Generation Of Cell-derived Microvesicles-Based Multifunctional Nanocarrier And Practice In Targeted Oncotherapy

Part 1 Magnetic and Folate Functionalization Enables Rapid Isolation and Enhanced TumorTargeting of Cell-Derived MicrovesiclesObjective:Cell-derived microvesicles(MVs)are nano-sized(100-1000 nm)membrane vesicles shed by various cell types.Recently,MVs have been received attention for useing as natural therapeutic platforms due to their capability of conveying bioactive molecules between cells.However,the lack of convenient and rapid methods for scalable isolation as well as the deficiencies in drug loading and unsatisfactory cancer-targeting capability has largely restricted their application in oncotherapy.The aim of this part is to develop a separation and targeting strategy to facilitate the transformation of MVs into multifunctional nanocarrier.Methods:Biotin and folate(FA)-modified MVs,which can be further magnetically functionalized by streptavidin conjugated iron oxide nanoparticles(SA-IONPs),were obtained via the self-assemble of phospholipid bilayer.SA-IONPs labeled MVs were separated from culture medium with a permanent magnet.Then,the biocompatibility and tumor targeting capability of FA/IONP-MVs were evaluated in vitro and in vivo.FA/IONP-MVs were further loaded with doxorubicin(DOX)via electroporation.The in vivo targeted antitumor efficacy was evaluated in tumor-bearing mice model.Reulsts:By conjugated with SA-IONP,biotin/FA-MVs were conveniently,efficiently and rapidly isolated from the culture medium with a permanent magnet.FA/IONP-MVs were biocompatible as nanovesicles without significant side effects.After loaded with DOX,FA/IONP-MVs-DOX exhibited excellent cancer targeted therapy properties in xenograft tumor mice model.Conclusion:Our study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors,thus facilitating the development of natural therapeutic nanoplatforms.Part 2 Generation of Natural Multifunctional Nanovectors for in vivo Bioimging and Therapeutics from Circulating MicrovesiclesObjective:So far,all the MVs used as theranostic vectors in previous studies have been produced in vitro from cell culture supernatants,which is still associated with several concerns regarding practical applications,such as difficulty in scalable isolation,low yield of MVs from in vitro cell cultures and undesirable cross-contamination may occur during the long-term culture and immunoreactions or interactions with host cells when applied in vivo.The presence of MVs has also been reported in various human body fluids,such as blood,saliva,urine and cerebrospinal fluid and they are referred to as body fluids derived MVs.The present study aims to transform the body fluids derived MVs into multifunctional therapeutic nanovectors.Methods:Circulating MVs(CMVs),which were freshly purified from the peripheral blood of patients with oral squamous cell carcinoma(OSCC)patients,were embedded with ultrasmall NIR-fluorescent magnetic quantum dots(Ag2Se@Mn QDs)via electroporation.The tissue distribution and natural tumor-targeting behavior of CMVs were evaluated in living mice with in vivo MR imaging system and 7.0 T small animal MRI scanner.To develope a dual-modally traceable nanoplatform for cancer theranostics,siRNA and Ag2Se@Mn QDs were simultaneously loaded into CMVs via electroporation.Results:CMVs were efficiently labeled with Ag2Se QDs through electroporation with favorable biocompatibility.Due to the favorable biocompatibility and superior dual-mode traceability of Ag2Se@Mn QD-labeled CMVs,we investigated the in vivo biodistribution patterns of CMVs and revealed the excellent natural tumor-targeting activity of CMVs in living mice.Importantly,the siRNA/Ag2Se QDs-loaded CMVs exhibited excellent therapeutic effects in an oral cancer-bearing mice model.Conclusion:This study not only realized the first efficient labeling and in vivo dual-mode tracking of CM Vs that were directly isolated from human body fluids but also transformed these naturally occurring MVs into multifunctional visualized nanovectors for targeted oncotherapy,opening new avenues for the development of real natural theranostic nanoplatforms.