Biology subjects

Duncan, G.

Publications and source records attributed to Duncan, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Sprayable in Situ Forming PEG Hydrogels for Intranasal Drug Delivery

Intranasal administration provides a noninvasive route for local and systemic drug delivery, yet its clinical effectiveness remains limited by rapid mucociliary clearance and short drug residence times within the nasal cavity. Existing stimulus-responsive nasal hydrogels seek to address this challenge but rely on physiological triggers such as temperature, pH, or ionic strength to induce gelation, resulting in variable performance due to natural differences in the nasal cavity. Here, we present a polyethylene glycol (PEG)-based hydrogel platform that overcomes these limitations through rapid in situ gel formation that is independent of physiological stimuli. The two liquid precursor solutions remain sprayable during administration and rapidly crosslink upon deposition to form a cohesive hydrogel depot. Although this dual-component design presents challenges for compatibility with conventional nasal spray devices, we demonstrate successful delivery using commercially available dual-barrel spray systems. The hydrogel maintained spray performance and regional nasal deposition comparable to conventional aqueous formulations while significantly reducing mucociliary transport to prolong mucosal residence. Importantly, reduced mucociliary transport was achieved without impairing ciliary function or compromising epithelial barrier integrity, addressing key safety considerations for repeated intranasal administration. In addition, the hydrogel altered epithelial uptake and transport of biologics, suggesting its potential to improve the nasal delivery of macromolecular therapeutics that typically exhibit poor bioavailability. The results of these study establish a versatile and clinically translatable approach for enhancing intranasal drug delivery across a broad range of therapeutic applications.

bioengineering↗

bioRxiv2026

MUC5B and MUC5AC function in combination to regulate mucociliary transport on human airway epithelium

Muco-obstructive lung diseases are characterized by impaired airway clearance and altered mucin composition. MUC5B and MUC5AC are the primary gel-forming mucins in airway mucus, yet how their relative abundance influences the physical and functional properties of mucus remains poorly understood. Here, we investigated how compositional variations in MUC5B and MUC5AC within mucus impact mucociliary transport. Mucus enriched in either MUC5B or MUC5AC was generated using human airway epithelial models depleted for each mucin via CRISPR/Cas9-targeted knockout. Defined mixtures of these mucins were generated at physiologically relevant ratios, for assessment of their microrheological properties and mucociliary transport behavior in differentiated primary human airway epithelial tissue cultures. We found that increasing MUC5AC content reduced network pore size and increased microviscosity, concomitant with reduced mucociliary transport, demonstrating that mucin composition alters the biophysical and functional properties of the mucus barrier. In normal airway tissue cultures with MUC5B-predominant mucus, overlay of MUC5AC on the apical surface impaired mucociliary transport, whereas MUC5B had minimal effect. Conversely, MUC5B supplementation uniquely improved mucociliary transport in IL-13 stimulated cultures exhibiting mucostasis, whereas additional MUC5AC did not alter transport. Together, these findings demonstrate that the ratio of MUC5B to MUC5AC can shape mucus organization at the microscale, which in turn governs mucociliary transport at the tissue scale.

cell biology↗

bioRxiv2026

Liquid crystalline mesophase spacing as a quantitative predictor of release kinetics for co-loaded hydrophilic and hydrophobic payloads

Liquid crystalline mesophases exhibit structurally programmable internal architectures that enable co-loading of chemically orthogonal molecules within a single composite material. Realizing the potential of these materials for drug delivery requires a quantitative understanding of how tuning the composition affects internal mesophase architecture and consequently performance metrics such as payload release. Here, Flash NanoPrecipitation with hydrophobic ion pairing is used to prepare nanocarriers containing liquid crystalline mesophases co-encapsulating two compounds from widely different chemical classes: hydrophilic polymyxin B (logP -6) with one of four hydrophobic co-core materials (logP 7-11), achieving >75% encapsulation efficiency and up to 32% and 50% mass loadings for polymyxin and co-core. Synchrotron SAXS is used to quantify characteristic mesophase repeat spacing, which is found to be tunable as a function of composition. A strong correlation between d-spacing and polymyxin release rate is presented. Co-core chemistry and weight fraction jointly govern mesophase architecture, and repeat distance emerges as a structural metric linking these to the hydrophilic payload release kinetics. Mucus diffusivity and antibacterial efficacy are assessed as independent performance metrics, and results corroborate the release behavior. These findings establish a quantitative framework connecting material composition, mesophase architecture, and functional performance that can be applied toward rational co-formulation design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/734853v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@a0c38eorg.highwire.dtl.DTLVardef@86d33eorg.highwire.dtl.DTLVardef@196c83dorg.highwire.dtl.DTLVardef@3423c3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOToC Graphic TextC_FLOATNO Flash NanoPrecipitation yields liquid crystalline nanocarriers co-encapsulating with high efficiency payloads with widely distinct physicochemical properties. Synchrotron SAXS establishes characteristic repeat spacing as a quantitative structural metric directly governing hydrophilic release kinetics, providing a rational design framework linking mesophase architecture to functional performance across a range of payload structures. C_FIG

