Rapid and Highly Efficient Purification of Extracellular Vesicles Enabled by a TiO2 Hybridized Spongy-like Polymer.

We developed a novel purification medium of extracellular vesicles (EVs) by constructing a spongy-like monolithic polymer kneaded with TiO2 microparticles (TiO2-hybridized spongy monolith, TiO2-SPM). TiO2-SPM was applied in a solid-phase extraction format and enabled simple, rapid, and highly efficient purification of EVs. This is due to the high permeability caused by the continuous large flow-through pores of the monolithic skeleton (median pore size; 5.21 µm) and the specific interaction of embedded TiO2 with phospholipids of the lipid bilayers. Our method also excels in efficiency and comprehensiveness, collecting small EVs (SEVs) from the same volume of a cell culture medium 130.7 times more than typical ultracentrifugation and 4.3 times more than affinity purification targeting surface phosphatidylserine by magnetic beads. The purification method was completed within 1 h with simple operations and was directly applied to serum SEVs. Finally, we demonstrated flexibility toward the shape and size of our method by depleting EVs from fetal bovine serum (FBS), which is a necessary process to prevent contamination of culture cell-derived EVs with exogenous FBS-derived EVs. Our method will eliminate the tedious and difficult purification processes of EVs, providing a universal purification platform for EV-based drug discovery and pathological diagnosis.

Efficient Selective Adsorption of SARS-CoV-2 via the Recognition of Spike Proteins Using an Affinity Spongy Monolith.

Since the outbreak of COVID-19, SARS-CoV-2, the infection has been spreading to date. The rate of false-negative result on a polymerase chain reaction (PCR) test considered the gold standard is roughly 20%. Therefore, its accuracy poses a question as well as needs improvement in the test. This study reports fabrication of a substrate of an anti-spike protein (AS)-immobilized porous material having selective adsorption toward a spike protein protruding from the surface of SARS-CoV-2. We have employed an organic polymer substrate called spongy monolith (SPM). The SPM has through-pores of about 10 µm and is adequate for flowing liquid containing virus particles. It also involves an epoxy group on the surface, enabling arbitrary proteins such as antibodies to immobilize. When antibodies of the spike protein toward receptor binding domain were immobilized, selective adsorption of the spike protein was observed. At the same time, when mixed analytes of spike proteins, lysozymes and amylases, were flowed into an AS-SPM, selective adsorption toward the spike proteins was observed. Then, SARS-CoV-2 was flowed into the BSA-SPM or AS-SPM, amounts of SARS-CoV-2 adsorption toward the AS-SPM were much larger compared to the ones toward the BSA-SPM. Furthermore, rotavirus was not adsorbed to the AS-SPM at all. These results show that the AS-SPM recognizes selectively the spike proteins of SARS-CoV-2 and may be possible applications for the purification and concentration of SARS-CoV-2.

A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers.

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.

Reversible conjugation of biomembrane vesicles with magnetic nanoparticles using a self-assembled nanogel interface: single particle analysis using imaging flow cytometry.

Nanoscale biomembrane vesicles such as liposomes and extracellular vesicles are promising materials for therapeutic delivery applications. However, modification processes that disrupt the biomembrane affect the performance of these systems. Non-covalent functionalization approaches that are facile and easily reversed by environmental triggers are therefore being widely investigated. In this study, liposomes were successfully hybridized with magnetic iron oxide particles using a cholesterol-modified pullulan nanogel interface. Both the magnetic nanoparticles and the hydrophobic core of the lipid bilayer interacted with the hydrophobic cholesteryl moieties, resulting in stable hybrids after simple mixing. Single particle analysis by imaging flow cytometry showed that the hybrid particles interacted in solution. Calcein loaded liposomes were not disrupted by the hybridization, showing that conjugation did not affect membrane stability. The hybrids could be magnetically separated and showed significantly enhanced uptake by HeLa cells when a magnetic field was applied. Differential scanning calorimetry revealed that the hybridization mechanism involved hydrophobic cholesteryl inserting into the biomembrane. Furthermore, exposure of the hybrids to fetal bovine serum proteins reversed the hybridization in a concentration dependent manner, indicating that the interaction was both reversible and controllable. This is the first example of reversible inorganic material conjugation with a biomembrane that has been confirmed by single particle analysis. Both the magnetic nanogel/liposome hybrids and the imaging flow cytometry analysis method have the potential to significantly contribute to therapeutic delivery and nanomaterial development.

Two cases of symptomatic common carotid artery occlusion treated by carotid endarterectomy with L-shaped ministernotomy.

BACKGROUND: Common carotid artery occlusion (CCAO) is rare. Symptomatic lesions are resistant to medical treatment and revascularization are often required, but there is no consensus on the treatment of CCAO. In this paper, two cases of symptomatic CCAO treated by carotid endarterectomy (CEA) with L-shaped ministernotomy, in which the lesions extended to the beginning part of the CCA, are reported. CASE DESCRIPTION: Case 1 involved a 74-year-old man who presented with transient left limb numbness and an abnormal right visual field. Cerebrovascular angiography showed that the right CCA was occluded immediately after its origin and blood was supplied from the posterior circulation. CEA was performed with an L-shaped ministernotomy that allowed exposure of the CCA origin with minimal invasion. There were no complications associated with the sternal incision and he was discharged with a modified Rankin Scale (mRS) score of 0. Case 2 involved a 70-year-old man who presented with left half-blindness. Magnetic resonance imaging showed infarction in the right posterior cerebral artery region and neck echo showed CCA pseudo occlusion just before the carotid bulb. A new infarction in the right middle cerebral artery region developed during hospitalization. CEA with partial sternotomy was performed. The patient was rehabilitated with no deterioration of neurological findings and transferred with an mRS score of 3. CONCLUSION: There were no complications resulting from partial sternotomy in the two cases presented. CEA with partial sternotomy could be an effective treatment option for CCAO in which the internal carotid artery is patent and thrombus extends to the proximal CCA.

Nanogel hybrid assembly for exosome intracellular delivery: effects on endocytosis and fusion by exosome surface polymer engineering.

Surface polymer engineering was applied with a carrier of exosomes, namely, the amphiphilic cationic CHP (cCHP) nanogel, to improve the delivery of exosome content by forming complexes with the exosomes. Mouse macrophage cells were used to produce the exosomes, which were then mixed with the cCHP nanogel to form a hybrid. Transmission electron microscopy revealed that the surface of each exosome was coated with cCHP nanogel particles. Flow cytometry also showed a significant uptake of this exosome/nanogel hybrid by HeLa cells, with the main mechanism behind this internalization being endocytosis. A range of different molecules that inhibit different types of endocytosis were also applied to determine the particular pathway involved, with a caveolae-mediated endocytosis inhibitor being revealed to markedly affect the hybrid uptake. Next, we evaluated the fate of the internalized hybrid using fluorescent labeling, with the results suggesting fusion between endosomes and exosomes. Finally, revealing the functional efficacy of this approach, we showed that the nanogel system could successfully deliver functional exosomes into cells, as indicated by its ability to induce neuron-like differentiation in the recipient cells. Overall, our findings show the potential of using this hybrid nanocarrier system for transporting various contents in exosomes and ensuring their effective delivery in a functionally intact state.

Magnetically Navigated Intracellular Delivery of Extracellular Vesicles Using Amphiphilic Nanogels.

Various cells in vivo secrete exosomes consisting of lipid bilayers. They carry mRNAs and miRNAs capable of controlling cellular functions and can be used as drug delivery system nanocarriers. There is the current need to further improve the efficiency of exosome uptake into target cells. In this study, we prepared a hybrid of exosomes and magnetic nanoparticles, which could be guided to target cells by a magnetic field for efficient uptake. Magnetic nanogels were prepared and hybridized to fluorescently labeled exosomes isolated from PC12 cells. By applying a magnetic field to a hybrid with magnetic nanogel, exosomes were efficiently transferred into target cells as confirmed by confocal laser microscopy. Finally, we found that differentiation of adipose-derived stem cells to neuron-like cells was enhanced by magnetic induction of the exosome-magnetic nanogel hybrid, indicating maintenance of the intrinsic functions of the exosomes in the differentiation of adipose-derived stem cells.