Drug Delivery with Extracellular Vesicles: From Imagination to Innovation

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

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Natural and synthetic nanoparticles for delivery of biologics

Nano is a prefix derives from the Greek word ‘νᾶνος’, which means ‘drawf’. Nanomedicine is the healthcare application of nanoscale or nanostructure materials. It is an inter-disciplinary field involving chemistry, biology, physics, materials sciences and clinical medicine.Nanomedicines can be used for both the diagnosis and therapy of diseases. There are several attractive features of nanomedicines as drug delivery systems when compared with traditional small molecular weight drugs. For example, 1) they can increase the solubility of hydrophobic drugs; 2) effectively deliver drugs intracellularly and across barriers such as the blood-brain barrier; 3) be ‘smart’ by active targeting the site of disease, thus increase therapeutic outcome and simultaneously reduce side effects of drugs caused by accumulation in healthy tissues; 4) prolong circulation time by surface decoration drug-loaded nanoparticles with polyethylene glycol (PEG) that prevent aggregation, opsonization, and phagocytosis; 5) overcome drug resistance as drug-loaded nanoparticles are uptake in a stealth endocytosis process that drugs become ‘invisible’ to drug efflux pumps. Nanomedicine can be mainly divided into two categories, i.e. synthetic nanomedicine system and natural nanomedicine system. Synthetic systems are mainly obtained by chemical synthesis methods, which can be further divided into 1) polymer-based nanocarriers such as micelles, nanogels and dendrimers; and 2) liposomes and lipidbased nanocarriers. Natural nanomedicine systems include, viruses, lipoproteins and extracellular vesicles (EVs). EVs can be released by different domains of life including eukaryotes, bacteria and fungi. (Deatherage and Cookson 2012, Brown, Wolf et al. 2015) In this thesis, two nanomedicine systems are investigated. One is a synthetic system: i.e. a nanogel. The characterization of this novel nanogel is presented, and we also investigated the possibility of using this nanogel for nucleic acid delivery in chapter 2 in both in vitro and in vivo models. EVs have shown to be an interesting emerging nanomedicine system over the past decade. They can be released by all domains of life which includes eukaryotes, bacteria, fungi and archaea. EVs released by mammalian cells and bacteria are studied in this thesis. We discuss the potential application of mammalian EVs as drug carriers for gene delivery purposes in chapter 3, and discuss the application of bacterial EVs as vaccine candidates in chapter 4. In addition, we investigated the possibility of using bacterial EVs as ‘nano-weapons’ for combating bacterial infections. As EVs from Gram-positive bacteria have received less attention than their counterpart from Gram-negative bacteria, in chapter 5, we studied the isolation, characterization and immuno-modulatory aspects of membrane vesicles (MVs) from Gram-positive bacteria Enterococcus faecium E1162 and its isogenic mutants. To improve the targeting and immunogenicity properties of EVs from both mammalian cells and bacteria, we investigated a post-insertion approach for EVs modification in chapter 6. Finally, a co-culture system which mimic the intestinal environment was set up to evaluate the immunogenicity of OMVs in chapter 7 with the aim to develop an in-vitro platform to study the oral vaccination evaluation of OMVs. In chapter 8 the main results of this thesis are summarized and future research directions are discussed

Natural and synthetic nanoparticles for delivery of biologics

Nano is a prefix derives from the Greek word ‘νᾶνος’, which means ‘drawf’. Nanomedicine is the healthcare application of nanoscale or nanostructure materials. It is an inter-disciplinary field involving chemistry, biology, physics, materials sciences and clinical medicine.Nanomedicines can be used for both the diagnosis and therapy of diseases. There are several attractive features of nanomedicines as drug delivery systems when compared with traditional small molecular weight drugs. For example, 1) they can increase the solubility of hydrophobic drugs; 2) effectively deliver drugs intracellularly and across barriers such as the blood-brain barrier; 3) be ‘smart’ by active targeting the site of disease, thus increase therapeutic outcome and simultaneously reduce side effects of drugs caused by accumulation in healthy tissues; 4) prolong circulation time by surface decoration drug-loaded nanoparticles with polyethylene glycol (PEG) that prevent aggregation, opsonization, and phagocytosis; 5) overcome drug resistance as drug-loaded nanoparticles are uptake in a stealth endocytosis process that drugs become ‘invisible’ to drug efflux pumps. Nanomedicine can be mainly divided into two categories, i.e. synthetic nanomedicine system and natural nanomedicine system. Synthetic systems are mainly obtained by chemical synthesis methods, which can be further divided into 1) polymer-based nanocarriers such as micelles, nanogels and dendrimers; and 2) liposomes and lipidbased nanocarriers. Natural nanomedicine systems include, viruses, lipoproteins and extracellular vesicles (EVs). EVs can be released by different domains of life including eukaryotes, bacteria and fungi. (Deatherage and Cookson 2012, Brown, Wolf et al. 2015) In this thesis, two nanomedicine systems are investigated. One is a synthetic system: i.e. a nanogel. The characterization of this novel nanogel is presented, and we also investigated the possibility of using this nanogel for nucleic acid delivery in chapter 2 in both in vitro and in vivo models. EVs have shown to be an interesting emerging nanomedicine system over the past decade. They can be released by all domains of life which includes eukaryotes, bacteria, fungi and archaea. EVs released by mammalian cells and bacteria are studied in this thesis. We discuss the potential application of mammalian EVs as drug carriers for gene delivery purposes in chapter 3, and discuss the application of bacterial EVs as vaccine candidates in chapter 4. In addition, we investigated the possibility of using bacterial EVs as ‘nano-weapons’ for combating bacterial infections. As EVs from Gram-positive bacteria have received less attention than their counterpart from Gram-negative bacteria, in chapter 5, we studied the isolation, characterization and immuno-modulatory aspects of membrane vesicles (MVs) from Gram-positive bacteria Enterococcus faecium E1162 and its isogenic mutants. To improve the targeting and immunogenicity properties of EVs from both mammalian cells and bacteria, we investigated a post-insertion approach for EVs modification in chapter 6. Finally, a co-culture system which mimic the intestinal environment was set up to evaluate the immunogenicity of OMVs in chapter 7 with the aim to develop an in-vitro platform to study the oral vaccination evaluation of OMVs. In chapter 8 the main results of this thesis are summarized and future research directions are discussed

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

Effects of Freeze-Thaw Cycles on Phosphorus from Sediments in the Middle Reaches of the Yarlung Zangbo River

The effect of the freeze-thaw process is an important factor in soil nutrient changes and erosion enhancement. Sediments in the middle reaches of the Yarlung Zangbo River are likely affected by the daily freeze-thaw cycles in winter. Examining the freeze-thaw effects of phosphorus from sediments in this area is of great significance for protecting the structure and safety of the ecosystem. The freeze-thaw process of sediments in the middle reaches of the Yarlung Zangbo River was simulated through laboratory experiments, and different phosphorus contents and particle states were synchronously detected and analyzed. The results show that freeze-thaw cycles can accelerate phosphorus migration and release in the sediments, and the total amount of phosphorus release increases by 12%. After being subjected to freeze-thaw cycles, the sediment particles were broken, and the competition between ions for adsorption sites reduced phosphorus adsorption onto the sediments from the middle reaches of the Yarlung Zangbo River. The organic matter on the sediment surface was also broken down, and the energy dispersive spectroscopy (EDS) results showed that the combined ions that were released competed for the adsorption sites on the particle surfaces, thereby promoting phosphorus release. Among the different forms of phosphorus, aluminum-bound phosphorus (Al-P) and iron-bound phosphorus (Fe-P) are the two most released phosphorus forms by the freeze-thaw process. Although the contents of Al-P and Fe-P only account for 2.41% of the total phosphorus content, both phosphorus forms are biologically available, and freeze-thaw cycles may increase the risk of nutrient loss. This research may provide information for the study of phosphorus in river ecosystems in areas subjected to freeze-thaw cycles

ELISA‐based detection of immunoglobulins against extracellular vesicles in blood plasma

Abstract 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

Preparation and Solid-State Characterization of Dapsone Drug–Drug Co-Crystals

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(3<i>H</i>)-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

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