Harnessing Self-Repairing and Crystallization Processes for Effective Enzyme Encapsulation in Covalent Organic Frameworks.

Immobilization of fragile enzymes in crystalline porous materials offers new opportunities to expand the applications of biocatalysts. However, limited by the pore size and/or harsh synthesis conditions of the porous hosts, enzymes often suffer from dimension limitation or denaturation during the immobilization process. Taking advantage of the dynamic covalent chemistry feature of covalent organic frameworks (COFs), herein, we report a preprotection strategy to encapsulate enzymes in COFs during the self-repairing and crystallization process. Enzymes were first loaded in the low-crystalline polymer networks with mesopores formed at the initial growth stage, which could offer effective protection for enzymes from the harsh reaction conditions, and subsequently the encapsulation proceeded during the self-repairing and crystallization of the disordered polymer into the crystalline framework. Impressively, the biological activity of the enzymes can be well-maintained after encapsulation, and the obtained enzyme@COFs also show superior stability. Furthermore, the preprotection strategy circumvents the size limitation for enzymes, and its versatility was verified by enzymes with different sizes and surface charges, as well as a two-enzyme cascade system. This study offers a universal design idea to encapsulate enzymes in robust porous supports and holds promise for developing high-performance immobilized biocatalysts.

Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line.

Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.