Extracellular Delivery Research Articles

Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells

Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (~1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (~30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.

Phytotoxic Tryptoquialanines Produced In Vivo by Penicillium digitatum Are Exported in Extracellular Vesicles.

Penicillium digitatum is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by P. digitatum It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by P. digitatum. To investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of Citrus sinensis seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in P. digitatum We detected extracellular vesicles (EVs) released by P. digitatum both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity in vitro and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the P. digitatum EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of P. digitatum EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection.IMPORTANCE During the postharvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes green mold disease and is responsible for up to 90% of total citrus losses. Chemical fungicides are widely used to prevent green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the P. digitatum model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by P. digitatum during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how P. digitatum causes damage to its primary hosts.

Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle for miRNA-Based Therapy

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Non-viral reprogramming of human nucleus pulposus cells with FOXF1 via extracellular vesicle delivery: an in vitro and in vivo study.

Intervertebral disc (IVD) degeneration is characterized by decreased cellularity and proteoglycan synthesis and increased inflammation, catabolism, and neural/vascular ingrowth. Regenerative methods for IVD degeneration are largely cell-therapy-based or involve viral vectors, which are associated with mutagenesis and undesired immune responses. The present study used bulk electroporation and engineered extracellular vesicles (EVs) to deliver forkhead-box F1 (FOXF1) mRNA to degenerate human nucleus pulposus (NP) cells as a minimally invasive therapeutic strategy for IVD regeneration. Bulk electroporation was used to investigate FOXF1 effects on human NP cells during a 4-week culture in 3D agarose constructs. Engineered EV delivery of FOXF1 into human IVD cells in monolayer was determined, with subsequent in vivo validation in a pilot mouse IVD puncture model. FOXF1 transfection significantly altered gene expression by upregulating healthy NP markers [FOXF1, keratin 19 (KRT19)], decreasing inflammatory cytokines [interleukin (IL)-1β, −6], catabolic enzymes [metalloproteinase 13 (MMP13)] and nerve growth factor (NGF), with significant increases in glycosaminoglycan accumulation in human NP cells. Engineered EVs loaded with FOXF1 demonstrated successful encapsulation of FOXF1 cargo and effective uptake by human NP cells cultured in monolayer. Injection of FOXF1-loaded EVs into the mouse IVD in vivo resulted in a significant upregulation of FOXF1 and Brachyury, compared to controls at 7 d post-injection, with no evidence of cytotoxicity. This is the first study to demonstrate non-viral delivery of FOXF1 and reprogramming of human NP cells in vitro and mouse IVD cells in vivo. This strategy represents a non-addictive approach for treating IVD degeneration and associated back pain.

Evidence for the extracellular delivery of influenza NS1 protein

We constructed a reporter influenza A/Puerto Rico/8/1934 virus expressing truncated 124aa N-terminal NS1 protein fused to a luciferase reporter sequence (NanoLuc) without signal peptide. The reproduction activity of the vector correlated well with the luminescent activity in the lysates of infected cell cultures or mouse respiratory organ suspensions. Surprisingly, we found that luciferase enzymatic activity was present not only in the intracellular compartments but also in cell culture supernatants as well as in the sera or bronchiolar lavages of infected mice. This fact allowed us to formulate a working hypothesis about the extracellular delivery mechanism of the NS1 protein. To test this idea, we conducted co-transfection experiments in Vero cells with different combinations of plasmids encoding influenza genomic segments and chimeric NS1-NanoLuc encoding plasmid. We found that the emergence of the luciferase reporter in the extracellular compartment was promoted by the formation of the ribonucleoprotein complex (RNP) from the co-transfection of plasmids expressing PB1, PB2, PA, and NP proteins. Therefore, influenza NS1 protein may be delivered to the extracellular compartment together with the nascent RNP complexes during the maturation of virus particles.

Socially Distanced Intercellular Communication: Mechanisms for Extracellular Vesicle Cargo Delivery.

