Extracellular Membrane Vesicles Research Articles

Abstract 3985: S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos

Abstract Growing evidence indicates that tumor cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that of liver progenitor genes. However, the role of S100A10 in hepatocellular carcinoma (HCC) progression is unclear. Furthermore, extracellular vesicles (EVs) or exosomes are critical mediator of tumorigenesis and metastasis, but the extracellular functions of S100A10, particularly those related to EVs (EV-S100A10), are unknown. In this study, we observed that S100A10 was upregulated at mRNA level in patients’ HCC tumors as compared to the corresponding non-tumorous livers, associated with a copy-number gain. Overexpression of S100A10 was correlated with more aggressive tumor features and poor prognosis. Furthermore, functional assays demonstrated it promoted HCC initiation and progression, including increased sphere forming ability in vitro, enhanced chemoresistance, upregulated liver cancer stemness markers and activated AKT and ERK pathways. In addition, in orthotopic injection mouse model, S100A10 enhanced both the intrahepatic and pulmonary metastasis of HCC xenografts, with upregulated epithelial-mesenchymal transition (EMT) changes. Of significance, we found that S100A10 was present in EVs secreted by HCC cells. Functionally, the EVs secreted by HCC cells promoted the migratory and invasive abilities of recipient HCC cells. Consistent with the oncogenic role of S100A10 in HCC, EVs from S100A10-overexpressing HCC cells enhanced the tumor promoting effects, while the S100A10-depleted HCC cells abrogated the effects, as compared to the vector control group. To further consolidate the tumor promoting role of S100A10-EV, we blocked the effects using neutralizing antibody (NA) by pretreating HCC cells with S100A10 NA together with EV. Indeed, the NA significantly abrogated the stemness and metastatic properties induced by S100A10-EVs both in vitro and in vivo. In addition, the EMT changes and activation of AKT and ERK were also validated in the recipient HCC cells with the S100A10-EV treatment, while they were abolished with the NA treatment. Moreover, we found that S100A10 governed the protein cargos in EVs and mediated the binding of MMP2, fibronectin and EGF to EV membranes through physical binding with integrin αV to promote the motility of recipient cells. Of note, we observed a simultaneous change of p-EGFR, indicating potential activation of AKT and ERK induced by EGFR. On the other hand, we found the plasma EV-S100A10 level was relatively higher in HCC patients than healthy donors. Collectively, the data showed that S100A10 could be transferred among cells through EVs and activated signaling pathways to facilitate metastasis. Targeting S100A10 may be a potential therapeutic strategy for HCC. Citation Format: Lu TIAN, Xia WANG, Jingyi LU, HongYang Huang, Goofy Yu-Man Tsui, Karen Man-Fong Sze, Tan-To Cheung, Irene Oi-Lin NG. S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3985.

The Emerging Biological Functions of Exosomes from Dental Tissue-Derived Mesenchymal Stem Cells.

Exosomes are one kind of small-cell extracellular membranous vesicles that can regulate intercellular communication and give rise to mediating the biological behaviors of cells, involving in tissue formation, repair, the modulation of inflammation, and nerve regeneration. The abundant kinds of cells can secret exosomes, among them, mesenchymal stem cells (MSCs) are very perfect cells for mass production of exosomes. Dental tissue-derived mesenchymal stem cells (DT-MSCs), including dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, stem cells from human periodontal ligament (PDLSCs), gingiva-derived mesenchymal stem cells, dental follicle stem cells, tooth germ stem cells, and alveolar bone-derived mesenchymal stem cells, are now known as a potent tool in the area of cell regeneration and therapy, more importantly, DT-MSCs can also release numerous types of exosomes, participating in the biological functions of cells. Hence, we briefly depict the characteristics of exosomes, give a detailed description of the biological functions and clinical application in some respects of exosomes from DT-MSCs through systematically reviewing the latest evidence, and provide a rationale for their use as tools for potential application in tissue engineering.

Recent advances in extracellular vesicles in gastrointestinal cancer and lymphoma.

Extracellular vesicles (EVs) are intercellular communication agents that transfer microRNAs (miRNAs), other non-coding RNAs (ncRNAs), messenger RNAs (mRNAs), proteins, lipids, metabolites, and other molecules from donor cells (e.g., cancer cells) to recipient cells (e.g., stromal cells). In 2007, miRNAs were reported to be abundant among the ncRNAs present in EVs. Since then, many studies have investigated the functions of miRNAs and have attempted to apply these molecules to aid in the diagnosis and treatment of cancer. Research on EVs has expanded, particularly in the field of cancer, because cancer cells heavily secrete EVs. The cargo of these EVs, especially those in small EVs, such as exosomes, is assumed to work cooperatively and significantly in the tumor microenvironment and to promote metastasis. In this review, we first summarize recent studies on EVs in gastrointestinal cancer and highlight studies on human satellite II RNAs, which are a type of ncRNA found in EVs that possess repetitive sequences. Second, since several recent studies have revealed that phospholipids, which are components of EV membranes, play important roles in intercellular communication and the generation of lipid mediators in the tumor microenvironment, we review the reported roles of these molecules and discuss their potential use in the design of new cancer treatments.

Mussel settlement mediated by bacterial VgrG proteins via extracellular outer membrane vesicles

T6SS plays an important role in biofilm formation, but how bacterial T6SS regulates biofilm formation and its effect on marine invertebrate settlement is still unclear. Here, the role of T6SS spike protein VgrG and VgrG protein synthesis gene on biofilm formation and Mytilus coruscus settlement was examined. Deletion of the core gene vgrG of T6SS resulted in a decrease in the growth capability of Leisingera aquaemixtae and inhibition of the ability of biofilm formation. The OMV content and inducing activity of ΔvgrG biofilms were decreased respectively by 20.72% and 59.70% compared with the wild-type biofilms. OMVs induced the settlement of M. coruscus, while VgrG proteins did not induce mussel settlement directly. Addition of VgrG proteins resulted in the upregulation of OMVs in the ΔvgrG biofilms. Adding OMVs caused no increasing inducing activity of ΔvgrG biofilms. Interestingly, the inducing activity of ΔvgrG biofilms both by adding VgrG proteins alone, and by adding VgrG proteins and OMVs in combination increased significantly, and restored to the inducing capability of wild-type biofilms. Thus, bacterial T6SS spike protein VgrG could mediate mussel settlement by increasing the OMV content of biofilms. The present findings shed new light on the interaction between T6SS and marine invertebrate settlement.

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery.

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Small extracellular vesicles have distinct CD81 and CD9 tetraspanin expression profiles in plasma from rheumatoid arthritis patients.

Extracellular vesicles (EVs) are implicated in the pathogenesis of rheumatoid arthritis (RA) but little is known about the composition of specific small EV (sEV) subpopulations. This study aimed to characterize the CD63, CD81 and CD9 tetraspanin profile in the membrane of single EVs in plasma from treatment naïve RA patients and assess potential discrepancies between methotrexate (MTX) responder groups. EVs isolated from plasma were characterized using transmission electron microscopy, and detection of surface markers (CD63, CD81 and CD9) on single EVs was performed on the ExoView platform. All RA patients (N = 8) were newly diagnosed, treatment naïve, females, ACPA positive and former smokers. The controls (N = 5) were matched for age and gender. After three months of MTX treatment, responders (N = 4) were defined as those with ΔDAS28 > 1.2 and DAS28 ≤ 3.2 post-treatment. The isolated EVs were 50-200nm in size. The RA patients had a higher proportion of both CD9 and CD81 single positive sEVs compared to healthy controls, while there was a decrease in CD81/CD9 double positive sEVs in patients. Stratification of RA patients into MTXresponders and non-responders revealed a distinctly higher proportion of CD81 single positive sEVs in the responder group. The proportion of CD81/CD9 double positive sEVs (anti-CD9 captured) was lower in the non-responders, but increased upon 3months of MTXtreatment. Our exploratory study revealed distinct tetraspanin profiles in RA patients suggesting their implication in RA pathophysiology and MTX treatment response.

