Fetal microchimerism cells suppress arthritis progression by inducing CD14+ IL-10+ cells in pregnant experimental mice.

BACKGROUND: Fetal microchimerism occurs in the mother after a pregnancy. To investigate the role of fetal microchimerism cells (FMCs) in rheumatoid arthritis, we analyzed the population of fetal cells in pregnant experimental arthritis mice. METHODS: We used EGFP+ fetuses, which were mated with either healthy female mice or CIA mice, and male C57BL/6J-Tg (Pgk1-EGFP)03Narl mice, to detect the population of FMCs in maternal circulation. The disease progression was determined by measuring the clinical score and histological stains during pregnancy. The fetal cells have been analyzed if expressing EGFP, CD45, and Scal by flow cytometry. We also detected the expression of CD14+ IL-10+ cells in vivo and in vitro. RESULTS: Our data showed that the pregnancy ameliorated the arthritis progression of CIA mice. The IHC stains showed the CD45 -Sca-1+ EGFP+ FMCs were expressed in the bone marrow and peripheral blood mononuclear cells (PBMC) at 14 gestation days. However, Treg and Tc cell populations showed no significant change in the bone marrow. The data showed the H2Kb + fetal cells induced CD14+ IL10+ cell populations increased in the bone marrow in vitro and in vivo. CONCLUSION: Our investigations demonstrated that the FMCs protected the CIA mice from cartilage damage and triggered an immunosuppressive response in them by increasing the number of CD14+ IL10+ cells. In conclusion, the FMCs could potentially exhibit protective properties within the context of inflammatory arthritis that arises during pregnancy.

Aggregate-selective removal of pathological tau by clustering-activated degraders.

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.

Monitoring Circulating Myeloid Cells in Peritonitis with an In Vivo Imaging Flow Cytometer.

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases.

Immune Responses Induced by a Recombinant Lactiplantibacillus plantarum Surface-Displaying the gD Protein of Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the herpes viruses that can infect a wide range of animals including pigs, cattle, sheep, mice, and wild animals. PRV is a neurotropic alphaherpesvirus capable of infecting a variety of mammals. There is a rising interest in the targeted application of probiotic bacteria to prevent viral diseases, including PRV. In this study, the surface expression of enhanced green fluorescent protein (EGFP) on recombinant Lactiplantibacillus plantarum NC8 (rNC8) through the LP3065 LPxTG motif of Lactobacillus plantarum WCFS1 was generated. The surface expression was observed through confocal microscopy. Dendritic cell targeting peptides (DCpep) were also fused with LPxTG that help to bind with mouse DCs. The PRV-gD was cloned in LP3065 LPxTG, resulting in the generation of rNC8-LP3065-gD. Inactivated rNC8-LP3065-gD was administered intravenously in mice on days 1 and 7 at a dose of 200 µL (109 CFU/mouse) for monitoring immunogenicity. Subsequently, a challenge dose of PRV TJ (104 TCID50) was administered intramuscularly at 14 days post-immunization. The survival rate of the immunized mice reached 80% (4/5) with no significant signs of illness. A significant rise in anti-gD antibodies was detected in the immunized mice by ELISA. Quantitative PCR (qPCR) results showed decreased viral loading in different body tissues. Flow cytometry of lymphocytes derived from mice spleen indicated an increase in CD3+CD4+ T cells, but CD3+CD8+ T cells were not detected. Moreover, it offers a model to delineate immune correlates with rNC8-induced immunity against swine viral diseases.

RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

BEAM: A combinatorial recombinase toolbox for binary gene expression and mosaic genetic analysis.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

AlphaFold2-guided engineering of split-GFP technology enables labeling of endogenous tubulins across species while preserving function.

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

EL4 Murine-Lymphoma-Stromal-Cell Fusion Hybrids Observed With Multiple Distinct Morphologies in the Primary Tumor and Metastatic Organs of a Syngeneic Mouse Model.

BACKGROUND/AIM: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. MATERIALS AND METHODS: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. RESULTS: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. CONCLUSION: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression.

Revealing cis- and trans-regulatory elements underlying nuclear distribution and function of the Arabidopsis histone H2B.8 variant.

The H2B.8 variant has been diverged from other variants by its extended N-terminal region that possesses a conserved domain. We generated transgenic Arabidopsis plants expressing H2B.9 (class I), H2B.5 (class II) and H2B.8 (class III) fused to GFP under the 35 S promoter and studied their nuclear distribution and function. H2B.8-GFP showed peculiar nuclear localization at chromocenters in all cell types examined, while H2B.5-GFP and H2B.9-GFP displayed various patterns often dependent on cell types. H2B variants faithfully assembled onto nucleosomes showing no effect on nuclear organization; H2B.8-GFP appeared as three distinct isoforms in which one isoform appeared to be SUMOylated. Interestingly, transient expression in protoplasts revealed H2B.8 nuclear localization distinct from transgenic plants as it was restricted to the nuclear periphery generating a distinctive ring-like appearance accompanied by nuclear size reduction. This unique appearance was abolished by deletion of the N-terminal conserved domain or when H2B.8-GFP is transiently expressed in ddm1 protoplasts. GFP-TRAP-coupled proteome analysis uncovered H2B.8-partner proteins including H2A.W.12, which characterizes heterochromatin. Thus, our data highlight H2B.8 as a unique variant evolved in angiosperms to control chromatin compaction/aggregation and uncover cis- and trans-regulatory elements underlying its nuclear distribution and function.