Extracellular vesicles (EVs) are increasingly being recognised as players in intercellular communication within the human body. EVs are nano-sized vesicles that are secreted by virtually all cells, primarily arising from either the plasma membrane or the endocytic system. They contain a wide range of proteins and nucleic acids in their lumen, as well as cell surface proteins on their exterior. The proteins and nucleic acids within are the 'cargo' that EVs deliver into the cytosol of recipient cells to elicit a response or phenotypic change. For delivery to occur, the cargo needs to cross two lipid bilayers; one that makes up the vesicle itself, and the other of the recipient cell. Exactly how this process works is a topic that is poorly understood, despite being pivotal for their function. Furthermore, extracellular vesicles have therapeutic potential as drug delivery vehicles. Therefore, understanding their delivery mechanism and harnessing its action for drug delivery is of great importance. This chapter will focus on the proposed mechanisms for cargo delivery and discuss existing evidence for cargo delivery from EVs into the cytosol of recipient cells.

A pH-sensitive T7 peptide-decorated liposome system for HER2 inhibitor extracellular delivery: an application for the efficient suppression of HER2+ breast cancer.

HER2+ breast cancer is highly aggressive and proliferative even after multiple chemotherapy regimens. At present, the available clinical treatment duration of chemotherapeutic agents is limited by severe toxicity to noncancerous tissues, which are attributed to insufficient targeting. Here, we designed an active-targeted and pH-responsive liposome to improve the treatment. The ideas were as follows: (1) using liposome as a nano-delivery system for HER2 inhibitor (lapatinib; LAP) to reduce the toxicity; (2) modifying the capsule with T7 peptide for specific targeted delivery to the tumor cells, and (3) enabling the capsule with the pH-sensitive ability and triggering sustained drug release at extracellular weakly acidic microenvironment to emerge toxicity in tumors and to improve curative effects. It was found that T7 peptide-modified pH-sensitive liposome (T7-LP) was more effective and safer than free drug and unmodified liposome, and reduced drug-induced side effects and noncancerous toxicity. These results support the application potential of T7-LP in improving the efficacy of LAP in HER2+ breast cancer treatment. It might be a novel LAP formulation as a clinical agent.

Recent trends and development in targeted delivery of therapeutics through enzyme responsive intelligent nanoplatform

Stimuli responsive nanocarriers proved to be an efficient system in accurate diagnosis and targeted delivery of therapeutic moieties. Enzymes have been researched widely as successful targeting agents among various endogenous and exogenous stimuli responsive systems and employed as bio-nanotechnological tool due to their intrinsic presence, higher specificity and catalytic action in various biological processes. Nanosystems are fabricated possessing unique responsiveness toward enzymes that are elevated at pathological sites leading to targeted release of drugs at diseased site. This review discusses various enzymes and explores recently developed nanocarriers responsive to enzymes with versatile extracellular and intracellular drug delivery applications.

Intense cardiac-targeted small extracellular vesicles-mediated delivery of RAGE siRNA attenuates inflammation in rat myocarditis model

