A self-healing microencapsulation process involves mixing preformed porous microspheres in an aqueous solution containing the desired protein and converting them into closed-pore microspheres. Spongelike poly-d,l-lactide-co-glycolide (PLGA) microspheres are expected to be advantageous to protein loading through self-healing. This study aimed to identify and assess relevant critical parameters, using lysozyme as a model protein. Several parameters governed lysozyme loading. The pore characteristics (open-pore, closed-pore, and porosity) of the preformed microspheres substantially affected lysozyme loading efficiency. The type of surfactant present in the aqueous medium also influenced lysozyme loading efficiency. For instance, cetyltrimethylammonium bromide showing a superior wetting functionality increased the extent of lysozyme loading more than twice as compared to Tween 80. Dried preformed microspheres were commonly used before, but our study found that wet microspheres obtained at the end of the microsphere manufacturing process displayed significant advantages in lysozyme loading. Not only could an incubation time for hydrating the microspheres be shortened dramatically, but also a much more considerable amount of lysozyme was encapsulated. Interestingly, the degree of microsphere hydration determined the microstructure and morphology of closed-pore microspheres after self-healing. Understanding these critical process parameters would help tailor protein loading into spongelike PLGA microspheres in a bespoke manner.

A unique approach using methylamine as a chemical trigger leads to the formation of sponge-like PLGA microspheres with extreme porosity.

Unwanted drug crystals often form on the surface of PLGA microspheres or in an aqueous phase when a hydrophobic drug undergoes emulsion‐templated microencapsulation processes. In our study, over 70% of progesterone crystallizes in the aqueous phase when microencapsulation proceeds with a typical oil‐in‐water solvent evaporation process. During filtration employed for microsphere recovery, unentrapped drug crystals are collected alongside with progesterone‐containing microspheres. This phenomenon accompanies unfavorable consequences on the microsphere quality. In contrast, when microspheres are prepared with a new solvent extraction‐evaporation hybrid process, it is possible to completely avoid drug crystallization. Consequently, the new microencapsulation technique yields high drug encapsulation efficiencies of ≥ 90.8%, and the resultant microspheres show a homogeneous size distribution pattern. Also, the microsphere surface is free of drug crystals. For loading hydrophobic drugs into PLGA microspheres, the new microencapsulation process reported in this study has distinct advantages over commonly used emulsion‐templated solvent evaporation processes. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43768.

CRISPR-Cas12a system has been widely utilized for genome editing and molecular diagnostics applications. However, existing CRISPR-Cas12a systems still have much room for improving its functionality to expand their applications. To achieve this goal, a thorough understanding of the conformational dynamics of CRISPR-Cas12a system is essentially required. In this regard, single-molecule fluorescence assays have been recently applied to study the dynamics of CRISPR-Cas12a. In this review, we introduce single-molecule fluorescence studies on CRISPR-Cas12a system and provide practical guidelines required for reliable implementation of the single-molecule fluorescence methods to study the molecular mechanism of DNA cleavage reaction of CRISPR-Cas12a system.