Chemically Tagging Cargo for Specific Packaging inside and on the Surface of Virus-like Particles.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Engineered intrinsically fluorescent galectin-8 variants with altered valency, ligand recognition and biological activity.

Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.

Construction of pVAX-1-based linear covalently closed vector with improved transgene expression.

INTRODUCTION: This study presents a Mammalian Linear Expression System (MLES), a linear covalently closed (LCC) vector based on pVAX-1. The purpose of this system was to improve gene expression in mammalian cells and to test the efficacy of MLES in transient transfection and transgene expression using in vitro and in vivo models. Additionally, we aimed to evaluate potential inflammatory responses in vivo. MATERIALS AND METHODS: MLES was developed by modifying pVAX-1, and the construct was confirmed by gel electrophoresis. Lipofectamine®2000 was used to assess the transfection efficiency and expression of MLES in various cell lines. In vivo studies were conducted in mice injected with MLES/EGFP, and the resulting transfection efficiency, gene expression, and inflammatory responses were analyzed. RESULTS: MLES exhibited higher transfection efficiency and expression levels compared to pVAX-1 when tested on HEK-293, CHO-K1, and NIH-3T3 cells. When tested in vivo, MLES/EGFP showed elevated expression in the heart, kidney, liver, and spleen compared with pVAX-1/EGFP. Minimal changes are observed in the lungs. Additionally, MLES induced a reduced inflammatory response in mice compared with pVAX-1/EGFP. CONCLUSIONS: MLES offer improved transfection efficiency and reduced inflammation, representing a significant advancement in gene therapy and recombinant protein production. Further research on MLES-mediated gene expression and immune modulation will enhance gene therapy strategies.

Generation of green fluorescent protein reporter knock-in iPSC line at the 3'UTR region of the KLOTHO locus.

We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.

Generation and characterization of a human iPSC line expressing EGFP-tagged CDH1, KSCBi002-A-3.

E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell-cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.

Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters.

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Quantifying biomolecular organisation in membranes with brightness-transit statistics.

Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.

pJoseph2: a family of plasmids as positive controls for bacterial protein expression, transfections, and western blots.

Epitope tagging represents a powerful strategy for expedited identification, isolation, and characterization of proteins in molecular biological studies, including protein-protein interactions. We aimed to improve the reproducibility of epitope-tagged protein expression and detection by developing a range of plasmids as positive controls. The pJoseph2 family of expression plasmids functions in diverse cellular environments and cell types to enable the evaluation of transfection efficiency and antibody staining for epitope detection. The expressed green fluorescent proteins harbor five unique epitope tags, and their efficient expression in Escherichia coli, Drosophila Schneider's line 2 cells, and human SKOV3 and HEK293T cells was demonstrated by fluorescence microscopy and western blotting. The pJoseph2 plasmids provide versatile and valuable positive controls for numerous experimental applications.

Epitope tagging, a fundamental technique in molecular biology, involves attaching short amino acid sequences (epitope tags) to target proteins for their efficient identification and study. This technique has evolved since its inception, enabling diverse applications in protein research. Notably, CRISPR/Cas9 gene editing has enhanced epitope tagging by enabling the tagging of endogenous genes, expanding its versatility. However, reproducibility challenges exist, demanding positive controls for troubleshooting. The pJoseph2 family of plasmids was developed to address this need, providing robust positive controls for various epitope-based experiments, from bacterial expression to Drosophila and mammalian cell studies. This resource enhances the reliability and accuracy of epitope tagging, benefiting researchers across disciplines.

Retinal Penetrating Adeno-Associated Virus.

Purpose: The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection. Methods: Recently, we described a molecular chaperone (Nuc1) that enhanced the penetration of small and large molecules, including AAV, into the retina. The Nuc1 amino acid sequence or a truncated version of Nuc1 (IKV) was genetically incorporated into an exposed loop of AAV2/9 VP1 protein. These novel recombinant AAV vectors expressing green fluorescent protein (GFP) or nuclear factor erythroid 2 p45-related factor 2 (Nrf2) were injected into the vitreous of C57Bl/6J or Nrf2 knockout mice, respectively. The amount of GFP expression or oxidative stress as measured by 8-Hydroxy-2'-deoxyguanosine staining in C57Bl/6J or Nrf2 knockout mice, respectively, was quantified. Results: Incorporation of Nuc1 into AAV2/9 did not lead to significant expression of GFP in the murine retina. However, incorporation of IKV into AAV2/9 led to robust expression of GFP in photoreceptors and retinal pigment epithelium (RPE) via the intravitreal and subretinal routes of delivery. Furthermore, expression of Nrf2 using an IKV vector led to a reduction in oxidative stress in the retina of C57Bl/6J and Nrf2 knockout mice. Conclusions: We have developed a novel AAV vector that enables delivery of transgenes to the outer retina of mice, including photoreceptors and RPE following intravitreal injection.

Polyphenol-Enabled 2D Nanopatch for Enhanced Nasal Mucoadhesion and Immune Activation.

The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.