Formulation and Evaluation of Polymer-Based Nanoparticles for Intravitreal Gene-Delivery Applications.

The advent of the first-ever retinal gene therapy product, involving subretinal administration of a virus-based gene delivery platform, has garnered hope that this state-of-the-art therapeutic modality may benefit a broad spectrum of patients with diverse retinal disorders. On the other hand, clinical studies have revealed limitations of the applied delivery strategy that may restrict its universal use. To this end, intravitreal administration of synthetic gene-delivery platforms, such as polymer-based nanoparticles (PNPs), has emerged as an attractive alternative to the current mainstay. To achieve success, however, it is imperative that synthetic platforms overcome key biological barriers in human eyes encountered following intravitreal administration, including the vitreous gel and inner limiting membrane (ILM). Here, we introduce a series of experiments, from the fabrication of PNPs to a comprehensive evaluation in relevant experimental models, to determine whether PNPs overcome these barriers and efficiently deliver therapeutic gene payloads to retinal cells. We conclude the article by discussing a few important considerations for successful implementation of the strategy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation and characterization of PNPs Basic Protocol 2: Evaluation of in vitro transfection efficacy Basic Protocol 3: Evaluation of PNP diffusion in vitreous gel Basic Protocol 4: Ex vivo assessment of PNP penetration within vitreoretinal explant culture Basic Protocol 5: Assessment of in vivo transgene expression mediated by intravitreally administered PNPs.

Freeze-Thawing-Induced Macroporous Catechol Hydrogels with Shape Recovery and Sponge-like Properties.

Catechol-containing hydrogels have been exploited in biomedical fields due to their adhesive and cohesive properties, hemostatic abilities, and biocompatibility. Catechol moieties can be oxidized to o-catecholquinone, a chemically active intermediate, in the presence of oxygen to act as an electrophile to form catechol-catechol or catechol-amine/thiol adducts. To date, catechol cross-linking chemistry to fabricate hydrogels has been mostly performed at room temperature. Herein, we report large increases in catechol cross-linking reaction kinetics by the freeze-thawing process. The formation of ice crystals during freezing steps spatially condenses catechol-containing polymers into nearly frozen (yet unfrozen) regions, resulting in decreases in the polymeric chain distances. This environment allows great increases in catechol cross-linking kinetics, a phenomenon that can also occur during thawing steps. The increased cross-linking rate and spatial condensation in the cryogels provide unique wall and pore structures, which result in elastic, spongelike hydrogels. The moduli of the cryogels prepared by glycol-chitosan-catechol (g-chitosan-c) were improved by 3-6-fold compared to room temperature-cured conventional hydrogels, and the degree of improvement increased depending on the freezing time and the number of freeze-thawing cycles. Unlike typical cell encapsulations before cross-linking, which have often been a source of cytotoxicity, the macroporosity of cryogels allows nontoxic cell seeding with ease. This research offers a new way to utilize catechol cross-linking chemistry by freeze-thawing processes to simultaneously regulate mechanical strength and porous structures in catechol-containing hydrogels.

VATA: A Poly(vinyl alcohol)- and Tannic Acid-Based Nontoxic Underwater Adhesive.

Few studies aiming to develop a glue with an underwater reusable adhesive property have been reported because combining the two properties of reusable adhesion and underwater adhesion into a single glue formulation is a challenging issue. Herein, preparation of a simple mixture of poly(vinyl alcohol) (PVA) and a well-known phenolic compound, namely, tannic acid (TA), results in an underwater glue exhibiting reusable adhesion. We named the adhesive VATA (PVA + TA). Using VATA, two stainless steel objects (0.77 kg each) are able to be instantly attached. In addition to the high adhesive strength, surface-applied VATA in water retains its adhesive capability even after 24 h. In contrast, cyanoacrylate applied under the same water condition rapidly loses its adhesive power. Another advantage is that VATA's adhesion is reusable. Bonded objects can be forcibly detached, and then the detached ones can be reattached by the residual VATA. VATA maintains nearly 100% of its initial adhesive force, even after 10 repetitions of attach-detach cycles. VATA bonds various materials ranging from metals and polymers to ceramics. Particularly, we first attempt to test the toxicity of the underwater adhesives using an invertebrate nematode, Caenorhabditis elegans and gold fish (vertebrate) due to potential release to the environment.