Abstract Background Despite advances in the field and new therapeutics being developed, heart disease remains the leading cause of mortality worldwide. Small extracellular vesicles (sEV) are 30–150 nm in diameter and ferry RNA and proteins to cells. sEV are a natural carrier of many signaling molecules and play an important role in heart disease. However, the high probability of off-target effects associated with these carriers is a major barrier to translation into clinical application. Purpose Our aim was to evaluate whether sEVs engineered to express potent cardiac targeting peptides (CTPs) could deliver siRNA to the heart and exert a therapeutic effect. Methods We use vectors encoding LAMP2B (sEV) or CTP-LAMP2B (PsEV) into HEK 293 cells expressing. sEVs were purified from culture media of HEK 293 cells by serial centrifugation followed by tangential flow filtration (TFF) system. sEV and PsEV were loaded with siRNAs by Exo-Fect™ exosome transfection reagent, and were treated into H9C2 rat cardiomyocyte. TNF-alpha were then added to the cells to induce inflammation. And sEVs were intravenously injected into myocarditis rat. Inflammation factors of in vitro and in vivo inflammation model were identified by western blot. Echocardiographic examination was also performed in rat. Results The successful development of PsEVs was analyzed by Western blot and TEM. We observed a 4 fold increase from that of the previously developed sEVs in H9C2 cells and a 200% increase in cardiac-specific delivery without toxicity in rat model. AGE is involved in proinflammatory/pro-apoptotic processes. To block RAGE, we loaded RAGE siRNA (siRAGE) in sEV that had high expression of PsEV. Characteristics of sEVs were maintained despite siRNA load.In inflammatory cell models and rat disease models, PsEV-treated groups significantly reduced molecular levels associated with inflammatory responses such as RAGE, IL-6, TNF-alpha, COX2, HMGB1, and p-p65 / p65. Conclusions Our results suggest that PsEV can potentially serve as a treatment delivery vehicle for heart disease. Scheme Funding Acknowledgement Type of funding source: None

Abstract PO-049: Exploiting tumor acidic microenvironment for improved therapeutics

Abstract The tumor acidic microenvironment is a fundamental characteristic of solid tumors which distinguishes the tumor from adjacent normal tissues. This unique tumor microenvironment (TME) feature directly contributes to various aspect of tumor progression including gene expression, immune suppression, drug resistance, invasion and metastasis. Acidic TME poses great challenges for the efficacy of therapeutics; extracellular acidity of cancer cells provides less permissive conditions for optimal target engagement and drug efficacy. In addition targeting the effectors of acid-base balance enriched in hypoxia and acidic regions of the tumor provide valuable tools for normalizing the extracellular acidity and delivery of therapeutics agents to areas of the tumor less malleable to conventional therapies. Thus in the case of therapeutic antibodies, strategies that specifically integrates this TME specific feature in the design could improve binding to the target under low pH conditions and serve as delivery moiety that improve tumor specificity and functional efficacy. Here we describe two examples of these therapeutic strategies that leverage the acidic and hypoxic microenvironment to overcome the challenges posed by this hostile environment with the aim of generating improved therapeutic antibodies. A. Characterization of a set of function blocking therapeutic antibodies against key target expressed in response to hypoxia which contributes to tumor acidification, we demonstrate functional efficacy of this antibody to block the activity of the target in vitro and consequently reduce tumor spheroid growth. This and similar antibodies provide tools for specific targeting of the tumor areas that are often inaccessible to most therapeutics. B. Identification of targets enriched in acidic tumor microenvironment and design of pH selective therapeutic antibodies that preferentially bind to the target and exert their function under tumor acidic conditions. We demonstrate the functional selectivity of a variant of antibody targeting HER 2, with optimized binding under low pH conditions, on the growth of BT474 spheroids. The pH selective antibody variant blocked the tumor spheroid growth when spheroids were grown in TME-relevant- low pH conditions but it remained ineffective under normal physiological pH conditions. Suggesting increased selectivity of this antibody variant towards the acidic TME that in turn lower the on-target -off-tumor toxicity. In conclusion here we provide two examples of the strategies which exploit the targeting of hypoxia induced genes and pH selective design of the therapeutic antibodies in order to improve tumor targeting capacity and reduce systemic toxicity of conventional therapeutics. Citation Format: Nazanin Rohani Larijani, Traian Sulea, Mehdi Arbabi Ghahroudi, Beatrice Paul Roc, Mylene Gosselin, Joey Sheff, John C. Zwaagstra, Anne E.G. Lenferink. Exploiting tumor acidic microenvironment for improved therapeutics [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-049.

Extracellular vesicles-based drug delivery systems for cancer immunotherapy

Extracellular vesicles (EVs), a heterogeneous population of membrane vesicles, are secreted by almost all living cells and contain different proteins, lipids and nucleic acid depending on their sources. Recently, numerous reports indicated EVs could interact with diverse cells in tumor microenvironment and influence both physiological and pathological conditions. Given that EVs regulate tumor immune microenvironment, EVs-based strategies are explored for anti-tumor immunotherapy. In this review, about nine kinds of EVs derived from diverse cells are indicated. The biological functions in tumor immunity, strategies for anti-tumor immunotherapy and further clinical trials of these EVs will be introduced in detail.

Development of Triggerable, Trackable, and Targetable Carbon Monoxide Releasing Molecules.

Carbon monoxide (CO) is a gaseous signaling molecule produced in humans via the breakdown of heme in an O2-dependent reaction catalyzed by heme oxygenase enzymes. A long-lived species relative to other signaling molecules (e.g., NO, H2S), CO exerts its physiological effects via binding to low-valent transition metal centers in proteins and enzymes. Studies involving the administration of low doses of CO have shown its potential as a therapeutic agent to produce vasodilation, anti-inflammatory, antiapoptotic, and anticancer effects. In pursuit of developing tools to define better the role and therapeutic potential of CO, carbon monoxide releasing molecules (CORMs) were developed. To date, the vast majority of reported CORMs have been metal carbonyl complexes, with the most well-known being Ru2Cl4(CO)6 (CORM-2), Ru(CO)3Cl(glycinate) (CORM-3), and Mn(CO)4(S2CNMe(CH2CO2H)) (CORM-401). These complexes have been used to probe the effects of CO in hundreds of cell- and animal-based experiments. However, through recent investigations, it has become evident that these reagents exhibit complicated reactivity in biological environments. The interpretation of the effects produced by some of these complexes is obscured by protein binding, such that their formulation is not clear, and by CO leakage and potential redox activity. An additional weakness with regard to CORM-2 and CORM-3 is that these compounds cannot be tracked via fluorescence. Therefore, it is unclear where or when CO release occurs, which confounds the interpretation of experiments using these molecules. To address these weaknesses, our research team has pioneered the development of metal-free CORMs based on structurally tunable extended flavonol or quinolone scaffolds. In addition to being highly controlled, with CO release only occurring upon triggering with visible light (photoCORMs), these CO donors are trackable via fluorescence prior to CO release in cellular environments and can be targeted to specific cellular locations.In the Account, we highlight the development and application of a series of structurally related flavonol photoCORMs that (1) sense characteristics of cellular environments prior to CO release; (2) enable evaluation of the influence of cytosolic versus mitochondrial-localized CO release on cellular bioenergetics; (3) probe the cytotoxicity and anti-inflammatory effects of intracellular versus extracellular CO delivery; and (4) demonstrate that albumin delivery of a photoCORM enables potent anticancer and anti-inflammatory effects. A key advantage of using triggered CO release compounds in these investigations is the ability to examine the effects of the molecular delivery vehicle in the absence and presence of localized CO release, thus providing insight into the independent contributions of CO. Overall, flavonol-based CO delivery molecules offer opportunities for triggerable, trackable, and targetable CO delivery that are unprecedented in terms of previously reported CORMs and, thus, offer significant potential for applications in biological systems.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This articleprovides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic. Current challenges limiting development of EVs for drug delivery are the lack of: standardized cell-based platforms for the production of EV-based therapeutics; EV reference materials that allow researchers/manufacturers to validate EV measurementsand standardized measurement systems for determining the molecular composition of EVs.

