Preparation Of Vesicles Research Articles

Free flow electrophoresis allows quick and reproducible preparation of extracellular vesicles from conditioned cell culture media

Aim: Despite intensive research during the last decade, it remains challenging to prepare extracellular vesicles (EVs) of high purity, especially from primary body liquids or protein-rich conditioned media. For now, time-consuming combinations of at least two orthogonal methods, e.g., density and size separation, are required to enrich EVs to high purity, often at the expense of processing time. Therefore, novel technologies are required that allow EV preparation in acceptable time intervals and to fair purities. Free-flow electrophoresis (FFE) constitutes a well-established semi-preparative method to separate and prepare analytes, e.g., by inherent differences in their electric charges. FFE combines a flow-driven longitudinal transport of sample material with vertical electrophoresis and allows the separation of sample components into up to 96 different fractions. It was our aim to evaluate the potential of FFE for the separation of EVs from other sample components of EV-containing protein-rich conditioned cell culture media. Methods: Exemplarily, conditioned media of mesenchymal stem/stromal cells raised in the presence of EV-containing 10% human platelet lysate were processed. We analyzed the obtained fractions by different technologies, including imaging flow cytometry, western blot and nanoparticle tracking analysis. Results: We demonstrate that FFE quickly and reproducibly separates EVs from a huge proportion of molecules included in the original sample. Conclusion: Our results qualify FFE as a feasible, quick and reproducible technology for the preparation of bona fide EVs.

Physicochemical Characterization of Hyaluronic Acid and Chitosan Liposome Coatings

Hyaluronic acid (HA) and chitosan (CH) are biopolymers that are widely used in many biomedical applications and for cosmetic purposes. Their chemical properties are fundamental to them working as drug delivery systems and improving their synergistic effects. In this work, two different protocols were used to obtain zwitterionic liposomes coated with either hyaluronic acid or chitosan. Specifically, the methodologies used to perform vesicle preparation were chosen by taking into account the specific chemical properties of these two polysaccharides. In the case of chitosan, liposomes were first synthesized and then coated, whereas the coating of hyaluronic acid was achieved through lipidic film hydration in an HA aqueous solution. The size and the zeta-potential of the polysaccharide-coated liposomes were determined by dynamic light scattering (DLS). This approach allowed coated liposomes to be obtained with hydrodynamic diameters of 264.4 ± 12.5 and 450.3 ± 16.7 nm for HA- and CH-coated liposomes, respectively. The chemical characterization of the coated liposomal systems was obtained by surface infrared (ATR-FTIR) and nuclear magnetic resonance (NMR) spectroscopies. In particular, the presence of polysaccharides was confirmed by the bands assigned to amides and saccharides being in the 1500–1700 cm−1 and 800–1100 cm−1 regions, respectively. This approach allowed confirmation of the efficiency of the coating processes, evidencing the presence of HA or CH at the liposomal surface. These data were also supported by time-of-flight secondary ion mass spectrometry (ToF-SIMS), which provided specific assessments of surface (3–5 nm deep) composition and structure of the polysaccharide-coated liposomes. In this work, the synthesis and the physical chemistry characterization of coated liposomes with HA or CH represent an important step in improving the pharmacological properties of drug delivery systems.

An Update to Dialysis-Based Drug Release Testing-Data Analysis and Validation Using the Pharma Test Dispersion Releaser.

Currently, a wide variety of complex non-oral dosage forms are entering the global healthcare market. Although many assays have been described in recent research, harmonized procedures and standards for testing their in vitro performance remain widely unexplored. Among others, dialysis-based techniques such as the Pharma Test Dispersion Releaser are developed for testing the release of drugs from nanoparticles, liposomes, or extracellular vesicle preparations. Here, we provide advanced strategies and practical advice for the development and validation of dialysis-based techniques, including documentation, analysis, and interpretation of the raw data. For this purpose, key parameters of the release assay, including the hydrodynamics in the device at different stirring rates, the selectivity for particles and molecules, as well as the effect of excipients on drug permeation were investigated. At the highest stirring rate, a more than twofold increase in the membrane permeation rate (from 0.99 × 10−3 to 2.17 × 10−3 cm2/h) was observed. Additionally, we designed a novel computer model to identify important quality parameters of the dialysis experiment and to calculate error-corrected release profiles. Two hydrophilic creams of diclofenac, Voltaren® Emulgel, and Olfen® gel, were tested and provide first-hand evidence of the robustness of the assay in the presence of semisolid dosage forms.

Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

DEVELOPMENT AND IN-VITRO CHARACTERIZATION OF LIPOSOMAL GEL OF BIFONAZOLE FOR TOPICAL USE

The main objective of this study is to prepare and evaluate liposomes as a carrier for bifonazole. Liposomes are colloidal particles designed as concentric biomolecular layers that are capable of encapsulating drugs. Liposomes of soya lecithin and cholesterol were prepared in ten batches (L1- L10) with different ratio using thin film hydration technique. Prepared liposomes were evaluated for entrapment efficiency and particle size analysis. Liposomal gel formulations of prepared liposomes were formulated in six batches (L9F1- L9F6) and evaluated for drug content, in-vitro drug release study, particle size, zeta potential, polydispersity index (PDI) and stability study were performed. The entrapment efficiency, particle size, zeta potential and PDI of all liposome vesicle preparations were analyzed and it was found that the batch L9 liposome vesicles produced the entrapment efficiency 76.52 ± 0.175, particle size 119.1 nm, zeta potential -0.4, and PDI was found to be 0.406. Liposomal gel formulation was prepared. The drug content, in-vitro drug release study and stability study of all six batches of liposomal gel formulation were analyzed, and it was found that the L9F3 formulation shows better drug content, in-vitro drug release and stability at 5°C ± 2°C among all the formulations. Evaluation of prepared liposomal gel formulations was performed. After comparing all formulations of evaluation results on the basis of drug content and, in-vitro drug release and stability study, batch L9F3 formulation was considered as the best formulation.

A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues—which range in size and chemical composition—with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

Tropism of Extracellular Vesicles and Cell-Derived Nanovesicles to Normal and Cancer Cells: New Perspectives in Tumor-Targeted Nucleic Acid Delivery.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

Combination of multifunctional ursolic acid with kinase inhibitors for anti-cancer drug carrier vesicles

A sterically stabilized unilamellar nanocarrier vesicle (SSV) system containing dipalmitoylphosphatidylcholine, cholesterol, ursolic acid and PEGylated phospholipid has been developed by exploiting the structural advantages of ursolic acid: by spontaneously attaching to the lipid head groups, it induces curvature at the outer side of the bilayers, allowing the preparation of size-limited vesicles without extrusion. Ursolic acid (UA) also interacts with the PEG chains, supporting steric stabilization even when the amount of PEGylated phospholipid is reduced. Using fluorescence immunohistochemistry, vesicles containing ursolic acid (UA-SSVs) were found to accumulate in the tumor in 3 h on xenografted mouse, suggesting the potential use of these vesicles for passive tumor targeting.Further on, mono- and combination therapy with UA and six different kinase inhibitors (crizotinib, erlotinib, foretinib, gefitinib, refametinib, trametinib) was tested on seven cancer cell-lines. In most combinations synergism was observed, in the case of trametinib even at very low concentration (0.001 μM), which targets the MAPK pathway most often activated in human cancers. The coupled intercalation of UA and trametinib (2:1 molar ratio) into vesicles causes further structural advantageous molecular interactions, promoting the formation of small vesicles. The high drug:lipid molar ratio (~0.5) in the novel type of co-delivery vesicles enables their direct medical application, possibly also overcoming the multidrug resistance effect.

Scaled preparation of extracellular vesicles from conditioned media.

Extracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded; furthermore, EV-containing conditioned media are produced. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. CM are either stored until DSP or are directly processed. As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics.

Phytosomes as Innovative Delivery Systems for Phytochemicals: A Comprehensive Review of Literature.

Nowadays, medicinal herbs and their phytochemicals have emerged as a great therapeutic option for many disorders. However, poor bioavailability and selectivity might limit their clinical application. Therefore, bioavailability is considered a notable challenge to improve bio-efficacy in transporting dietary phytochemicals. Different methods have been proposed for generating effective carrier systems to enhance the bioavailability of phytochemicals. Among them, nano-vesicles have been introduced as promising candidates for the delivery of insoluble phytochemicals. Due to the easy preparation of the bilayer vesicles and their adaptability, they have been widely used and approved by the scientific literature. The first part of the review is focused on introducing phytosome technology as well as its applications, with emphasis on principles of formulations and characterization. The second part provides a wide overview of biological activities of commercial and non-commercial phytosomes, divided by systems and related pathologies. These results confirm the greater effectiveness of phytosomes, both in terms of biological activity or reduced dosage, highlighting curcumin and silymarin as the most formulated compounds. Finally, we describe the promising clinical and experimental findings regarding the applications of phytosomes. The conclusion of this study encourages the researchers to transfer their knowledge from laboratories to market, for a further development of these products.

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage.

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on cyclodextrin-catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is often accomplished by preloading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles prepared using methyl-α-cyclodextrin exchange were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at nonphysiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological ion concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein leakage out of the vesicles.

Injectable drug delivery systems of doxorubicin revisited: In vitro-in vivo relationships using human clinical data

A growing number of nanomedicines entered the clinical trials and improved our understanding of the in vivo responses expected in humans. The in vitro drug release represents an important critical quality attribute involved in pharmacokinetics. Establishing in vitro-in vivo relationships for nanomedicines requires a careful analysis of the clinical data with respect to the unique differences between drugs and nanomedicines. Also, the biorelevant assay must reflect the release mechanism of the carrier. Four drug delivery systems of doxorubicin were evaluated for their in vitro release behavior under biorelevant conditions using the dispersion releaser. The pharmacokinetics observed during the first-in-men clinical trials were analyzed using a custom-made physiologically-based nanocarrier biopharmaceutics model. The drug product Lipodox® and the clinical candidate NanoCore-7.4 were evaluated to validate the model. Afterward, the in vivo performances of the preclinical candidates NanoCore-6.4 and doxorubicin-loaded nano-cellular vesicle technology systems (an extracellular vesicle preparation) were predicted. In vitro and in vivo release were in good correlation as indicated by the coefficients of determination of 0.98648 (NanoCore-7.4) and 0.94107 (Lipodox®). The predictions required an estimation of the carrier half-life in blood circulation leading to considerable uncertainty. Still, the simulations narrow down the possible scenarios in the clinical evaluation of nanomedicines and provide a valuable addition to animal studies.

Osmotic Vesicle Collapse of Sealed Inside-Out Membrane Vesicles From Red Blood Cells.

The preparation of plasma membrane vesicles from a large variety of cells has contributed a wealth of information on the identity and vectorial properties of membrane transporters and enzymes. Vesicles from red blood cell (RBC) membranes are generated in media of extremely low tonicity. For functional studies, it is required to suspend the vesicles in higher tonicity media in order to bring the concentrations of the substrates of transporters and enzymes under investigation within the physiological ranges. We investigated the effects of hypertonic transitions on the vesicle morphology using transmission electron microscopy. The results show that hypertonic transitions cause an irreversible osmotic collapse of sealed membrane vesicles. Awareness of the collapsed condition of vesicles during functional studies is critical for the proper interpretation of experimental results.

Development of Drug-Resistant Klebsiella pneumoniae Vaccine via Novel Vesicle Production Technology.

Drug resistance of Klebsiella pneumoniae severely threatens human health. Overcoming the mechanisms of K. pneumoniae resistance to develop novel vaccines against drug-resistant K. pneumoniae is highly desired. Here, we report a technology platform that uses high pressure to drive drug-resistant K. pneumoniae to pass through a gap, inducing the formation of stable artificial bacterial biomimetic vesicles (BBVs). These BBVs had little to no bacterial intracellular protein or nucleic acid and had high yields. BBVs were efficiently taken up by dendritic cells to stimulate their maturation. BBVs as K. pneumoniae vaccines had the dual functions of inducing bacteria-specific humoral and cellular immune responses to increase animals' survival rate and reduce pulmonary inflammation and bacterial loads. We believe that BBVs are new-generation technology for bacterial vesicle preparation. Establishment of this BBV vaccine platform can maximally expand preparation technology for vaccines against drug-resistant K. pneumoniae.

