Nucleic Acid Cargo Research Articles

Microparticle Depots for Controlled and Sustained Release of Endosomolytic Nanoparticles.

Nucleic acids have gained recognition as promising immunomodulatory therapeutics. However, their potential is limited by several drug delivery barriers, and there is a need for technologies that enhance intracellular delivery of nucleic acid drugs. Furthermore, controlled and sustained release is a significant concern, as the kinetics and localization of immunomodulators can influence resultant immune responses. Here, we describe the design and initial evaluation of poly(lactic-co-glycolic) acid (PLGA) microparticle (MP) depots for enhanced retention and sustained release of endosomolytic nanoparticles that enable the cytosolic delivery of nucleic acids. Endosomolytic p[DMAEMA]10kD-bl-[PAA0.3-co-DMAEMA0.3-co-BMA0.4]25kD diblock copolymers were synthesized by reversible addition-fragmentation chain transfer polymerization. Polymers were electrostatically complexed with nucleic acids and resultant nanoparticles (NPs) were encapsulated in PLGA MPs. To modulate release kinetics, ammonium bicarbonate was added as a porogen. Release profiles were quantified in vitro and in vivo via quantification of fluorescently-labeled nucleic acid. Bioactivity of released NPs was assessed using small interfering RNA (siRNA) targeting luciferase as a representative nucleic acid cargo. MPs were incubated with luciferase-expressing 4T1 (4T1-LUC) breast cancer cells in vitro or administered intratumorally to 4T1-LUC breast tumors, and silencing via RNA interference was quantified via longitudinal luminescence imaging. Endosomolytic NPs complexed to siRNA were effectively loaded into PLGA MPs and release kinetics could be modulated in vitro and in vivo via control of MP porosity, with porous MPs exhibiting faster cargo release. In vitro, release of NPs from porous MP depots enabled sustained luciferase knockdown in 4T1 breast cancer cells over a five-day treatment period. Administered intratumorally, MPs prolonged the retention of nucleic acid within the injected tumor, resulting in enhanced and sustained silencing of luciferase relative to a single bolus administration of NPs at an equivalent dose. This work highlights the potential of PLGA MP depots as a platform for local release of endosomolytic polymer NPs that enhance the cytosolic delivery of nucleic acid therapeutics.

Nonviral Gene Delivery with Cationic Glycopolymers

The field of gene therapy, which aims to treat patients by modulating gene expression, has come to fruition and has landed several landmark FDA approvals. Most gene therapies currently rely on viral vectors to deliver nucleic acid cargo into cells, but there is significant interest in moving toward chemical-based methods, such as polymer-based vectors, due to their low cost, immunocompatibility, and tunability. The full potential of polymer-based delivery systems has yet to be realized, however, because most polymeric transfection reagents are either too inefficient or too toxic for use in the clinic. In this Account, we describe developments in carbohydrate-based cationic polymers, termed glycopolymers, for enhanced nonviral gene delivery. As ubiquitous components of biological systems, carbohydrates are a rich class of compounds that can be harnessed to improve the biocompatibility of non-native polymers, such as linear polyamines used for promoting transfection. Reineke et al. developed a new class of carbohydrate-based polymers called poly(glycoamidoamine)s (PGAAs) by step-growth polymerization of linear monosaccharides with linear ethyleneamines. These glycopolymers were shown to be both efficient and biocompatible transfection reagents. Systematic modifications of the structural components of the PGAA system revealed structure-activity relationships important to its function, including its ability to degrade in situ. Expanding upon the development of step-growth glycopolymers, monosaccharides, such as glucose, were functionalized as vinyl-based monomers for the formation of diblock copolymers via radical addition-fragmentation chain-transfer (RAFT) polymerization. Upon complexation with plasmid DNA, the glucose-containing block creates a hydrophilic shell that promotes colloidal stability as effectively as PEG functionalization. An N-acetyl-d-galactosamine variant of this diblock polymer yields colloidally stable particles that show increased receptor-mediated uptake by liver hepatocytes in vitro and promotes liver targeting in mice. Finally, the disaccharide trehalose was incorporated into polycationic structures using both step-growth and RAFT techniques. It was shown that these trehalose-based copolymers imparted increased colloidal stability and yielded plasmid and siRNA polyplexes that resist aggregation upon lyophilization and reconstitution in water. The aforementioned series of glycopolymers use carbohydrates to promote effective and safe delivery of nucleic acid cargo into a variety of human cells types by promoting vehicle degradation, tissue-targeting, colloidal stabilization, and stability toward lyophilization to extend shelf life. Work is currently underway to translate the use of glycopolymers for safe and efficient delivery of nucleic acid cargo for gene therapy and gene editing applications.