Tuning the Extracellular Vesicles Membrane through Fusion for Biomedical Applications.

Membrane fusion is one of the key phenomena in the living cell for maintaining the basic function of life. Extracellular vesicles (EVs) have the ability to transfer information between cells through plasma membrane fusion, making them a promising tool in diagnostics and therapeutics. This study explores the potential applications of natural membrane vesicles, EVs, and their fusion with liposomes, EVs, and cells and introduces methodologies for enhancing the fusion process. EVs have a high loading capacity, bio-compatibility, and stability, making them ideal for producing effective drugs and diagnostics. The unique properties of fused EVs and the crucial design and development procedures that are necessary to realize their potential as drug carriers and diagnostic tools are also examined. The promise of EVs in various stages of disease management highlights their potential role in future healthcare.

A Versatile Design-Enabled Analysis of Circulating Extracellular Vesicles in Disease Diagnosis.

Circulating extracellular vesicles (EVs) are considered as potential biomarkers for treatment and diagnosis of many diseases. Most of the existing methods for the EV analysis only have a single function and thus reveal limited information carried by EVs. Herein, a phosphatidylserine-targeting peptide-facilitated design that enables the versatile analysis of circulating EVs for varying requirement is proposed. In the design, DNA probes are inserted into the EV membrane through hydrophobic interactions, and accelerate the removal of protective shielding from DNA-gated metal-organic framework, thereby releasing a large number of methylene blue molecules to amplify the electrochemical signal. Electrochemical results demonstrate equally high sensitivities toward the quantification of EVs derived from different cell sources using an indiscriminative DNA probe. More importantly, the probe can be endowed with extended function for more accurate classification of cell-specific features through the identification of specific EV biomarkers, and demonstrates the potential use in the diagnosis of cardiovascular in a principle-of-proof study for clinical application. Therefore, the method provides a versatile design for the identification of EV features, and may address the needs of clinical diagnosis in the future.

Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Contribution of Extracellular Membrane Vesicles To the Secretome of Staphylococcus aureus.

The microbial secretome modulates how the organism interacts with its environment. Included in the Staphylococcus aureus secretome are extracellular membrane vesicles (MVs) that consist of cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. The extent to which MVs contribute to the diverse composition of the secretome is not understood. We performed a proteomic analysis of MVs purified from the S. aureus strain MRSA252 along with a similar analysis of the whole secretome (culture supernatant) before and after depletion of MVs. The MRSA252 secretome was comprised of 1,001 proteins, of which 667 were also present in MVs. Cell membrane-associated proteins and lipoteichoic acid in the culture supernatant were highly associated with MVs, followed by cytoplasmic and extracellular proteins. Few cell wall-associated proteins were contained in MVs, and capsular polysaccharides were found both in the secretome and MVs. When MVs were removed from the culture supernatant by ultracentrifugation, 54 of the secretome proteins were significantly depleted in abundance. Proteins packaged in MVs were characterized by an isoelectric point that was significantly higher than that of proteins excluded from MVs. Our data indicate that the generation of S. aureus MVs is a mechanism by which lipoteichoic acid, cytoplasmic, and cell membrane-associated proteins are released into the secretome. IMPORTANCE The secretome of Staphylococcus aureus includes soluble molecules and nano-sized extracellular membrane vesicles (MVs). The protein composition of both the secretome and MVs includes cytoplasmic and membrane proteins, as well as exoproteins, some cell wall-associated proteins, and glycopolymers. How the MV cargo differs from the protein composition of the secretome has not yet been addressed. Although the compositions of the secretome and MVs were strikingly similar, we identified 54 proteins that were specifically packaged in MVs. Proteins highly associated with MVs were characterized by their abundance in the secretome, an association with the bacterial membrane, and a basic isoelectric point. This study deepens our limited understanding about the contribution of MVs to the secretome of S. aureus.

Extra-Cellular Vesicles Derived from Thyroid Cancer Cells Promote the Epithelial to Mesenchymal Transition (EMT) and the Transfer of Malignant Phenotypes through Immune Mediated Mechanisms.

Thyroid cancer is the most common endocrine cancer, and its incidence is increasing in many countries around the world. Among thyroid cancers, the papillary thyroid cancer (PTC) histotype is particularly prevalent. A small percentage of papillary tumors is associated with metastases and aggressive behavior due to de-differentiation obtained through the epithelial-mesenchymal transition (EMT) by which epithelial thyroid cells acquire a fibroblast-like morphology, reduce cellular adhesion, increase motility and expression of mesenchymal proteins. The tumor microenvironment plays an important role in promoting an aggressive phenotype through hypoxia and the secretion of HMGB1 and other factors. Hypoxia has been shown to drastically change the tumor cell phenotype and has been associated with increasing metastatic and migratory behavior. Cells transfer information to neighboring cells or distant locations by releasing extracellular membrane vesicles (EVs) that contain key molecules, such as mRNAs, microRNAs (miRNAs), and proteins, that are able to modify protein expression in recipient cells. In this study, we investigated the potential role of EVs released by the anaplastic cancer cell line CAL-62 in inducing a malignant phenotype in a papillary cancer cell line (BCPAP).

Construction of Fusion Protein for Enhanced Small RNA Loading to Extracellular Vesicles.

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs.

Escherichia coli-Derived Outer Membrane Vesicles Relay Inflammatory Responses to Macrophage-Derived Exosomes.

Extracellular vesicles are considered to be an inflammatory factor in several acute and chronic inflammatory diseases. The present study shows that exosomes from macrophages (Mφ) infected with live Escherichia coli induced secretion of proinflammatory factors by uninfected Mφ. Inflammatory responses induced by exosomes derived from Mφ infected with heat-inactivated E. coli or lipopolysaccharide were significantly weaker than those elicited by outer membrane vesicles (OMVs) released from live E. coli. Proteome analysis of exosomes from Mφ infected with live or heat-inactivated E. coli revealed that E. coli proteins OmpA, GroL1, DegP, CirA, and FepA are candidate triggers of exosome-mediated inflammatory responses. OMVs from a cirA-deleted strain suppressed exosome-mediated inflammatory responses by uninfected Mφ. The C terminus of the CirA protein (residues 158 to 633), which was relayed from E. coli-derived OMV to Mφ-derived exosomes, promoted exosome-mediated inflammatory responses by uninfected Mφ. These results suggest an alternative mechanism by which extracellular vesicles from E. coli OMV-elicited Mφ transmit proinflammatory responses to uninfected Mφ. IMPORTANCE Recently, extracellular membrane vesicles (EVs) were regarded as drivers that carry cargo such as proteins, lipids, metabolites, RNA, and DNA for intracellular signaling transduction. Mammalian cells release various types of EVs, including microvesicles shed from the plasma membrane, exosomes from endosomes, apoptotic bodies, and others. EVs have been reported to mediate inflammatory signals between mammalian cells. In addition, bacteria are also known to release EVs to carry various bacterial factors. In this study, we show that bacterial EVs lead host mammalian cells to release stimulatory EVs that enhance inflammatory responses. Our results provide a novel example that bacterial EVs transduce biological signals to mammalian EVs.

Loading Nanoceria Improves Extracellular Vesicle Membrane Integrity and Therapy to Wounds in Aged Mice.

