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In this study, we demonstrate the fabrication of polymersomes, protein-blended polymersomes, and polymeric microcapsules using droplet microfluidics. Polymersomes with uniform, single bilayers and controlled diameters are assembled from water-in-oil-in-water double-emulsion droplets. This technique relies on adjusting the interfacial energies of the droplet to completely separate the polymer-stabilized inner core from the oil shell. Protein-blended polymersomes are prepared by dissolving protein in the inner and outer phases of polymer-stabilized droplets. Cell-sized polymeric microcapsules are assembled by size reduction in the inner core through osmosis followed by evaporation of the middle phase. All methods are developed and validated using the same glass-capillary microfluidic apparatus. This integrative approach not only demonstrates the versatility of our setup, but also holds significant promise for standardizing and customizing the production of polymer-based artificial cells., (© 2024. The Author(s).)

Abstract Cerebrovascular dysfunction following traumatic brain injury (TBI) is a well-characterized phenomenon. Given the therapeutic potential of xenon, we aimed to study its effects after localized delivery to the brain using microbubbles. We designed xenon-containing microbubbles stabilized by dibehenoylphosphatidylcholine (DBPC) and polyethylene glycol (PEG) attached to saturated phospholipid (DPSE-PEG5000). Using a pig model of TBI, these microbubbles were intravenously injected, and ultrasound was used to release xenon at the level of the carotid artery. The control group received perfluorobutane containing microbubbles. Diffusion tensor imaging (DTI) showed areas of higher fractional anisotropy for pigs receiving xenon microbubbles compared to the control group at 1 day after injury. Radial diffusivity analysis showed that this effect was mainly the result of acute edema. Pigs were euthanized at 5 days, and the brain tissues of xenon-treated animals showed reduction of perivascular inflammation and blood-brain barrier disruption. Endothelial cell culture experiments showed that glutamate reduces tight junction protein zona occludens-1 (ZO-1), but treatment with xenon microbubbles attenuates this effect. Xenon treatment protects cerebrovasculature and reduces astroglial reactivity after TBI. Further, these data support the future use of localized delivery of various therapeutic agents for brain injury using microbubbles in order to limit systemic side effects and reduce costs.

The continuing emergence of variants of the SARS-CoV-2 virus requires the development of modular molecular therapies. Here, we engineered a recombinant amphiphilic protein, oleosin, to spontaneously self-assemble into multivalent micellar nanostructures which can block the Spike S1 protein of SARS-CoV-2 pseudoviruses (PVs). Short recombinant proteins like oleosin can be formulated more easily than antibodies and can be functionalized with precision through genetic engineering. We cloned S1-binding mini-protein genes called LCB x, previously designed by David Baker's laboratory (UW Seattle), to the N-terminus of oleosin, expressing Oleo-LCB x proteins in E. coli . These proteins largely formed 10-100 nm micelles as verified by dynamic light scattering. Two proteins, Oleo-LCB1 and Oleo-LCB3, were seen to completely and irreversibly block transduction by both wild-type and delta variant PVs into 293T-hsACE2 cells at 10 μM. Presented in multivalent micelles, these proteins reduced transduction by PVs down to a functional protein concentration of 5 nM. Additionally, Oleo-LCB1 micelles outperformed corresponding synthetic LCB1 mini-proteins in reducing transduction by PVs. Tunable aqueous solubility of recombinant oleosin allowed incorporation of peptides/mini-proteins at high concentrations within micelles, thus enhancing drug loading. To validate the potential multifunctionality of the micelles, we showed that certain combinations of Oleo-LCB1 and Oleo-LCB3 performed much better than the individual proteins at the same concentration. These micelles, which we showed to be non-toxic to human cells, are thus a promising step toward the design of modular, multifunctional therapeutics that could bind to and inactivate multiple receptors and proteins necessary for the infection of the SARS-CoV-2 virus.

Traumatic brain injury (TBI) is associated with high mortality and morbidity in children and adults. Unfortunately, there is no effective management for TBI in the acute setting. Rodent studies have shown that xenon, a well-known anesthetic gas, can be neuroprotective when administered post-TBI. Gas inhalation therapy, however, the approach typically used for administering xenon, is expensive, inconvenient, and fraught with systemic side effects. Therapeutic delivery to the brain is minimal, with much of the inhaled gas cleared by the lungs. To bridge major gaps in clinical care and enhance cerebral delivery of xenon, this study introduces a novel xenon delivery technique, utilizing microbubbles, in which a high impulse ultrasound signal is used for targeted cerebral release of xenon. Briefly, an ultrasound pulse is applied along the carotid artery at the level of the neck on intravenous injection of xenon microbubbles (XeMBs) resulting in release of xenon from microbubbles into the brain. This delivery technique employs a hand-held, portable ultrasound system that could be adopted in resource-limited environments. Using a high-fidelity porcine model, this study demonstrates the neuroprotective efficacy of xenon microbubbles in TBI for the first time., Competing Interests: No competing financial interests exist., (© Misun Hwang et al., 2022; Published by Mary Ann Liebert, Inc.)