Pharmaceutical Research

Nakajima, Miki; Tamai, Ikumi

doi: 10.1007/s11095-023-03519-8pmid: 37079150

Shimoda, Asako; Akiyoshi, Kazunari

doi: 10.1007/s11095-023-03511-2pmid: 37038008

BackgroundExtracellular vesicles (EVs) are a group of cell-derived membrane vesicles that carry a variety of cargo such as protein, nucleic acids, and lipids, and are secreted by almost all cell types. Functionally, EVs play important roles in physiological and pathological processes such as immune responses and tumor growth through intercellular communication by transferring this molecular information between cells. Therefore, they have potential versatile clinical applications as disease biomarkers and drug delivery carriers.ProblemNotably, subpopulations of EVs exhibit distinct characteristics depending on their cell of origin, including the expression of surface glycans, which have been implicated in a variety of cellular processes such as field cancerization, cell recognition, and signal transduction. However, these are features have not been fully exploited because of the difficulty in analyzing these proteins.ApproachIn this paper, we summarize the advancements in glycoengineering and high-performance lectin microarray for high-throughput analysis of EV glycans to generate an index of heterogeneity to identify disease biomarkers, and describe how understanding the function of EVs in disease can enhance their potential application in the clinic.

Arthur, Peggy; Kandoi, Sangeetha; Sun, Li; Kalvala, Anil; Kutlehria, Shallu; Bhattacharya, Santanu; Kulkarni, Tanmay; Nimma, Ramesh; Li, Yan; Lamba, Deepak A.; Singh, Mandip

doi: 10.1007/s11095-022-03350-7pmid:

Zapała, Barbara; Kamińska, Agnieszka; Piwowar, Monika; Paziewska, Agnieszka; Gala-Błądzińska, Agnieszka; Stępień, Ewa Ł.

doi: 10.1007/s11095-023-03481-5pmid: 36859746

BackgroundThe aim of this study was to investigate the role of urine-derived extracellular vesicles (uEVs) in diabetic kidney disease (DKD) in patients diagnosed with type 2 diabetes mellitus (T2DM).MethodsUEVs were characterized by size distribution and microRNA content by next-generation small RNA sequencing and quantitative reverse transcription PCR.ResultsA subset of sixteen miRNAs enriched in T2DM patients with DKD, including hsa-miR-514a-5p, hsa-miR‑451a, hsa-miR-126-3p, hsa-miR-214, or hsa-miR‑503 was identified. Eight miRNAs as hsa-miR-21-3p, hsa-miR-4792, hsa-miR‑375, hsa-miR-1268a, hsa-miR-501-5p, or hsa-miR-582 were downregulated. Prediction of potential target genes and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed possible functions related to cellular processes such as apoptosis, inflammation, and tissue remodeling, that promote diabetic complications, such as DKD. Among them, hsa-miR-375, hsa-miR-503, and hsa-miR-451a make important contribution. Additionally, downregulated hsa-miR-582-5p has not been reported so far in any diabetes-related pathways.ConclusionsThis study revealed the most significant miRNAs in uEVs of patients with T2DM. However, as this is a bioinformatic prediction that we performed based on the putative targets of the identified miRNAs. Thus, further in vitro functional studies are needed to confirm our findings. Knowing the fact that EVs are crucial in transferring miRNAs, there is a great need toto discover their involvement in the pathomechanism of T2DM-related kidney disease.

Erwin, Nina; Serafim, Maria Fernanda; He, Mei

doi: 10.1007/s11095-022-03420-wpmid: 36319886

Extracellular vesicles (EVs) have various advantageous properties, including a small size, high biocompatibility, efficient cargo loading, and precise cell targeting ability, making them promising tools for therapeutic development. EVs have been increasingly explored for applications like drug delivery. However, due to limited cellular secretion rates of EVs, wide-scale clinical applications are not achievable. Therefore, substantial strategies and research efforts have been devoted to increasing cellular secretion rates of EVs. This review describes various studies exploring different methods to increase the cellular production of EVs, including the application of electrical stimulus, pharmacologic agents, electromagnetic waves, sound waves, shear stress, cell starvation, alcohol, pH, heat, and genetic manipulation. These methods have shown success in increasing EV production, but careful consideration must be given as many of these strategies may alter EV properties and functionalities, and the exact mechanisms causing the increase in cellular production of EVs is generally unknown. Additionally, the methods’ effectiveness in increasing EV secretion may diverge with different cell lines and conditions. Further advancements to enhance EV biogenesis secretion for therapeutic development is still a significant need in the field.

Kobayashi, Yuki; Kitamura, Shimpei; Takahashi, Yuki; Takakura, Yoshinobu

doi: 10.1007/s11095-022-03405-9pmid: 36195822

PurposePhosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS(−) sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS(−) sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed.MethodsBulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS(−) sEVs was estimated using magnetic beads that bind to PS(+) sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated.ResultsEnzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS(−) sEVs. PSD treatment hardly changed the physicochemical properties of PS(−) sEVs. Moreover, the serum concentration profile and PK parameters of the PS(−) sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life.ConclusionsTreatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS(−) sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS(−) sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.