Wound healing is a programmed process through which tissue restores its integrity after an injury. Advancing age is a risk factor for delayed cutaneous wound healing; however, ideal therapeutic approaches for aged wound have not been developed yet. By dissecting the harsh microenvironment of aged wound, we propose an integrated chemical and biological strategy to mitigate two main hostile factors including oxidative stress and ischemia. Mesenchymal stem cell-derived extracellular vesicles (EVs) are a rising star in regenerative medicine due to their powerful facilitation in tissue repair and regeneration. However, the fragile lipid membrane limits their function under the oxidative stress microenvironment. Nanoceria is an antioxidative nanozyme; here, we reveal that nanoceria-loaded EVs derived from mesenchymal stem cells facilitate cutaneous wound healing in aged mice. DG-CeO2 was prepared via coating CeO2 covalently with d-glucose to promote their cellular endocytosis. DG-CeO2 was packaged into EVs under optimized hypoxic conditions (DG-CeO2 EVsHyp). We further demonstrated that DG-CeO2 EVsHyp had favorable biocompatibility and antioxidative and proangiogenic effects during the cutaneous wound healing in both young and aged mice. Further evidence revealed that DG-CeO2 EVsHyp-transferred miR-92a-3p/125b-5p and their targets associated with aging degeneration may be the potential mechanisms. Collectively, these findings highlight that nanoceria-loaded EVs released by engineered stem cells may represent a potential therapeutic approach for tissue regeneration in aged population.

S100A10 promotes HCC development and progression via transfer in extracellular vesicles and regulating their protein cargos

ObjectiveGrowing evidence indicates that tumour cells exhibit characteristics similar to their lineage progenitor cells. We found that S100 calcium binding protein A10 (S100A10) exhibited an expression pattern similar to that...

Microfluidic Platform for Profiling of Extracellular Vesicles from Single Breast Cancer Cells.

Extracellular vesicles (EVs) are considered as valuable biomarkers to discriminate healthy from diseased cells such as cancer. Passing cytosolic and plasma membranes before their release, EVs inherit the biochemical properties of the cell. Here, we determine protein profiles of single EVs to understand how much they represent their cell of origin. We use a microfluidic platform which allows to immobilize EVs from completely isolated single cells, reducing heterogeneity of EVs as strongly seen in cell populations. After immunostaining, we employ four-color total internal reflection fluorescence microscopy to enumerate EVs and determine their biochemical fingerprint encoded in membranous or cytosolic proteins. Analyzing single cells derived from pleural effusions of two different human adenocarcinoma as well as from human embryonic kidney (SkBr3, MCF-7 and HEK293, respectively), we observed that a single cell secretes enough EVs to extract the respective tissue fingerprint. We show that overexpressed integral plasma membrane proteins are also found in EV membranes, which together with populations of colocalized proteins, provide a cell-specific, characteristic pattern. Our method highlights the potential of EVs as a diagnostic marker and can be directly employed for fundamental studies of EV biogenesis.

Study on biofilm formation and heterogeneity in Clostridium perfringens

Many bacteria form biofilms and survive in the actual environment. Biofilms are not only a major form of bacteria but are also involved in tolerance to environmental stresses and antibiotics, suggesting the association with bacterial pathogenesis. Cells within biofilms display phenotypic heterogeneity; thus, even bacteria, unicellular organisms, can functionally differentiate and show multicellular behavior. Therefore, it is necessary to understand bacteria as a population to control their survival and pathogenesis in the actual environment. Previously, we found that Clostridium perfringens, an anaerobic pathogenic bacterium, form different structures in different temperatures and phenotypic heterogeneity on biofilm matrix gene expression within the biofilm. In this article, I summarize the results of our research on biofilms and their heterogeneity, the mechanisms of post-transcriptional gene expression regulation of virulence genes, and bacteria-host interactions mediated by extracellular membrane vesicles.

Biogenesis of Mesoporous Silica Nanoparticles Enclosed in Extracellular Vesicles by Mouse Renal Adenocarcinoma Cells.

Integrating the versatility of synthetic nanoparticles to natural biomaterials, such as cells or cell membranes, has gained considerable attention as promising alternative cargo delivery platforms in recent years. Extracellular vesicles (EVs), natural nanomaterials composed of a protein-rich lipid bilayer secreted by cells, have also shown advantages and great potential as a nano delivery platform in combination with synthetic particles due to their specific natural properties in overcoming several biology hurdles possessed in the recipient cell. Therefore, the preservation of EV's origin properties is critical for their application as nanocarriers. This chapter will describe the encapsulation procedure of MSN encapsulated in EV membrane derived from mouse renal adenocarcinoma (Renca) cells through biogenesis. The FMSN-enclosed EVs produced through this approach still contain preserved EV's natural membrane properties.

