Nanobody-Functionalized Cellulose for Capturing SARS-CoV-2.

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 253 million people, claiming ∼5.1 million lives to date. Although mandatory quarantines, lockdowns, and vaccinations help curb viral transmission, there is a pressing need for cost-effective systems to mitigate the viral spread. Here, we present a generic strategy for capturing SARS-CoV-2 through functionalized cellulose materials. Specifically, we developed a bifunctional fusion protein consisting of a cellulose-binding domain and a nanobody (Nb) targeting the receptor-binding domain of SARS-CoV-2. The immobilization of the fusion proteins on cellulose substrates enhanced the capture efficiency of Nbs against SARS-CoV-2 pseudoviruses of the wild type and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography with highly porous cellulose to capture viruses from complex fluids in a continuous fashion. By capturing and containing viruses through the Nb-functionalized cellulose, our work may find utilities in virus sampling and filtration through the development of paper-based diagnostics, environmental tracking of viral spread, and reducing the viral load from infected individuals. IMPORTANCE The ongoing efforts to address the COVID-19 pandemic center around the development of diagnostics, preventative measures, and therapeutic strategies. In comparison to existing work, we have provided a complementary strategy to capture SARS-CoV-2 by functionalized cellulose materials through paper-based diagnostics as well as virus filtration in perishable samples. Specifically, we developed a bifunctional fusion protein consisting of both a cellulose-binding domain and a nanobody specific for the receptor-binding domain of SARS-CoV-2. As a proof of concept, the fusion protein-coated cellulose substrates exhibited enhanced capture efficiency against SARS-CoV-2 pseudovirus of both the wild type and the D614G variant, the latter of which has been shown to confer higher infectivity. Furthermore, the fusion protein was integrated into a customizable chromatography for binding viruses from complex biological fluids in a highly continuous and cost-effective manner. Such antigen-specific capture can potentially immobilize viruses of interest for viral detection and removal, which contrasts with the common size- or affinity-based filtration devices that bind a broad range of bacteria, viruses, fungi, and cytokines present in blood (https://clinicaltrials.gov/ct2/show/NCT04413955). Additionally, since our work focuses on capturing and concentrating viruses from surfaces and fluids as a means to improve detection, it can serve as an "add-on" technology to complement existing viral detection methods, many of which have been largely focusing on improving intrinsic sensitivities.

Constitutive expression of spliced X-box binding protein 1 inhibits dentin formation in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated, which subsequently converts an unspliced X-box binding protein 1 (XBP1U) mRNA to a spliced mRNA that encodes a potent XBP1S transcription factor. XBP1S is essential for relieving ER stress and secretory cell differentiation. We previously established Twist2-Cre;Xbp1 CS/+ mice that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells. In this study, we analyzed the dental phenotype of Twist2-Cre;Xbp1 CS/+ mice. We first generated a mutant Xbp1s minigene that corresponds to the recombinant Xbp1 Δ26 allele (the Xbp1 CS allele that has undergone Cre-mediated recombination) and confirmed that the Xbp1s minigene expressed XBP1S that does not require IRE1α activation in vitro. Consistently, immunohistochemistry showed that XBP1S was constitutively expressed in the odontoblasts and other dental pulp cells in Twist2-Cre;Xbp1 CS/+ mice. Plain X-ray radiography and µCT analysis revealed that constitutive expression of XBP1S altered the dental pulp chamber roof- and floor-dentin formation, resulting in a significant reduction in dentin/cementum formation in Twist2-Cre;Xbp1 CS/+ mice, compared to age-matched Xbp1 CS/+ control mice. However, there is no significant difference in the density of dentin/cementum between these two groups of mice. Histologically, persistent expression of XBP1S caused a morphological change in odontoblasts in Twist2-Cre;Xbp1 CS/+ mice. Nevertheless, in situ hybridization and immunohistochemistry analyses showed that continuous expression of XBP1S had no apparent effects on the expression of the Dspp and Dmp1 genes. In conclusion, these results support that sustained production of XBP1S adversely affected odontoblast function and dentin formation.

Comparison of bacterial nanocellulose produced by different strains under static and agitated culture conditions.