Suzuki, Kohei; Nakano, Masataka; Nakashima, Shimon; Fukami, Tatsuki; Nakajima, Miki

doi: 10.1007/s11095-022-03368-xpmid: 36002612

PurposeSmall extracellular vesicles (sEV) containing proteins and RNAs play important roles as intercellular signal mediators. A critical issue is that there are multiple methods to prepare sEV fractions. The purpose of this study was to examine whether cancer cell-derived sEV fractions prepared by different isolation methods show similar responses for the induction of inflammatory cytokines in macrophages.MethodssEV fractions from the conditioned medium of MCF-7 cells were prepared by ultracentrifugation (UC), the MagCapture Exosome Isolation Kit PS (PS), or the ExoQuick-TC kit (EQ). The mRNA levels of inflammatory cytokines in differentiated THP-1 cells treated with the sEV fractions were evaluated.ResultsThe yields of sEV fractions obtained from 1 mL conditioned medium by UC, PS, or EQ were 3.2×108 particles (0.27 μg protein), 12.8×108 particles (0.87 μg protein) and 23.5 ×108 particles (4.50 μg protein), respectively. The average particle sizes in the UC, PS, and EQ fractions were 184.8 ± 1.8 nm, 157.8 ± 1.3 nm and 165.8 ± 1.1 nm, respectively. CD9 and CD81, markers of sEV, were most highly detected in the PS fraction, followed by the EQ and UC fractions. These results suggest that PS gave sEV with relatively high purity, and many protein contaminants appear to be included in the EQ fraction. The mRNA levels of inflammatory cytokines in THP-1 macrophages were most prominently increased by treatment with the UC fraction, followed by the EQ and PS fractions, suggesting that contaminants rather than sEV may largely induce an inflammatory response.ConclusionThe isolation method affects the evaluation of sEV function.

Zeng, Weiping; Wen, Zhengbo; Chen, Honglin; Duan, Yuyou

doi: 10.1007/s11095-022-03224-ypmid: 35352281

Exosomes are extracellular vesicles secreted by cells with a particle size of 30–150 nm in diameter. Exosomes can be used as natural drug carriers. The treatment of cancer with drug-loaded exosomes is an area of high interest. This review introduces the composition, function, isolation and characterization of exosomes, and briefly describes the selection of exosome donor cells and methods for drug loading. Through studies on therapies with drug-loaded exosomes in gastric cancer, lung cancer, brain cancer and other cancers, the advantages and disadvantages of drug-loaded exosomes have been analyzed.

Okamura, Akihiko; Yoshioka, Yusuke; Saito, Yoshihiko; Ochiya, Takahiro

doi: 10.1007/s11095-022-03463-zpmid: 36577860

Cardiac diseases such as myocardial infarction and heart failure have been the leading cause of death worldwide for more than 20 years, and new treatments continue to be investigated. Heart transplantation, a curative treatment for severe cardiac dysfunction, is available to only a small number of patients due to the rarity of donors and high costs. Cardiac regenerative medicine using embryonic stem cells and induced pluripotent stem cells is expected to be a new alternative to heart transplantation, but it has problems such as induction of immune response, tumor formation, and low survival rate of transplanted cells. On the other hand, there has been a focus on cell-free therapy using extracellular vesicles (EVs) due to their high biocompatibility and target specificity. Exosomes, one type of EV, play a role in the molecular transport system in vivo and can be considered a drug delivery system (DDS) innate to all living things. Exosomes contain nucleic acids and proteins, which are transported from secretory cells to recipient cells. Molecules in exosomes are encapsulated in a lipid bilayer, which allows them to exist stably in body fluids without being affected by nuclease degradation enzymes. Therefore, the therapeutic use of exosomes as DDSs has been widely explored and is being used in clinical trials and other clinical settings. This review summarizes the current topics of EVs as DDSs in cardiac disease.

Munir, Javaria; Ngu, Alice; Wang, Haichuan; Ramirez, Denise M. O.; Zempleni, Janos

doi: 10.1007/s11095-022-03404-wpmid: 36198923

Small extracellular vesicles (sEVs, “exosomes”) in milk have attracted considerable attention for use in delivering therapeutics to diseased tissues because of the following qualities. The production of milk sEVs is scalable, e.g., more than 1021 sEVs may be obtained annually from a single cow. Milk EVs protect their cargo against degradation in the gastrointestinal tract and during industrial processing. Milk sEVs and their cargo are absorbed following oral administration and they cross barriers such as intestinal mucosa, placenta and the blood–brain barrier in humans, pigs, and mice. Milk sEVs do no alter variables of liver and kidney function, or hematology, and do not elicit immune responses in humans, rats, and mice. Protocols are available for loading milk sEVs with therapeutic cargo, and a cell line is available for assessing effects of milk sEV modifications on drug delivery. Future research will need to assess and optimize sEV shelf-life and storage and effects of milk sEV modifications on the delivery of therapeutic cargo to diseased tissues.