Dissecting Out the Molecular Mechanism of Insecticidal Activity of Ostreolysin A6/Pleurotolysin B Complexes on Western Corn Rootworm.

Ostreolysin A6 (OlyA6) is a protein produced by the oyster mushroom (Pleurotus ostreatus). It binds to membrane sphingomyelin/cholesterol domains, and together with its protein partner, pleurotolysin B (PlyB), it forms 13-meric transmembrane pore complexes. Further, OlyA6 binds 1000 times more strongly to the insect-specific membrane sphingolipid, ceramide phosphoethanolamine (CPE). In concert with PlyB, OlyA6 has potent and selective insecticidal activity against the western corn rootworm. We analysed the histological alterations of the midgut wall columnar epithelium of western corn rootworm larvae fed with OlyA6/PlyB, which showed vacuolisation of the cell cytoplasm, swelling of the apical cell surface into the gut lumen, and delamination of the basal lamina underlying the epithelium. Additionally, cryo-electron microscopy was used to explore the membrane interactions of the OlyA6/PlyB complex using lipid vesicles composed of artificial lipids containing CPE, and western corn rootworm brush border membrane vesicles. Multimeric transmembrane pores were formed in both vesicle preparations, similar to those described for sphingomyelin/cholesterol membranes. These results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from specific interactions of OlyA6 with CPE, and the consequent formation of transmembrane pores in the insect midgut.

α-Synuclein in Plasma-Derived Extracellular Vesicles Is a Potential Biomarker of Parkinson's Disease.

Extracellular vesicles are small vesicles that are released from many cells, including neurons. α-Synuclein has recently been described in extracellular vesicles derived from the central nervous system and may contribute to the spreading of disease pathology in α-synuclein-related neurodegeneration. We aimed to examine the potential diagnostic value of α-synuclein in plasma extracellular vesicles from patients with Parkinson's disease (PD). Preanalytical variables were studied to establish an optimized assay for preparation of plasma extracellular vesicles and detection of extracellular vesicle-derived α-synuclein. Plasma samples were obtained from 2 independent cohorts. The Tübingen cohort contained 96 patients with PD, 50 patients with dementia with Lewy bodies, 50 patients with progressive supranuclear palsy (PSP), and 42 healthy controls; the Kassel cohort included 47 patients with PD, 43 patients with dementia with Lewy bodies, and 36 controls with secondary parkinsonian syndromes. Extracellular vesicles were prepared from total plasma by size exclusion chromatography and quantified by nanoparticle tracking analysis, α-synuclein content was measured by an electrochemiluminescence assay. α-Synuclein concentration in plasma extracellular vesicles provided the best discrimination between PD, dementia with Lewy bodies, PSP, and healthy controls, with an area under the curve of 0.804 (PD vs dementia with Lewy bodies), 0.815 (PD vs. PSP), and 0.769 (PD vs healthy controls) in the Tübingen cohort. Results were validated in the Kassel cohort. The concentration of α-synuclein in plasma extracellular vesicles may serve as a potential diagnostic biomarker for PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

High-grade extracellular vesicles preparation by combined size-exclusion and affinity chromatography

Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.

Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma

Free flow electrophoresis allows quick preparation of extracellular vesicles from cell culture supernatants and human plasma

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

Analysis of peptides and small proteins in preparations of horse milk exosomes, purified on anti-CD81-Sepharose

Exosomes are nanovesicles (40–100 nm) involved in intracellular communication. Milk is a unique source of exosomes. In our previous papers, our group was the first to describe horse milk exosomes. Here we show how crude vesicle preparations were obtained from 18 horse milk samples by standard centrifugation, ultracentrifugation, and gel-filtration methods. Transmission electron microscopy and flow cytometry confirmed that exosomes contain tetraspanins on their surface. Affinity chromatography of vesicle preparations on anti-CD81-Sepharose produced extra-purified exosomes with more than 20 peptides and small proteins of 0.8–10 kDa as detected by MALDI-TOF mass spectrometry. The peptides are natural intrinsic components of horse milk exosomes and are not formed either as a result of or during sample preparation. We assume some of the hundreds and thousands of exosome proteins previously described by other authors in milk exosomes by highly sensitive liquid chromatography-mass spectrometry to have been misidentified as full-length proteins.