bioengineering↗

bioRxiv2026

Lung surfactants as a component of lipid nanoparticles for pulmonary mRNA delivery

Pulmonary delivery of lipid nanoparticles (LNPs) remains an area of significant interest, given the broad range of genetic disorders that could be addressed through localized administration of therapeutic nucleic acids to the lung. In this study, we investigated how incorporation of the clinically used lung surfactant cocktail Poractant alfa affects the in vitro and in vivo transfection performance of mRNA-loaded LNPs. The resulting lung surfactant-enhanced LNPs (Surf-LNPs) exhibited substantial improvements in particle assembly, yielding an order of magnitude higher particle concentration at equivalent input conditions compared to conventional (Onpattro-like) LNP formulations. In vitro, Surf-LNPs demonstrated several-fold increases in mRNA transfection efficiency and protein expression while maintaining excellent cytocompatibility. These enhancements are attributed to an elevated apparent pKa and the surface-active properties of surfactant protein B (SP-B), which promote more rapid and efficient endosomal escape relative to conventional LNPs. In vivo evaluation following intranasal administration further revealed enhanced mCherry expression in the lungs of mice treated with Surf-LNPs compared to conventional LNPs. Ultimately, these findings establish lung surfactant incorporation as a simple yet powerful formulation strategy to improve pulmonary gene delivery using LNPs, with the potential to significantly advance the translation of inhaled nucleic acid therapeutics.

bioengineering↗

bioRxiv2026

Airway mucins function as endogenous inhibitors of neutrophil extracellular traps

Neutrophils recruited to the airways are important for innate lung defense and can release neutrophil extracellular traps (NETs) to capture and eliminate microbes. While NETs are not abundant in healthy airways, uncontrolled NETosis is a known pathological feature and contributor to both chronic and acute respiratory diseases. Prior studies have shown that mucin glycoproteins secreted in the oral cavity and cervicovaginal tract can modulate NETosis, but it remains unknown whether mucins secreted in the respiratory tract influence NET formation. In these studies, we discovered that human airway mucus strongly inhibits NETosis in primary human neutrophils in a sialic acid dependent manner. In comparison, mucus produced by human airway epithelial cells genetically engineered to lack either MUC5B or MUC5AC secreted airway mucins showed a reduced ability to suppress NETosis. To assess how the lung microenvironment in obstructive lung diseases may influence mucus-dependent NET formation, we engineered a synthetic, mucin-laden hydrogel model with physical properties resembling that of mucus in a healthy lung and a disease-affected lung. When neutrophils were cultured on these gel substrates, we found that increasing gel stiffness led to a significantly greater extent of NETosis. Together these data demonstrate a new functional role of airway mucus in modulating neutrophil homeostasis in the respiratory tract and provide evidence that mucus dysfunction in disease can impair its ability to regulate NETosis.

immunology↗

bioRxiv2025

Functional characterization of the 9q34.13 locus identifies RAPGEF1 as modulating risk for melanoma and nevi via RAS activation

Genome-wide association studies identified a melanoma- and nevus count-associated locus on chromosome band 9q34.13. Fine-mapping and melanocyte expression data collectively suggest two potential causal genes with opposite association with risk: higher levels of Rap guanine nucleotide exchange factor 1 (RAPGEF1) and lower levels of uridine-cytidine kinase 1 (UCK1). Colocalization analyses and conditional TWAS suggest multiple causal cis-regulatory sequence variants in partial linkage disequilibrium (LD) to each other. Melanocyte capture-HiC and CRISPR-inhibition demonstrated regulatory interactions between fine-mapped variants and the RAPGEF1 and UCK1 promoters. Focusing on RAPGEF1, we demonstrate RAPGEF1 expression promotes melanocyte growth and drives malignant transformation of human immortalized melanocytes. Following treatment with human EGF, RAPGEF1 overexpression activated both RAP1 and RAS. Further, we show RAPGEF1 expression is significantly enriched in melanomas lacking strongly activating RAS-MAPK mutations, suggesting that RAPGEF1 may promote oncogenic RAS-MAPK signaling in melanomas. Furthermore, in these tumors, we provide preliminary evidence to support the prognostic relevance of RAPGEF1 expression in patients lacking RAS or BRAF mutations. Together with other recent studies, these data suggest that germline variation influencing RAS activation may play a key role in nevus development and melanoma risk.

genetics↗

bioRxiv2025

Synthetic Mucus Biomaterials Enable Localized Therapeutic Antibody Delivery in Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic condition characterized by recurrent gastrointestinal inflammation that requires long-term therapeutic intervention. While anti-TNF- monoclonal antibodies (mAbs) are effective in maintaining remission in IBD, systemic delivery is associated with immunosuppression, poor targeting efficiency, and high cost. To address these limitations, we developed a synthetic mucin-based hydrogel for localized delivery of TNF--targeting mAbs. Mucins are heavily glycosylated biopolymers that naturally bind antimicrobial and anti-inflammatory proteins, making them well-suited for local biologic drug delivery at mucosal sites. Synthetic mucin-based hydrogels were formed by crosslinking mucin harvested from porcine small intestine with a 4-arm PEG-thiol and loaded with mAbs to evaluate biocompatibility, antibody release kinetics, and therapeutic efficacy. In vitro studies confirmed cytocompatibility of mucin-based hydrogels and demonstrated sustained release of full-length IgG antibodies, with enhanced release under proteolytic conditions simulating the gastrointestinal environment. Moreover, mucin-based hydrogels alone were found to modulate macrophage activation and dampen inflammation in LPS-stimulated macrophages. Treatment of LPS-stimulated macrophages with mAb-loaded hydrogels reduced pro-inflammatory cytokine production and macrophage activation, confirming retention of mAb bioactivity. Compared to antibodies administered in solution, in vivo biodistribution studies revealed greater absorption of antibodies when loaded in mucin-based hydrogels and administered via enema in TNBS-induced colitis mice likely due to enhanced adhesion to mucosal epithelium and slowed intestinal clearance. This study demonstrates the potential of mucin-based hydrogels as a platform for local mAb delivery in IBD, enabling targeted immunosuppression while minimizing systemic exposure.

bioengineering↗

bioRxiv2025

Female sex hormones enhance gonococcal colonization at the endocervix by modifying cervical mucus

Neisseria gonorrhoeae is a human-exclusive pathogen that causes gonorrhea. Gonococci (GC) initiate female infections by colonizing the cervix, which can remain asymptomatic, cause cervicitis, or ascend to the upper female reproductive tract (FRT), leading to severe tissue damage. The FRT undergoes sex hormone-mediated changes during the menstrual cycle, which have long been implicated in the vulnerability to GC infection. One of the major changes is the increase and decrease in the production of the gel-forming mucin MUC5B by the endocervix in response to the level of estradiol (E2). This study examined the impact of sex hormones on GC infection of the human cervix, utilizing a human cervical tissue explant model. Tissue explants were treated without and with E2 alone or in combination with progesterone (E2+P4) to mimic various menstrual cycle phases. Treatment of E2 or E2+P4 enhanced GC colonization at the endocervix exclusively, but did not affect epithelial transmigration. While both treatments increased the number of GC microcolonies, E2+P4 increased GC colony size on the endocervical epithelium. These increases were independent of GC host receptors, carcinoembryonic antigen-related cell adhesion molecules. GC effectively diffused through cervical mucus to interact with the cervical epithelium under all hormone conditions and through mucin hydrogels with different MUC5B and MUC5AC compositions. Mucus gels collected from cervical explants and animal mucin mixtures enhanced GC aggregation in vitro. GC diffusion through mucin-hydrogels and aggregation in the presence of cervical mucus or animal mucins decreased as the MUC5B concentration increased. Our results suggest that female sex hormones promote GC colonization at the human endocervix by modulating the cervical mucus production, regulating womens susceptibility to GC infection, and further reveal the ability of GC to evade the mucus defense barrier for infection. Author SummaryNeisseria gonorrhoeae is a bacterial pathogen that primarily infects the human genital and female reproductive tracts, causing gonorrhea. While this bacterium can infect both men and women, the infection can lead to severe and permanent damage to womens reproductive systems. Currently, the relationship of gonococcal infection with the menstrual cycle is unknown. Here, we utilize a human cervical tissue explant model that mimics gonococcal infection in women to examine the impact of female sex hormones that drive the menstrual cycle on gonococcal infection. We found that estrogen alone or in combination with progesterone enhanced gonococcal colonization, increasing both the number and size of bacterial microcolonies on the cervical luminal surface, through regulating mucus production. Gonococci effectively penetrate through mucus layers to reach cervical epithelial cells and also prefer to aggregate with each other in the presence of mucus. Our results reveal that hormone-regulated mucus production changes the vulnerability of women to gonococcal infection, and that gonococci convert the mucus defense barrier into a colonization facilitator.

microbiology↗

bioRxiv2025

Barrier Function of the Extracellular Matrix in AAV Gene Therapy

PurposeThe extracellular matrix (ECM) is a major component of the tissue microenvironment which may pose a barrier to the distribution of AAV in target organs, preventing delivery of therapeutic cargo. We sought to address this potential barrier to AAV gene therapy by furthering our understanding of AAV-ECM interactions. We hypothesized that both the AAV serotype and ECM composition will impact AAV transport and gene delivery. MethodsAAV2, AAV6, and AAV8 viral vectors were fluorescently labeled to allow for visualization of their diffusion through the ECM. Lung, liver, and small intestinal submucosal dECM hydrogels were formulated as models of the ECM with tissue-specific biomolecular content. We then characterized AAV and nanoparticle diffusion within decellularized ECM using fluorescent video microscopy and multiple particle tracking. Additionally, we evaluated AAV transduction in dECM-incorporated 2D and 3D spheroid tissue culture models. ResultsAll AAV displayed reduced diffusivity through ECM as compared to similarly sized nanoparticles. AAV2 diffusion was least affected by the presence of ECM across tissue types as compared to AAV6 and AAV8. AAV transduction in dECM incorporated in vitro models was significantly reduced in both a 2D and 3D setting. ConclusionsThese results suggest binding of AAV to the ECM may decrease their therapeutic effect in target tissues throughout the body. The barrier function of the ECM should be considered in development of AAV for gene therapy applications.

bioengineering↗

bioRxiv2025

RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identifies both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism. As proof-of-concept, CRASP-Seq analysis of an LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. High-throughput mutagenesis further showed that ZNF207s zinc finger domain broadly impacts alternative splicing through interactions with U1 snRNP factors. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-Seq to uncover key regulators and domains of alternative splicing. Main PointsO_LICRASP-Seq: RNA-coupled CRISPR screen quantifying gene and domain impact on splicing C_LIO_LIProfiling of five events identified 370 genes influencing alternative splicing C_LIO_LIZNF207 regulates splicing by interacting with U1 snRNP via its zinc-finger domains C_LIO_LIZNF207 depletion corrects LMNA aberrant splicing causing progeria C_LI

genetics↗

bioRxiv2025

AAV-mediated MUC5AC siRNA delivery to prevent mucociliary dysfunction in asthma

The main structural components of mucus produced in the lung are mucin 5B (MUC5B) and mucin 5AC (MUC5AC) where a relatively higher expression of MUC5B is typical in health. In the lungs of individuals with asthma, there is a shift from MUC5B to MUC5AC as the predominantly secreted mucin which has been shown to impair mucociliary clearance (MCC) and increase mucus plug formation in the airways. Given its role in asthmatic lung disease, MUC5AC represents a potential therapeutic target where a gene delivery approach could be leveraged to modulate its expression. For these purposes, we explored adeno-associated virus serotype 6 (AAV6), as a lung-tropic viral gene vector to target airway epithelial cells and reduce MUC5AC expression via siRNA delivery. We confirmed that AAV6 was able to transduce epithelial cells in the airways of healthy mice with high transgene expression in mucus-secreting goblet cells. Using multiple particle tracking analysis, we observed that AAV6 was capable of penetrating both normal and MUC5AC-enriched mucus barriers. Successful transduction with AAV6 was also achieved in IL-13 stimulated human airway epithelial (HAE) cells differentiated at air-liquid interface (ALI). AAV6 expressing MUC5AC-targeting siRNA was evaluated as a prophylactic treatment in HAE cell cultures before IL-13 challenge. IL-13 stimulated HAE cultures treated with AAV6-MUC5AC siRNA had significantly reduced MUC5AC mRNA and protein expression compared to untreated controls. Mucociliary transport in IL-13 stimulated HAE cultures was also maintained and comparable to healthy controls following AAV6-MUC5AC siRNA treatment. Together, these findings support that AAV6 may be used as an inhaled gene therapy to suppress MUC5AC overexpression and restore normal airway clearance function in asthma.

