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Extracellular vesicles (EVs) have emerged as a potential delivery vehicle for nucleic-acid-based therapeutics, but challenges related to their large-scale production and cargo-loading efficiency have limited their therapeutic potential. To address these issues, we developed a novel "shock wave extracellular vesicles engineering technology" (SWEET) as a non-genetic, scalable manufacturing strategy that uses shock waves (SWs) to encapsulate siRNAs in EVs. Here, we describe the use of the SWEET platform to load large quantities of KRAS G12C -targeting siRNA into small bovine-milk-derived EVs (sBMEVs), with high efficiency. The siRNA-loaded sBMEVs effectively silenced oncogenic KRAS G12C expression in cancer cells; they inhibited tumour growth when administered intravenously in a non-small cell lung cancer xenograft mouse model. Our study demonstrates the potential for the SWEET platform to serve as a novel method that allows large-scale production of cargo-loaded EVs for use in a wide range of therapeutic applications., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor-mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis-2-decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion., (© 2020 Wiley Periodicals LLC.)

As a new approach to islet transplantation, biocompatible monomethoxy-poly(ethylene glycol) (mPEG) was chemically grafted onto the pancreatic islet capsule. The aim of this study was to determine the optimal conditions for completely covering the islet by the mPEG while maintaining a high viability of islets according to the reaction time and the repeating number of the reaction. By grafting the fluorescein-PEG instead of mPEG, we determined the optimal mPEG grafting time as 1 h, during which time the procedure did not reduce islet viability. Insulin secretion from islets where the mPEG was grafted on for 3 times was similar to that of control islets. Moreover, the mPEG-grafted islets rapidly responded to the changes in the glucose concentration in the same pattern as did control islets. These results showed that mPEG grafting did not damage the function of islets. In conclusion, when the mPEG grafting was performed for 1 h and repeated twice with 1-day culture between each mPEG-grafting step, the mPEG completely covered the islet capsules without any damage to the viability and function of the islets. The main advantage of mPEG grafting on the islet capsule is that it can protect the islet against the host's immune system without increasing the islet size so that it can be administered into the portal vein by the catheter., (Copyright 2002 Wiley Periodicals, Inc.)

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis., (Copyright 2001 John Wiley & Sons, Inc.)

In order to develop a versatile model for blood-compatible materials, we studied morphology and platelet adhesion of a dipalmitoyl-phosphatidylcholine (DPPC)/dipalmitoyl-phosphatidylethanolamine-polyethylene glycol (PEG lipid) mixed monolayer. This monolayer, which mimics the cell membrane structure, consists of two heterogeneous layers, that is, a PEG layer lying on top of a phospholipid monolayer. The DPPC/PEG lipid mixed monolayer was prepared using the Langmuir-Blodgett (LB) Technique. The monolayer was transferred onto a silanized glass substrate by the down-stroke mode, at a surface pressure of 25 mN/m. The transfer efficiency achieved unity at all times. The morphologies of PEG chains on the phospholipid monolayer in water, in a dried state, and in a hydrated state were evaluated using Pi-A isotherm, ellipsometry, and atomic force microscopy (AFM), respectively. When the concentration of PEG lipid was below 1 mol %, the PEG chains could cover the DPPC surface completely in water, but not in the dried state. On the other hand, the PEG chains could cover the phospholipid surface completely in a dried state, as well as in water, when the PEG lipid concentration was above 3 mol %. These PEG chains, showing a brush-type conformation in water, were highly packed and had a bulky structure at the surface in the dried state as well as in the hydrated state. A bulky and extended PEG layer, above 3 mol % concentration, was greatly effective in the prevention of platelet adhesion., (Copyright 2000 John Wiley & Sons, Inc.)

The ability of heparin to interact with plasma proteins, in particular antithrombin III (ATIII) and thrombin, is its primary mechanism as an anticoagulant drug. Research efforts have focused on the biological activity of heparin under three conditions: in solution as a free molecule, chemically coupled directly onto a polymer surface, and coupled onto a polymer surface using hydrophilic spacer groups. Each of these conditions yields altered biological activity, presumably a result of differing binding interactions with ATIII and thrombin. In this report, insights into binding interaction of direct versus polyethylene oxide space immobilized heparin with ATIII, thrombin, and the generation of the thrombin-antithrombin complex will be presented.

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin.