ConspectusExtracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications.As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and on subsequent vesicle activity.We try to meet this challenge by careful experimental design and development of tools that enable robust readouts. By engineering the surface and cargo of extracellular vesicles through chemical and biological techniques, favorable characteristics can be enforced while unfavorable qualities can be overruled or masked. This is coupled to the precise evaluation of the interaction of extracellular vesicles with cells to determine the extracellular vesicle uptake routes and intracellular routing. Sensitive reporter assays enable reproducible analysis of functional delivery.This systematic evaluation and optimization of extracellular vesicles improves our insight into the critical determinants of extracellular vesicle activity and should improve translation into clinical application of engineered extracellular vesicles as a new class of drug delivery systems.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid after 30-60 min. EV association with peripheral blood mononuclear cells, especially B-cells, was observed as early as 1-min post-administration. EVs were detected in liver and spleen within 1 h of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.Tom Driedonks and Linglei Jiang contributed equally to this study.

Nucleic acid-based drugs offer a potentially effective tool for treatment of a variety of diseases, including cancer, cardiovascular diseases, neurological disorders and infectious diseases. However, clinical applications are hindered by instability of RNA molecules in the circulation and lack of efficient vectors that can deliver RNAs to target tissues and into diseased target cells. Synthetic polymer and lipids as well as virus-based vectors are among the most widely explored vehicles for RNA delivery, but clinical progress has been limited as a result of issues related to toxicity, immunogenicity and low efficiency. Most recently, the discovery that extracellular vesicles (EVs) are endogenous RNA carriers, which may display better biocompatibility and higher delivery efficiency as compared with the synthetic systems, has provided a ray of hope in coping with the delivery dilemma, and EV-based gene therapy has already sparked general interest both in academia and industry. In this review, the current knowledge on EV biology and their role in cell-cell communication will be summarized. Promises of EVs as drug carriers and recent technologies on tailoring EVs' biological attributes will be included, and preclinical studies in which EVs have shown promise for therapeutic RNA delivery will be discussed.

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, wellcharacterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titres of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. UponThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Pharmaceutical co-crystals involving two active pharmaceutical ingredients are rarely revealed in the literature. In this work, crystal engineering principles were exercised to guide the design and synthesis of the Biopharmaceutics Classification System class IV drug dapsone (DAP). We reported six drug−drug co-crystals of DAP with sulfanilamide, flavone, luteolin, caffeine in 1:1 stoichiometry, caffeine in 1:2 stoichiometry, and 2(3H)-benzothiazolone. Bioactive coformers were deliberately selected. The resulting co-crystals were fully characterized by a range of analytical technologies, including X-ray powder diffraction, Fourier transform infrared spectroscopy, polarized light microscopy, differential scanning calorimetry, and thermogravimetric analysis, etc. Single-crystal structure analysis reveals that reoccurring supramolecular synthons are observed in different DAP co-crystals. Equilibrium solubility and intrinsic dissolution rates were also compared with those of the parent drug. This work expands the pharmaceutically acceptable solid forms of DAP and supplements the successful cases of drug−drug co-crystals.

Extracellular vesicles (EVs) are mediators of intercellular communication by transferring functional biomolecules from their originating cells to recipient cells. This intrinsic ability has gained EVs increased scientific interest in their use as a direct therapeutic in the field of regenerative medicine or as vehicles for drug delivery. EVs derived from stem cells or progenitor cells can act as paracrine mediators to promote repair and regeneration of damaged tissues. Despite substantial research efforts into EVs for various applications, their use remains limited by the lack of highly efficient and scalable production methods. Here, we present the biofabrication of cell-derived nanovesicles (NVs) as a scalable, efficient, and cost-effective production alternative to EVs. We demonstrate that NVs have a comparable size and morphology as EVs, but lack standard EV (surface) markers. Additionally, in vitro uptake experiments show that human fetal cardiac fibroblast, endothelial cells, and cardiomyocyte progenitor cells internalize NVs. We observed that cardiac progenitor cell-derived NVs and EVs are capable of activating mitogen-activated protein kinase 1/2 (MAPK1/2)-extracellular signal-regulated kinase, and that both NVs and EVs derived from A431 and HEK293 cells can functionally deliver Cre-recombinase mRNA or protein to other cells. These observations indicate that NVs may have similar functional properties as EVs. Therefore, NVs have the potential to be applied for therapeutic delivery and regenerative medicine purposes.

Four dapsone-flavone cocrystals were synthesized, including three polymorphic 1:1 cocrystals and a 1:2 cocrystal. Single crystal structures were disclosed, and the differences in H-bonding interactions were illustrated by comparison of the crystal structures and Hirshfeld surface analysis. The various cocrystals were fully characterized by X-ray powder diffraction, FTIR and thermal analysis. The solubilities, dissolution rates and form transformations in solution were determined. The phase transformation experiments revealed complex phase transformations among the cocrystals. Solid-state transformations among different stoichiometries were also observed in the dapsone-flavone system.Four dapsone-flavone cocrystals were synthesized and fully characterized by thermal and crystallographic techniques. The solubilties and dissolution rates exhibited differences among various forms. In addition solid-state transformations among different stoichiometries were observed in the co-crystal system.

Microbubbles-assisted ultrasound (USMB) has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs). USMB was performed on a monolayer of head-and-neck cancer cells (FaDu) with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only) were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm). Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells.