVDMS-SDT.?EXO-DVDMS-SDT significantly inhibited tumor lung metastases with the markedly declined MMP-9 level in tumor tissues histological analyses of lung tissues receiving the different treatments further confirmed the superior anti-metastatic effect of EXO-DVDMS+US1+US2.?No major changes on the body weight and major organs of tumor-bearing mice were observed following any treatment,indicating EXO-DVDMS has satisfactory biocompatibility and could be considered as an effective drug delivery platform.Conclusions:We designed a functionalized smart nanoparticle system by loading sinoporphyrin sodium(DVDMS),an excellent porphyrin sonosensitizer with potential therapeutic and imaging applications onto homotypic tumor cell-derived exosomes.By using the tumor exosome camouflage strategy,the resulting EXO-DVDMS was harnessed as biological fluorescence-guided "spy drones" for enhanced drug stability and ROS production in the tumor microenvironment.Meanwhile,EXO-DVDMS exhibited controlled ultrasound-responsive drug release ability.Additionally,the dual-targeting strategy by combining guided-ultrasound and tumor derived exosomes overcomes the limitation of the heterogeneous EPR effect,which enhanced targeted accumulation,deep tissue penetration and fluorescence imaging ability of DVDMS,and further resulting in highly effective tumor-specific SDT along with dramatically inhibitory effect on lung metastasis inhibition effect and excellent biocompatibility.The ultrasound-manipulated EXO-DVDMS platform provides insights into the application of cell-derived vesicles as spatio-temporally controlled nanosensitizers and drug delivery platforms,in order to achieve safe,accurate,effective and personalized anti-cancer therapy.