Bacterial nanocellulose (BNC) has many advantages over plant cellulose, which make it widely used in many fields, especially in the food industry. In this study, three strains including BCA263, BCC529, and P1 were selected for characteristics analysis of BNCs under static and agitated culture conditions. The BNCs produced under static culture condition were in the shape of uniform membrane, while BNCs produced under agitated culture were in form of small agglomerates and fragments. BCA263 and BCC529 strains were more suitable for static culture, while P1 strain was more suitable for agitated culture. BNCs produced under static culture condition exhibited higher crystallinity, stronger tensile strength, denser network structure, higher temperature resistance and good flame retardancy; while BNCs produced under agitated culture condition exhibited larger porous and lower crystallinity. Furthermore, BNCs produced under agitated culture condition were more suitable as a stabilizer of coffee milk beverage.

Rationally Designed Polycationic Carriers for Potent Polymeric siRNA-Mediated Gene Silencing.

The delivery of small interfering RNA (siRNA) remains a major hurdle for the clinical translation of RNA interference (RNAi) therapeutics. Because of its low valency and rigid nature, siRNA typically requires high excesses of cationic delivery materials to package it stably and deliver it to the cytoplasm of target cells, resulting in high toxicities and inefficient gene silencing in vivo. To address these challenges, we pair a polymeric form of siRNA, p-shRNA, with optimized biodegradable polycations to form stable complexes that induce far more potent gene silencing than with siRNA complexes. Furthermore, we unveil a set of design rules governing p-shRNA delivery, using degradable polycations containing hydrophobic and stabilizing polyethylene glycol domains that enable both stable condensation and efficient release inside cells. We demonstrate the therapeutic potential of this approach by silencing the oncogene STAT3 in a well-established B16F10 mouse melanoma model to significantly prolong survival. By blending nucleic acid engineering and polymer design, our system provides a potentially translatable platform for RNAi-based therapies.

Synthetic Charge-Invertible Polymer for Rapid and Complete Implantation of Layer-by-Layer Microneedle Drug Films for Enhanced Transdermal Vaccination.

The utility of layer-by-layer (LbL) coated microneedle (MN) skin patches for transdermal drug delivery has proven to be a promising approach, with advantages over hypodermal injection due to painless and easy self-administration. However, the long epidermal application time required for drug implantation by existing LbL MN strategies (15-90 min) can lead to potential medication noncompliance. Here, we developed a MN platform to shorten the application time in MN therapies based on a synthetic pH-induced charge-invertible polymer poly(2-(diisopropylamino) ethyl methacrylate- b-methacrylic acid) (PDM), requiring only 1 min skin insertion time to implant LbL films in vivo. Following MN-mediated delivery of 0.5 µg model antigen chicken ovalbumin (OVA) in the skin of mice, this system achieved sustained release over 3 days and led to an elevated immune response as demonstrated by significantly higher humoral immunity compared with OVA administration via conventional routes (subcutaneously and intramuscularly). Moreover, in an ex vivo experiment on human skin, we achieved efficient immune activation through MN-delivered LbL films, demonstrated by a rapid uptake of vaccine adjuvants by the antigen presenting cells. These features, rapid administration and the ability to elicit a robust immune response, can potentially enable a broad application of microneedle-based vaccination technologies.

Super natural killer cells that target metastases in the tumor draining lymph nodes.

Tumor draining lymph nodes are the first site of metastasis in most types of cancer. The extent of metastasis in the lymph nodes is often used in staging cancer progression. We previously showed that nanoscale TRAIL liposomes conjugated to human natural killer cells enhance their endogenous therapeutic potential in killing cancer cells cultured in engineered lymph node microenvironments. In this work, it is shown that liposomes decorated with apoptosis-inducing ligand TRAIL and an antibody against a mouse natural killer cell marker are carried to the tumor draining inguinal lymph nodes and prevent the lymphatic spread of a subcutaneous tumor in mice. It is shown that targeting natural killer cells with TRAIL liposomes enhances their retention time within the tumor draining lymph nodes to induce apoptosis in cancer cells. It is concluded that this approach can be used to kill cancer cells within the tumor draining lymph nodes to prevent the lymphatic spread of cancer.