Drug Delivery with Extracellular Vesicles: From Imagination to Innovation.

Extracellular 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.

Microbubbles-Assisted Ultrasound Triggers the Release of Extracellular Vesicles.

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.

Thermodynamic and kinetic investigation of agomelatine polymorph transformation.

Thermodynamic properties of polymorphic forms I and II of Agomelatine were investigated and the bimorphism was determined to be monotropically related. The phase transition kinetics from metastable form I to thermodynamically stable form II was studied and a quantification method was developed based on X-ray powder diffraction technique. Various solid-state kinetic models were examined and the results were fit to the experimental data. The nucleation kinetic models were found to be the best fit to describe the experimental data across the temperature range. The activation energy of the form transformation was calculated in the range of 116-122 kJ mol(-1), irrespective of which kinetic model selected.

ELISA-based detection of immunoglobulins against extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell-derived EVs into pig-tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV-mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV-based therapies in preclinical studies.

Vaccines' New Era-RNA Vaccine.

RNA vaccines, including conventional messenger RNA (mRNA) vaccines, circular RNA (circRNA) vaccines, and self-amplifying RNA (saRNA) vaccines, have ushered in a promising future and revolutionized vaccine development. The success of mRNA vaccines in combating the COVID-19 pandemic caused by the SARS-CoV-2 virus that emerged in 2019 has highlighted the potential of RNA vaccines. These vaccines possess several advantages, such as high efficacy, adaptability, simplicity in antigen design, and the ability to induce both humoral and cellular immunity. They also offer rapid and cost-effective manufacturing, flexibility to target emerging or mutant pathogens and a potential approach for clearing immunotolerant microbes by targeting bacterial or parasitic survival mechanisms. The self-adjuvant effect of mRNA-lipid nanoparticle (LNP) formulations or circular RNA further enhances the potential of RNA vaccines. However, some challenges need to be addressed. These include the technology's immaturity, high research expenses, limited duration of antibody response, mRNA instability, low efficiency of circRNA cyclization, and the production of double-stranded RNA as a side product. These factors hinder the widespread adoption and utilization of RNA vaccines, particularly in developing countries. This review provides a comprehensive overview of mRNA, circRNA, and saRNA vaccines for infectious diseases while also discussing their development, current applications, and challenges.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

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 (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour 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.

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants.

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, well-characterized 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 titers 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. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2 protects against disease and elicits neutralizing antibodies to wild-type and Delta variants.

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, well-characterized 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. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titres in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

A post-insertion strategy for surface functionalization of bacterial and mammalian cell-derived extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles which are released by cells from all three domains of life: Archaea, Bacteria and Eukarya. They can mediate cell-cell communication by transferring cargoes such as proteins and nucleic acids between cells. EVs receive great interest in both academia and industry as they have the potential to be natural drug carriers or vaccine candidates. However, limitations to their clinical translation exist as efficient isolation, loading, labelling and surface-engineering methods are lacking. In this article, we investigate a 'post-insertion' approach, which is commonly used in the functionalization of liposomes in the pharmaceutical field, on two different EV types: mammalian cell-derived EVs and bacteria-derived EVs. We aimed to find an easy and flexible approach to functionalize EVs, thereby improving the labelling, isolation, and surface-engineering.

Bacterial membrane vesicles as promising vaccine candidates.

Both Gram-positive and Gram-negative bacteria can release nano-sized lipid bilayered structures, known as membrane vesicles (MVs). These MVs play an important role in bacterial survival by orchestrating interactions between bacteria and between bacteria and host. The major constituents of MVs are proteins, lipids and nucleic acids. Due to the immunogenicity of the membrane lipids and/or proteins of the MVs, in combination with adjuvant danger signals and the repeating patterns on the nanosized surface, MVs can effectively stimulate the innate and adaptive immune system. Since they are non-replicating, they are safer than attenuated vaccines. In addition, by genetic engineering of the donor cells, further improvements to their safety profile, immunogenicity and yield can be achieved. To date, one MV-based vaccine against Neisseria meningitidis (N. meningitidis) serogroup B was approved. Other (engineered) MVs in the pipeline study are mostly in the preclinical phase.

Biofabrication of Cell-Derived Nanovesicles: A Potential Alternative to Extracellular Vesicles for Regenerative Medicine.

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.

Supramolecular structures and physicochemical properties of norfloxacin salts.

Seven new molecular salts of norfloxacin (1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-1H-quinoline-3-carboxylic acid; abbreviated as NF) with various organic acids (adipic acid, mucic acid, o-OH-benzoic acid, m-OH-benzoic acid, p-OH-benzoic acid, naphthalene-1, 5-disulfonic acid and naphthalene-2-sulfonic acid) were synthesized and their crystal structures were determined by X-ray crystallography. Supramolecular structures and reccurring packing patterns are discussed to understand the influence of non-covalent interactions in determination of the crystal packing and hydrate inclusion. The formation of hydrates was commonly observed among various NF salts, except for the adipate salt which exists as an anhydrous form. The physicochemical properties of salts were fully characterized with a variety of analytical techniques, including powder X-ray diffraction (PXRD), Fourier transform IR (FT-IR), Raman spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), hot-stage microscopy (HSM) and dynamic vapour sorption (DVS) etc. The synthesized norfloxacin salts were found to have different physicochemical properties, superior solubility and hygroscopicity. Particularly, NF adipate was found to be a desirable candidate for further development.

Solid-state characterization and transformation of various creatine phosphate sodium hydrates.

Creatine phosphate sodium (CPS) salt is a first-line cardiovascular drug for severe diastolic heart failure. The drug exists in different hydrate forms. The marketed drug form was determined as CPS·4.5H2 O (H1); however, the reference standard was supplied as CPS·6H2 O (H2). In this work, we present two newly identified hydrate forms: a thermodynamically stable low hydrate form, CPS·1.5H2 O (H3), and a pressure-sensitive transit form, CPS·7H2 O (H4). The hydrate forms were discovered through a comprehensive solid-state screening experiment and fully characterized using a range of analytical techniques including X-ray powder diffraction (XRPD), FTIR, Raman spectroscopy, hot-stage microscopy (HSM), thermogravimetric analysis, and differential scanning calorimetry. Stability tests revealed that H3 was the most stable hydrate under thermal stimulation. H4 is a pressure-sensitive hydrate and easily transforms to H2 and then H1 upon grinding. The form transformation process was closely monitored using the HSM, variable-temperature XRPD (VT-XRPD), and VT-Raman spectroscopy techniques. Specifically, the transformation of H4 to H1 is characterized in a single-crystal-to-single-crystal transformation process. The newly discovered hydrate form H3 has superior physicochemical properties than the marketed forms and is worthy of further development.