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

Super-resolution Imaging of Proton Sponge-Triggered Rupture of Endosomes and Cytosolic Release of Small Interfering RNA.

The intracellular delivery of nucleic acids and proteins remains a key challenge in the development of biological therapeutics. In gene therapy, the inefficient delivery of small interfering RNA (siRNA) to the cytosol by lipoplexes or polyplexes is often ascribed to the entrapment and degradation of siRNA payload in the endosomal compartments. A possible mechanism by which polyplexes rupture the endosomal membrane and release their nucleic acid cargo is commonly defined as the "proton sponge effect". This is an osmosis-driven process triggered by the proton buffering capacityof polyplexes. Herein, we investigate the molecular basis of the "proton sponge effect" through direct visualization of the siRNA trafficking process, including analysis of individual polyplexes and endosomes, using stochastic optical reconstruction microscopy. We probe the sequential siRNA trafficking steps through single molecule super-resolution analysis of subcellular structures, polyplexes, and silencing RNA molecules. Specifically, individual intact polyplexes released in the cytosol upon rupture of the endosomes, the damaged endosomal vesicles, and the disassembly of the polyplexes in the cytosol areexamined. We findthat the architecture of the polyplex and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. We provide evidence that in highly branched and rigid cationic polymers, such as glycogen or polyethylenimine, immobilized on silica nanoparticles, the proton sponge effect is effective in inducing osmotic swelling and rupture of endosomes.

Endothelial Cell-Derived Extracellular Vesicles Mitigate Radiation-Induced Hematopoietic Injury

Hematopoietic stem cells (HSCs) reside in specialized microenvironments and adjacent to endothelial cells (ECs), from which they receive regulatory cues. Following injury from radiation or chemotherapy, transplanted ECs can facilitate hematopoietic regeneration. Since these ECs do not engraft in the marrow, we hypothesize that ECs facilitate hematopoietic regeneration through secretion of extracellular vesicles (EVs). EVs are becoming recognized as a mode of cell-to-cell communication through transfer of their cargo of nucleic acids and proteins. Using ultracentrifugation, we visualized these EVs by transmission electron microscopy and measured their size to be a mean size of 160 nm (range 121-217 nm, n=18). Using flow cytometric analysis with Syto13, we demonstrate that these EVs contain nucleic acids in 30% of the particles. Quantitative real-time PCR show that these EVs express CD31 and VECadherin, and not Fibroblast Specific Marker 1 (FSP1), indicating EVs bear EC markers. At day 7 following 300 cGy, C57BL/6 cKit+Sca-1+Lineage- (KSL) cells cultured with EVs demonstrated total cell expansion and colony-forming unit cells (CFCs) compared to cultures with cytokines alone (*p< 0.0001 and 0.01, respectively, n= 3-6/group). Following myelosuppressive, sub-lethal radiation dose of 500 cGy, C57BL/6 mice treated with EVs on days 1-4 display increased marrow cellularity (*p= 0.003, n= 2-3/group), preserved vascular endothelial cells by mouse endothelial cell antigen immunohistochemistry (*p=0.03, n=2-4/group), CD150+CD48-KSL cells (*p= 0.03, n= 5/group), and increased CFCs (*p= 0.03, n= 6/group) compared to saline-treated mice. No differences were detected in these parameters when EV-treated mice were compared to mice treated with primary, cultured C57BL/6 marrow ECs. Following lethal-dose irradiation of 800 cGy, mice that received EVs on day 1-4 display prolonged survival compared to saline-treated mice (*p= 0.008 by Log-rank Analysis, n= 10/group). At day 30 following irradiation, 5 of 10 mice were alive in the EV-treated group compared to 0 of 10 in the saline-treated group. No differences in survival were observed between EV-treated and EC-treated mice (p= 0.4). Treatment of irradiated C57BL/6 hematopoietic stem/progenitor cells (HSPCs) with EVs generated from a genetically distinct strain (BALB/c mice) showed similar levels of cell expansion and CFCs compared to treatment of HSPCs from syngeneic EVs. Our findings show that syngeneic or allogenic EVs could be cell-derived therapy to deliver physiologic doses of nucleic acids and growth factors to hematopoietic cells to accelerate hematopoietic regeneration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vesiclepedia 2019:a compendium of RNA, proteins, lipids and metabolites in extracellular vesicles.

Extracellular vesicles (EVs) are membranous vesicles that are released by both prokaryotic and eukaryotic cells into the extracellular microenvironment. EVs can be categorised as exosomes, ectosomes or shedding microvesicles and apoptotic bodies based on the mode of biogenesis. EVs contain biologically active cargo of nucleic acids, proteins, lipids and metabolites that can be altered based on the precise state of the cell. Vesiclepedia (http://www.microvesicles.org) is a web-based compendium of RNA, proteins, lipids and metabolites that are identified in EVs from both published and unpublished studies. Currently, Vesiclepedia contains data obtained from 1254 EV studies, 38 146 RNA entries, 349 988 protein entries and 639 lipid/metabolite entries. Vesiclepedia is publicly available and allows users to query and download EV cargo based on different search criteria. The mode of EV isolation and characterization, the biophysical and molecular properties and EV-METRIC are listed in the database aiding biomedical scientists in assessing the quality of the EV preparation and the corresponding data obtained. In addition, FunRich-based Vesiclepedia plugin is incorporated aiding users in data analysis.

Synthetic materials at the forefront of gene delivery

The delivery of nucleic acids with transient activity for genetic engineering is a promising methodology with potential applications in the treatment of diseases ranging from cancer and infectious diseases to heritable disorders. Restoring the expression of a missing protein, correcting defective splicing of transcripts and silencing or modulating the expression of genes are powerful approaches that could have substantial benefits in biological research and medicine. Impressive progress in improving gene delivery has been made in the past decade, and several products have reached the market. However, translating the results of in vitro and preclinical studies into functional therapies is hindered by the suboptimal performance of gene delivery vehicles in capturing, protecting and delivering nucleic acid cargoes safely and efficaciously. Chemistry has a key role in the development of innovative synthetic materials to overcome the challenges of producing next-generation gene delivery therapies and protocols. In this Review, we discuss the latest chemical advances in the production of materials for the delivery of nucleic acids to cells and for gene therapy. Non-viral vehicles for the delivery of nucleic acids have potential applications for the treatment of diseases by, for example, restoring, correcting or silencing the expression of genes. In this Review, the authors discuss the latest developments in synthetic materials used for gene delivery and the challenges that must be overcome to transfer these innovations into the clinic.

Intracellular DNA Cargo Release from a Gold Nanoparticle Modulated by the Nature of the Surface Coupling Functionality.

Covalently coupling nucleic acids to a gold nanoparticle (AuNP) surface has been demonstrated for generatingeffective gene therapy agents to modify cellular protein expression. The therapeutic efficacy of the approach is anticipated to be impacted by the length of time the nucleic acid sequence resides in the endolysosomal pathway once transfected into a cell. It is believed that the dynamics of the processing should reflect the linkage chemistry of the DNA to the AuNP surface. In this manuscript the dynamics of nanotherapeutic uptake, nucleic acid release, and gene processing are investigated in vitro for a AuNP-nucleic acid delivery platform transfected into A375 human melanoma cells, as a function of the nucleic acid-gold linkage chemistry. The dynamics of cell processing of the single monodentate thiol (SX), bidentate dual thiol (SS), or mixed bidentate thiol plus amine (SN) coordination of nucleic acids to the AuNP surface are evaluated using a multicolor nanosurface energy transfer bio-optical transponder (SET-BOT) technology. The use of live-cell fluorescence microscopy allows for the direct visualization of the uptake and localization of a lipofectamine-packaged SET-BOT using a dye (DL700) that is not quenched in the proximity of the AuNP, while fluorescence "turn on" of a dye that is proximally quenched by the AuNP (DL488) is used to report on the dynamics of release of the nucleic acid cargo within the cell. For protein expression following transcription of the gene, the emission signature of a red fluorescent protein, tdTomato, is monitored. The intracellular rates of DNA release from the AuNP surface once endosomally packaged within the A375 human melanoma cells were found to follow the binding activity series: bidentate thiol > bidentate thiol plus amine > monodentate thiol, consistent with the strength of multidentate chelation, paired with the stabilizing influence of π-backbonding of thiols compared to σ-donation in amines, when bound to a gold surface.

Clinical Evaluations of Toxicity and Efficacy of Nanoparticle-Mediated Gene Therapy.

Considerable efforts have been devoted to develop safe and efficient gene therapies for life-threatening or inherited diseases. The choice of gene delivery vehicle plays key roles in enhancing the therapeutic effect of nucleic acid cargo. To date, gene therapy approaches involving both viral vectors and nonviral vectors have been evaluated in clinical trials. With improvements in material science and nanotechnologies, positively charged nanoparticles have emerged as potential gene delivery vehicles. In this review, we highlight clinical trials that examined cationic nanocarrier-mediated gene therapy as well as discuss both the toxicity and efficacy of nanocarrier-based therapeutics.

Cytosolic nucleic acid sensors of the innate immune system promote liver regeneration after partial hepatectomy

Stimulation of cytosolic nucleic acid sensors of innate immunity by pathogen-derived nucleic acids is important for antimicrobial defence, but stimulation through self-derived nucleic acids may contribute to autoinflammation and cancer. DNA sensing in the cytosol requires the stimulator of interferon genes (STING), while cytosolic RNA sensors use mitochondrial antiviral-signalling protein (MAVS). In a murine model of two-thirds hepatectomy, combined deficiency of MAVS and STING resulted in strongly impaired hepatocyte proliferation and delayed recovery of liver mass. Whereas lack of MAVS and STING did not influence upregulation of the G1-phase cyclins D1 and E1, it substantially reduced the hyperphosphorylation of retinoblastoma protein, attenuated the activation of cyclin-dependent kinase (CDK)-2, delayed upregulation of CDK1 and cyclins A2 and B1, and impaired S-phase entry of hepatocytes. Mechanistically, lack of cytosolic nucleic acid sensors strongly upregulated the anti-proliferative mediators TGF-β2 and activin A, which was associated with an increased expression of the cell cycle inhibitors p15 and p21. Partial hepatectomy was followed by the release of exosomes with abundant nucleic acid cargo, which may provide ligands for the MAVS and STING pathways. Together, these findings identify a previously unrecognised function of cytosolic nucleic acid sensors of innate immunity for promoting liver regeneration.

Extracellular vesicles and their nucleic acids for biomarker discovery

Extracellular vesicles (EVs) are a heterogenous population of vesicles originate from cells. EVs are found in different biofluids and carry different macromolecules, including proteins, lipids, and nucleic acids, providing a snap shot of the parental cells at the time of release. EVs have the ability to transfer molecular cargoes to other cells and can initiate different physiological and pathological processes. Mounting lines of evidence demonstrated that EVs' cargo and machinery is affected in disease states, positioning EVs as potential sources for the discovery of novel biomarkers. In this review, we demonstrate a conceptual overview of the EV field with particular focus on their nucleic acid cargoes. Current knowledge of EV subtypes, nucleic acid cargo and pathophysiological roles are outlined, with emphasis placed on advantages against competing analytes. We review the utility of EVs and their nucleic acid cargoes as biomarkers and critically assess the newly available advances in the field of EV biomarkers and high throughput technologies. Challenges to achieving the diagnostic potential of EVs, including sample handling, EV isolation, methodological considerations, and bioassay reproducibility are discussed. Future implementation of 'omics-based technologies and integration of systems biology approaches for the development of EV-based biomarkers and personalized medicine are also considered.

Extracellular Vesicles and the Application of System Biology and Computational Modeling in Cardiac Repair.

Recent literature suggests that extracellular vesicles (EVs), secreted from most cells and containing cell-specific cargo of proteins, lipids, and nucleic acids, are major driver of intracellular communication in normal physiology and pathological conditions. The recent evidence on stem/progenitor cell EVs as potential therapeutic modality mimicking their parental cell function is exciting because EVs could possibly be used as a surrogate for the stem cell-based therapy, and this regimen may overcome certain roadblocks identified with the use of stem/progenitor cell themselves. This review provides a comprehensive update on our understanding on the role of EVs in cardiac repair and emphasizes the applications of stem/progenitor cell-derived EVs as therapeutics and discusses the current challenges associated with the EV therapy.

Therapeutic Prospects of Extracellular Vesicles in Cancer Treatment.

Extracellular vesicles (EVs) are released by all cells within the tumor microenvironment, such as endothelial cells, tumor-associated fibroblasts, pericytes, and immune system cells. The EVs carry the cargo of parental cells formed of proteins and nucleic acids, which can convey cell-to-cell communication influencing the maintenance and spread of the malignant neoplasm, for example, promoting angiogenesis, tumor cell invasion, and immune escape. However, EVs can also suppress tumor progression, either by the direct influence of the protein and nucleic acid cargo of the EVs or via antigen presentation to immune cells as tumor-derived EVs carry on their surface some of the same antigens as the donor cells. Moreover, dendritic cell-derived EVs carry major histocompatibility complex class I and class II/peptide complexes and are able to prime other immune system cell types and activate an antitumor immune response. Given the relative longevity of vesicles within the circulation and their ability to cross blood–brain barriers, modification of these unique organelles offers the potential to create new biological-tools for cancer therapy. This review examines how modification of the EV cargo has the potential to target specific tumor mechanisms responsible for tumor formation and progression to develop new therapeutic strategies and to increase the efficacy of antitumor therapies.

Dimeric Human β-Defensin 3 as a Universal Platform for Intracellular Delivery of Nucleic Acid Cargos

Functional nucleic acids including siRNA, mRNA, and plasmid DNA are promising bioactive molecules to regulate cellular functions uncontrollable by conventional small molecule regulators. To realize successful cellular applications of these nucleic acids, an intracellular gene delivery vehicle with high efficiency and low cytotoxicity is required. Here, we report the dimerization of human β-defensin 3 (DhBD3) promoted by the interaction between β-strands and the application of DhBD3 for efficient delivery of various nucleic acid cargos. DhBD3 with multiple cationic residues could be complexed with various types of polyanionic DNA and RNA. DhBD3 could intracellularly deliver both small and large nucleic acid cargos loaded by complexation to regulate the expression level of target proteins, showing its potential as a universal platform for nucleic acid delivery. In addition, as DhBD3 is a human-derived material with high biocompatibility and can be robustly prepared by an inexpensive method, it is a promisin...

Evaluating the Mechanisms of Light-Triggered siRNA Release from Nanoshells for Temporal Control Over Gene Regulation.

The ability to regulate intracellular gene expression with exogenous nucleic acids such as small interfering RNAs (siRNAs) has substantial potential to improve the study and treatment of disease. However, most transfection agents and nanoparticle-based carriers that are used for the intracellular delivery of nucleic acids cannot distinguish between diseased and healthy cells, which may cause them to yield unintended widespread gene regulation. An ideal delivery system would only silence targeted proteins in diseased tissue in response to an external stimulus. To enable spatiotemporal control over gene silencing, researchers have begun to develop nucleic acid-nanoparticle conjugates that keep their nucleic acid cargo inactive until it is released from the nanoparticle on-demand by externally applied near-infrared laser light. This strategy can overcome several limitations of other nucleic acid delivery systems, but the mechanisms by which these platforms operate remain ill understood. Here, we perform a detailed investigation of the mechanisms by which silica core/gold shell nanoshells (NSs) release conjugated siRNA upon excitation with either pulsed or continuous wave (CW) near-infrared (NIR) light, with the goal of providing insight into how these nanoconjugates can enable on-demand gene regulation. We demonstrate that siRNA release from NSs upon pulsed laser irradiation is a temperature-independent process that is substantially more efficient than siRNA release triggered by CW irradiation. Contrary to literature, which suggests that only pulsed irradiation releases siRNA duplexes, we found that both modes of irradiation release a mixture of siRNA duplexes and single-stranded oligonucleotides, but that pulsed irradiation results in a higher percentage of released duplexes. To demonstrate that the siRNA released from NSs upon pulsed irradiation remains functional, we evaluated the use of NSs coated with green fluorescent protein (GFP)-targeted siRNA (siGFP-NS) for on-demand knockdown of GFP in cells. We found that GFP-expressing cells treated with siGFP-NS and irradiated with a pulsed laser experienced a 33% decrease in GFP expression compared to cells treated with no laser. Further, we observed that light-triggered gene silencing mediated by siGFP-NS is more potent than using commercial transfection agents to deliver siRNA into cells. This work provides unprecedented insight into the mechanisms by which plasmonic NSs release siRNA upon light irradiation and demonstrates the importance of thoroughly characterizing photoresponsive nanosystems for applications in triggered gene regulation.

Extracellular vesicles: important collaborators in cancer progression.

Extracellular vesicles (EVs) are membrane vesicles that are released from cells and mediate cell-cell communication. EVs carry protein, lipid, and nucleic acid cargoes that interact with recipient cells to alter their phenotypes. Evidence is accumulating that tumor-derived EVs can play important roles in all steps of cancer progression. Here, we review recent studies reporting critical roles for EVs in four major areas of cancer progression: promotion of cancer invasiveness and motility, enhancement of angiogenesis and vessel permeability, conditioning premetastatic niches, and immune suppression.

The biology of extracellular microvesicles.

The study of extracellular vesicles (EVs) is a rapidly evolving field, owing in large part to recent advances in the realization of their significant contributions to normal physiology and disease. Once discredited as cell debris, these membrane vesicles have now emerged as mediators of intercellular communication by interaction with target cells, drug and gene delivery, and as potentially versatile platforms of clinical biomarkers as a result of their distinctive protein, nucleic acid and lipid cargoes. While there are multiple classes of EVs released from almost all cell types, here we focus primarily on the biogenesis, fate and functional cargoes of microvesicles (MVs). MVs regulate many important cellular processes including facilitating cell invasion, cell growth, evasion of immune response, stimulating angiogenesis, drug resistance and many others.

The role of extracellular vesicles in biomineralisation: current perspective and application in regenerative medicine

Extracellular vesicles comprise a heterogenous population of exosomes and microvesicles that have critical roles in intercellular signalling and tissue development. These complex particles have been implicated as mediators of the therapeutic effects of stem cells via the transfer of an assorted cargo of proteins and nucleic acids, which can modulate inflammation and enhance endogenous regeneration in a range of tissues. In addition, extracellular vesicles have the capacity to be loaded with therapeutic molecules for targeted delivery of pharmaceuticals. The versatility, biostability and biocompatibility of extracellular vesicles make them appealing for regenerative medicine and may endow considerable advantages over single molecule approaches. Furthermore, since production can be optimised and assessed ex vivo, extracellular vesicles present a decreased risk of neoplastic transformation when compared with cell-based methods. To date, the contribution of vesicles to tissue development has perhaps been most comprehensively defined within hard tissues, such as endochondral bone, where they were first identified in 1969 and henceforth referred to as matrix vesicles. Within developing bone, vesicles function as vehicles for the delivery of pro-osteogenic factors and initiate early nucleational events necessary for matrix mineralisation. However, advancement in our understanding of the biogenesis and characterisation of matrix vesicles has occurred largely in parallel to associated developments in wider extracellular vesicle biology. As such, there is a requirement to align current understanding of matrix vesicle–mediated mineralisation within the context of an evolving literature surrounding exosomes and microvesicles. In this review, we present an overview of current progress and opinion surrounding the application of vesicles in regenerative medicine with a primary focus on their potential as an acellular approach for enhancing hard tissue regeneration. This is balanced with an assessment of areas where further development is required to maximise their application for regenerative medicine.

Integrated nanoscale deterministic lateral displacement arrays for separation of extracellular vesicles from clinically-relevant volumes of biological samples.

Extracellular vesicles (EVs) offer many opportunities in early-stage disease diagnosis, treatment monitoring, and precision therapy owing to their high abundance in bodily fluids, accessibility from liquid biopsy, and presence of nucleic acid and protein cargo from their cell of origin. Despite their growing promise, isolation of EVs for analysis remains a labor-intensive and time-consuming challenge given their nanoscale dimensions (30-200 nm) and low buoyant density. Here, we report a simple, size-based EV separation technology that integrates 1024 nanoscale deterministic lateral displacement (nanoDLD) arrays on a single chip capable of parallel processing sample fluids at rates of up to 900 μL h-1. Benchmarking the nanoDLD chip against commonly used EV isolation technologies, including ultracentrifugation (UC), UC plus density gradient, qEV size-exclusion chromatography (Izon Science), and the exoEasy Maxi Kit (QIAGEN), we demonstrate a superior yield of ∼50% for both serum and urine samples, representing the ability to use smaller input volumes to achieve the same number of isolated EVs, and a concentration factor enhancement of up to ∼3× for both sample types, adjustable to ∼60× for urine through judicious design. Further, RNA sequencing was carried out on nanoDLD- and UC-isolated EVs from prostate cancer (PCa) patient serum samples, resulting in a higher gene expression correlation between replicates for nanoDLD-isolated EVs with enriched miRNA, decreased rRNA, and the ability to detect previously reported RNA indicators of aggressive PCa. Taken together, these results suggest nanoDLD as a promising alternative technology for fast, reproducible, and automatable EV-isolation.

Role of Extracellular Vesicles in Viral and Bacterial Infections: Pathogenesis, Diagnostics, and Therapeutics.

Extracellular vesicles (EVs), or exosomes, are nanovesicles of endocytic origin that carry host and pathogen-derived protein, nucleic acid, and lipid cargos. They are secreted by most cell types and play important roles in normal cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses and pathophysiological processes. New research is beginning to decipher how EVs influence viral and bacterial pathogenesis. In this review, we will describe how EVs influence viral and bacterial pathogenesis by spreading pathogen-derived factors and how they can promote and inhibit the immune response to these pathogens. We will also discuss the emerging potential of EVs as diagnostic and therapeutic tools.