Abstract 6242: Development of alphalex™-toxin low pH targeting conjugates for the treatment of solid tumors

Abstract Maytansines and their derived maytansinoid DMx compounds are high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Unacceptable dose limiting systemic toxicity has limited the therapeutic potential of these potent anti-oncogenic compounds. Targeting maytansinoids to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. To date Trastuzumab-DM1 (Kadcyla®) remains the only approved antibody-maytansinoid conjugate on the market. Most preclinical maytansinoid conjugates to date face the same issues encountered by Kadcyla® - tumor restriction by target antigen and the potential for off target release of payload. alphalexTM is a tumor targeting technology consisting of a unique variant of pH-Low Insertion Peptide (pHLIP®; references 1-3), cleavable small molecule linker and anti-cancer agent warhead. alphalexTM thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we demonstrate the ability to conjugate the maytansinoids DM1 and DM4 to alphalexTM via both direct and linker-mediated conjugation. We have demonstrated the ability of our alphalexTM-DM4 conjugate candidates (CBX-13) to have single digit nanomolar potency in vitro as well as exquisitely potent and long-lasting anti-tumor activity in a HER2-negative xenograft model that is un-targetable by competing therapies. In particular we have demonstrated that CBX-13 safely delivers amounts of maytansinoid in vivo that otherwise result in systemic toxicity and death when dosed as free warhead. Based on the SAR of this first generation of maytansinoid conjugates we are further optimizing our alphalexTM - maytansinoid conjugation strategy with the goal of moving forward with IND-enabling studies in the near future.

Extracellular Vesicles-Based Drug Delivery Systems: A New Challenge and the Exemplum of Malignant Pleural Mesothelioma.

Research for the most selective drug delivery to tumors represents a fascinating key target in science. Alongside the artificial delivery systems identified in the last decades (e.g., liposomes), a family of natural extracellular vesicles (EVs) has gained increasing focus for their potential use in delivering anticancer compounds. EVs are released by all cell types to mediate cell-to-cell communication both at the paracrine and the systemic levels, suggesting a role for them as an ideal nano-delivery system. Malignant pleural mesothelioma (MPM) stands out among currently untreatable tumors, also due to the difficulties in achieving an early diagnosis. Thus, early diagnosis and treatment of MPM are both unmet clinical needs. This review looks at indirect and direct evidence that EVs may represent both a new tool for allowing an early diagnosis of MPM and a potential new delivery system for more efficient therapeutic strategies. Since MPM is a relatively rare malignant tumor and preclinical MPM models developed to date are very few and not reliable, this review will report data obtained in other tumor types, suggesting the potential use of EVs in mesothelioma patients as well.

The effect of zwitterionic surface content on blood circulation time of nanocapsule.

Zwitterionic modification can prolong the blood circulation time of nanocarrier in vivo, but zwitterionic content will affect the functions of nanocarrier such as enzyme-responsive and intracellular or extracellular delivery. Therefore, it is necessary to explore the relationship between the zwitterionic content and circulation time of nanocarrier so as to figure out what content of zwitterion can enable the nanocarrier to obtain both the long blood circulation ability and other functions mentioned above. Herein, using nanocapsule as a research model, we investigated the nanocapsule modified with zwitterion of phosphorylcholine (PC) or carboxybetaine (CB) respectively, and through 1H-NMR quantification we determined the zwitterionic surface content, so as to study the effect of PC or CB surface content on blood circulation performance of nanocapsule. In vivo study showed that the nanocapsule possessed an optimal surface filling ratios range for blood circulation of 43-68% for PC and of 20-68% for CB, with the longest t1/2=37.35 h for PC-nanocapsule and t1/2=45.27 h for CB-nanocapsule. Furthermore, the protein adsorption and macrophage endocytosis experiments indicated that when the surface filling ratio reached 43% for PC-nanocapsule and 20% for CB-nanocapsule, it could effectively reduce the protein adsorption and weaken macrophage endocytosis, thus explaining the phenomenon of long circulation time of nanocapsules from the point of protein adsorption and interaction with immune cells. This study proposes a new direction for designing long-circulating nanocarrier, and provides basis for constructing enzyme-responsive and intracellular or extracellular delivery platform.

