Construction Of A Targeted Therapeutic System Based On Extracellular Nanovesicles And Its In Vitro And In Vivo Experimental Studies

BackgroundExtracellular vesicles（EVs）are cell-derived lipid-bilayer enclosed vesicles of sub-micrometer sizes that are secreted by various cells.EVs can mediate intercellular communication through transferring donor cell derived proteins and nucleic acids.Currently,extracellular nanoscale vesicles（ENV,30-220 nm）including exosomes are under intense investigation.Currently,ENV have been exploited as drug vehicles for drug delivery.Compared with micelles,liposomes,and polymeric nanoparticles,ENV as a natural delivery system harboring natural membrane proteins can evade phagocytosis,have extended blood half-life,easily cross the blood-brain-barrier,directly fuse with membrane of recipient cells,and exhibit optimal biocompatibility without potential long-term safety issues.Although ENV-based drug delivery is promising,two major challenges exist.First the yield of ENV is low.Second,the preparation procedure for current ENV-based targeted drug delivery systems is tedious and labor-intensive.The technique we developed can properly address these two problems.In this study,Immature dendritic cells（imDCs）were selected as donor cells because of their low immunogenicity and anti-inflammation.However,the low production efficacy and no targeting effect make the ENV^s（ENV spontaneously secreted from cell,ENV^s）derived from imDCs cannot meet the requirements of clinical therapeutics.Therefore,imDC-derived ENV^s cannot be translated into clinical use.Here,we reported a new method for preparation of ENV for targeted cancer therapy.In brief,bone marrow derived imDCs were isolated from mouse and then expanded in vitro.Meanwhile,lipid tails and targeting ligands（AS1411）were covalently fused forming AS1411-Cholesterol.The lipidated AS1411 molecules were incorporated into membrane of imDCs through self-assembly of hydrophobic lipid tails.Next,the AS1411 grafted imDCs were extruded through filters with nanopores to generate ENV^me(ENV formed by mechanical extrusion,ENV^me)bearing AS1411followed by loading of paclitaxel（PTX）with sonication.The drug loaded ENV^mewere tested with cell assay in vitro and xenografted tumors in vivo for investigation of the treatment efficacy.PurposeTo develop a cancer-targeted ENV as novel drug delivery vesicles toward clinical translation,and to comprehensively investigate the treatment efficacy.Methods1.Immature mouse dendritic cells（imDC）were isolated from bone marrow of BALB/c mouse and cultured in vitro.imDC were analyzed by flow cytometry as well as fluorescence microscope.2.To measure the performance of lipid inserting into cellular lipid bilayer by the average fluorescence intensity of imDC.3.The morphology of AS1411-ENV^me was observed with a super-resolution microscopy,the size and generation efficiency by extrusion of AS1411-ENV^me were measured by Nanosight.The stability of AS1411-ENV^me was examined.4.PTX was loaded into AS1411-ENV^me and ENV^s by using either sonication or simple incubation.The average PTX loading efficiency was obtained with HPLC.The size and mean zeta potential of AS1411-ENV^me and ENV^s were analyzed.The release kinetics of PTX from AS1411-ENV^me and ENV^s was investigated with HPLC.5.Cargo proteins extracted from AS1411-ENV^me and ENV^s were analyzed by western blot.The targeting effect of AS1411-ENV^me with MDA-MB-231 cancer cells was validated in the study.6.MDA-MB-231 cells were treated with 200 n M PTX loaded AS1411-ENV^me,PTX loaded ENV^s,bare PTX and three negative controls,respectively.The cell viability was determined by MTT assay,and the apoptosis of cells was analyzed by flow cytometry as well as Image Xpress Micro XL widefield high-content analysis system.7.The MDA-MB-231 xenograft BALB/c mouse model was developed.Mice were treated with drugs as described before by intravenous injection.The volume and weight of tumors were measured.Furthermore,tumor proliferation and the histological structures of tumors and organs from all groups were analyzed.Results1.In vitro,colony-forming imDC were successfully cultured and maintained normal dendritic morphology.2.The average fluorescence intensity of cells labeled with chol was significantly higher than that of C18 and DSPE groups（p<0.01）.4 nmol of chol for labeling of～10⁷ imDC cells exhibited excellent fluorescence signals.3.The morphology of AS1411-ENV^me was observed with a super-resolution microscopy.Nanosight revealed the size distribution of AS1411-ENV^me ranged from50 nm to 270 nm with peak concentration at 103 nm.In addition,approximately8.7×10¹⁰ AS1411-ENV^me were prepared by extrusion of～10⁷ cells in 25 min.The production efficiency of AS1411-ENV^me using our modified protocol was nearly 30%,which is was approximately 2000-fold more efficient than that of spontaneous secretion from cells.AS1411-ENV^me and ENV^s were characterized by electron microscopy.Both displayed a typical saucer-shaped morphology under the transmission electron microscopy（TEM）.The fluorescence intensity of AS1411-ENV^me prepared by extrusion（～5×10⁸ in 10μl）was higher than that of equivalent AS1411-ENV^me prepared by direct incubation（p<0.05）.Furthermore,the ease of operation of ENV^s labeling is not comparable to cell labeling.The cryopreserved AS1411-ENV^me and ENV^s did not show significant aggregation or degradation at-80℃over 6 months.4.The average PTX loading efficiency with sonication was 21.33±1.53%in AS1411-ENV^me and 26.4±2.67%in ENV^s.In comparison,PTX loading efficiency with simple mixing,stirring and incubation was 5.06±1.12%in AS1411-ENV^me and7.73±2.38%in ENV^s,respectively.After PTX loading,the size of AS1411-ENV^meand ENV^s at peak concentration increased to 111 nm and 109 nm;the mean zeta potential of AS1411-ENV^me decreased from-23.7 mv to-25.6 mv,and that of ENV^sdecreased from-11.8 mv to-16.1 mv.5.Three cytosolic proteins including Annexin II,TSG101,and HSC70 and three surface proteins including CD59,CD9 and CD55 were identified in both AS1411-ENV^me and ENV^s with western blot.The three cytosolic proteins including Annexin II,TSG101,and HSC70 were barely detectable after PTX loading.After 30min a few ENV^s attached and fused with MDA-MB-231 cell membrane,however,AS1411-ENV^me efficiently bound MDA-MB-231 cells in only 10 min.6.Both AS1411-ENV^me PTX+and ENV^s PTX+showed burst release within the first one hour.At 24 h timepoint,PTX loaded AS1411-ENV^me and ENV^s released approximately 71.47%and 86.29%PTX,respectively.After cell treatment for 24 h,the respective IC50 of AS1411-ENV^me PTX+、ENV^s PTX+、PTX+was 14.24 n M,35.08 n M,and 212.69 n M.According to flow cytometry data,in groups of PTX loaded AS1411-ENV^me and PTX loaded ENV^s approximately 90%of cells were effectively blocked in G2-M phase,while in bare PTX group approximately 60%of cells had been retained in G2-M phase.The high-content screening also confirmed significantly more dead cells in groups of PTX loaded AS1411-ENV^me and PTX loaded ENV^s in comparison to that of bare PTX group.7.The MDA-MB-231 xenograft BALB/c mouse model was successfully developed.The mice were then randomly divided in to 6 experimental groups(PBS,ENV^s PTX-,AS1411-ENV^me PTX-,PTX+,ENV^s PTX+,AS1411-ENV^me PTX+).Drug was administrated by intravenous injection.At the end of treatment,the relative tumor volume of each group was 6.19±1.01（without PTX）,6.24±1.49（ENV^s without PTX）,6.58±1.4(AS1411-ENV^me without PTX),1.66±0.43（bare PTX）,0.57±0.15（PTX loaded ENV^s）,and 0.21±0.04(PTX loaded AS1411-ENV^me),respectively.Average tumor weight of PTX-treated three groups was 0.18 g（bare PTX）,0.05 g（PTX loaded ENV^s）,and 0.02 g(PTX loaded AS1411-ENV^me),respectively.The in vivo therapeutic efficacy of PTX loaded AS1411-ENV^me was improved approximately 9-and 3-fold compared to that of bare PTX and PTX loaded ENV^s,respectively（p<0.05）.Conclusions1.Compared to the other lipids,PEG-C18 and PEG-DSPE,PEG-Cholesterol can more efficiently incorporate into cellular membrane.Moreover,physical extrusion for ENV production is more efficient than natural release.2.Loading PTX with sonication is more efficient more traditional direct incubation.Compared to PTX loaded ENV^s and bare PTX,PTX loaded AS1411-ENV^me can more efficiently kill tumor cells in vitro and decrease the tumor volume in vivo.3.The imDC-derived ENV for targeted cancer therapy could be translated into clinical use in future.