Compartmentalized drug localization studies in extracellular vesicles for anticancer therapy.

In the development of therapeutic extracellular vesicles (EVs), drug encapsulation efficiencies are significantly lower when compared with synthetic nanomedicines. This is due to the hierarchical structure of the EV membrane and the physicochemical properties of the candidate drug (molecular weight, hydrophilicity, lipophilicity, and so on). As a proof of concept, here we demonstrated the importance of drug compartmentalization in EVs as an additional parameter affecting the therapeutic potential of drug-loaded EVs. In human adipose mesenchymal stem cell (hADSC) derived EVs, we performed a comparative drug loading analysis using two formulations of the same chemotherapeutic molecule - free doxorubicin (DOX) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid-conjugated doxorubicin (L-DOX) - to enhance the intracellular uptake and therapeutic efficacy. By nano surface energy transfer (NSET) and molecular simulation techniques, along with cryo-TEM analysis, we confirmed the differential compartmentalization of these two molecules in hADSC EVs. L-DOX was preferentially adsorbed onto the surface of the EV, due to its higher lipophilicity, whereas free DOX was mostly encapsulated within the EV core. Also, the L-DOX loaded EV (LDOX@EV) returned an almost three-fold higher DOX content as compared to the free DOX loaded EV (DOX@EV), for a given input mass of drug. Based on the cellular investigations, L-DOX@EV showed higher cell internalization than DOX@EV. Also, in comparison with free L-DOX, the magnitude of therapeutic potential enhancement displayed by the surface compartmentalized L-DOX@EV is highly promising and can be exploited to overcome the sensitivity of many potential drugs, which are impermeable in nature. Overall, this study illustrates the significance of drug compartmentalization in EVs and how this could affect intracellular delivery, loading efficiency, and therapeutic effect. This will further lay the foundation for the future systematic investigation of EV-based biotherapeutic delivery platforms for personalized medicine.

Improved extracellular vesicle-based mRNA delivery for familial hypercholesterolemia treatment.

Extracellular vesicle (EV)-based low-density lipoprotein receptor (Ldlr) mRNA delivery showed excellent therapeutic effects in treating familial hypercholesterolemia (FH). Nevertheless, the loading inefficiency of EV-based mRNA delivery presents a significant challenge. Recently, RNA-binding proteins (RBPs) have been fused to EV membrane proteins for selectively encapsulating targeted RNAs to promote loading efficiency. However, the strong interaction between therapeutic RNAs and RBPs prevents RNA release from endosomes to the cytosol in the recipient cells. In this study, an improved strategy was developed for efficient encapsulation of Ldlr mRNA into EVs in donor cells and controllable release in recipient cells. Methods: The MS2 bacteriophage coat protein (CD9-MCP) fusion protein, Ldlr mRNA, and a customized MS2 containing RNA aptamer base-pair matched with Ldlr mRNA were expressed in donor cells. Cells receiving the above therapeutic EVs were simultaneously treated with EVs containing "Ldlr releaser" with a sequence similar to the recognition sites in Ldlr mRNA. Therapeutic effects were analyzed in Ldlr-/- mice receiving EV treatments via the tail vein. Results: In vitro experiments demonstrated improved loading efficiency of Ldlr mRNA in EVs via MS2-MCP interaction. Treatment of "Ldlr releaser" competitively interacted with MS2 aptamer with higher affinity and released Ldlr mRNA from CD9-MCP for efficient translation. When the combinatory EVs were delivered into recipient hepatocytes, the robust LDLR expression afforded therapeutic benefits in Ldlr-/- mice. Conclusion: We proposed an EV-based mRNA delivery strategy for enhanced encapsulation of therapeutic mRNAs in EVs and RNA release into the cytosol for translation in recipient cells with great potential for gene therapy.