Microfluidic fabrication of stable collagen microgels with aligned microstructure using flow-driven co-deposition and ionic gelation

The controlled biofabrication of stable, aligned collagen hydrogels within microfluidic devices is critically important to the design of more physiologically accurate, longer-cultured on-chip models of tissue and organs. To address this goal, collagen-alginate microgels were formed in a microfluidic channel by calcium crosslinking of a flowing collagen-alginate solution through a cross-channel chitosan membrane spanning a pore allowing ion diffusion but not convection. The gels formed within seconds as isolated islands in a single channel, and their growth was self-limiting. Total gel thickness was controlled by altering the concentration of calcium and collagen-alginate flow rate to reach an equilibrium of calcium diffusion and solution convection at the gel boundary, for a desired thickness of 30–200 μm. Additionally, less calcium and higher flow produced greater compression of the gel, with regions farther from the pore compressing more. An aligned, stable collagen network was demonstrated by collagen birefringence, circumferential texture orientation, and little change in gel dimensions with de-chelation of calcium from alginate by prolonged flow of EDTA in the channel. Resultant gels were most stable and only slightly asymmetric when formed from solutions containing 8 mg ml−1 collagen. Diffusion of 4 kDa and 70 kDa fluorescently-labeled dextran indicated size-dependent diffusion across the gel, and accessibility of the construct to appropriately-sized bioactive molecules. This work demonstrates the physicochemical parameter control of collagen gel formation in microfluidic devices, with utility toward on-chip models of dense extracellular matrix invasion, cancer growth and drug delivery to cells within dense extracellular matrix bodies.

Evidence of the Mechanism by Which Polyomaviruses Exploit the Extracellular Vesicle Delivery System during Infection.

Increasing evidence suggests that human viruses can hijack extracellular vesicles (EVs) to deliver proteins, mRNAs, microRNAs (miRNAs) and whole viral particles during viral persistence in the host. Human polyomavirus (PyV) miRNAs, which downregulate large T-antigen expression and target host factors, help the virus escape immune elimination and may have roles in the success of viral persistence/replication and the development of diseases. In this context, several investigations have detected PyV miRNAs in EVs obtained from cell culture supernatants after viral infection, demonstrating the ability of these vesicles to deliver miRNAs to uninfected cells, potentially counteracting new viral infection. Additionally, PyV miRNAs have been identified in EVs derived from the biological fluids of clinical samples obtained from patients with or at risk of severe PyV-associated diseases and from asymptomatic control healthy subjects. Interestingly, PyV miRNAs were found to be circulating in blood, urine, cerebrospinal fluid, and saliva samples from patients despite their PyV DNA status. Recently, the association between EVs and PyV viral particles was reported, demonstrating the ability of PyV viral particles to enter the cell without natural receptor-mediated entry and evade antibody-mediated neutralization or to be neutralized at a step different from that of the neutralization of naked whole viral particles. All these data point toward a potential role of the association between PyVs with EVs in viral persistence, suggesting that further work to define the implication of this interaction in viral reactivation is warranted.

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term ‘extracellular vesicles’ refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell–cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.

Extracellular Vesicle- and Extracellular Vesicle Mimetics-Based Drug Delivery Systems: New Perspectives, Challenges, and Clinical Developments.

Extracellular vesicles (EVs) are small membrane-based nanovesicles naturally released from cells. Extracellular vesicles mimetics (EVMs) are artificial vesicles engineered from cells or in combination with lipid materials, and they mimic certain characteristics of EVs. As such, EVs facilitate intracellular communication by carrying and delivering biological materials, such as proteins, lipids, and nucleic acids, and they have been found to find organ tropism in preclinical studies. Because of their native structure and characteristics, they are considered promising drug carriers for future clinical use. This review outlines the origin and composition of natural EVs and EVM engineering and internalization. It then details different loading approaches, with examples of the drug delivery of therapeutic molecules. In addition, the advantages and disadvantages of loading drugs into EVs or EVMs as a drug delivery system are discussed. Finally, the advantages of EVMs over EVs and the future clinical translation of EVM-based drug delivery platforms are outlined.