cell biology↗

bioRxiv2024

Scalable Extraction of Airway Mucins from Porcine Trachea

Mucins are a major component of the innate defense system in the airways and their biological functions are important to consider in pulmonary disease research. However, the available mucus models for basic research relevant to the lung can be difficult to acquire in sufficient quantity to conduct such studies. Here, we present a new strategy to isolate airway mucins from pig trachea at the milligram to gram scale for use in pulmonary disease research. Using this protocol, we were able to isolate mucins with minimal DNA contamination consisting of [~]70% by weight protein. Compared to porcine gastric mucins extracted with the same procedure, the porcine tracheal extract possessed significantly greater O-linked glycoprotein (mucin) content. Particle tracking microrheology was used to evaluate the biophysical properties of porcine trachea mucins. We found porcine tracheal mucins formed a much tighter mesh network and possessed a significantly greater microviscosity compared to lab extracted porcine gastric mucins. In comparison to mucus harvested from human airway tissue cultures, we found porcine tracheal mucins also possessed a greater microviscosity suggesting these mucins can form into a gel-like material at physiological total solids concentrations. These studies establish an accessible means to isolate airway mucins from porcine trachea at large scale for use in pulmonary disease research.

biochemistry↗

bioRxiv2024

Synthetic mucus biomaterials synergize with antibiofilm agents to combat Pseudomonas aeruginosa biofilms

Bacterial biofilms are often highly resistant to antimicrobials causing persistent infections which when not effectively managed can significantly worsen clinical outcomes. As such, alternatives to standard antibiotic therapies have been highly sought after to address difficult-to-treat biofilm-associated infections. We hypothesized a biomaterial-based approach using the innate functions of mucins to modulate bacterial surface attachment and virulence could provide a new therapeutic strategy against biofilms. Based on our testing in Pseudomonas aeruginosa biofilms, we found synthetic mucus biomaterials can inhibit biofilm formation and significantly reduce the thickness of mature biofilms. In addition, we evaluated if synthetic mucus biomaterials could work synergistically with DNase and/or -amylase for enhanced biofilm dispersal. Combination treatment with these antibiofilm agents and synthetic mucus biomaterials resulted in up to 3 log reductions in viability of mature P. aeruginosa biofilms. Overall, this work provides a new bio-inspired, combinatorial approach to address biofilms and antibiotic-resistant bacterial infections.

bioengineering↗

bioRxiv2024

The Extracellular Matrix Limits Nanoparticle Diffusion and Cellular Uptake in a Tissue-Specific Manner

Overexpression and remodeling of the extracellular matrix (ECM) in cancer and other diseases may significantly reduce the ability of nanoparticles to reach target sites, preventing effective delivery of therapeutic cargo. Here, we evaluate how tissue-specific properties of the ECM affect nanoparticle diffusion using fluorescence video microscopy and cellular uptake via flow cytometry. In addition, we determined how PEGylation chain size and branching influence the ability of nanoparticles to overcome the ECM barrier from different tissues. We found that purified collagen, in the absence of other ECM proteins and polysaccharides, presented a greater barrier to nanoparticle diffusion as compared to decellularized ECM from the liver, lung, and small intestine submucosa. Nanoparticles with dense PEG coatings achieved up to [~]2000-fold enhancements in diffusion rate and cellular uptake up to [~]5-fold greater than non-PEGylated nanoparticles in the presence of ECM. We also found nanoparticle mobility in the ECM varied significantly between tissue types and the optimal nanoparticle PEGylation strategy to enhance ECM penetration was strongly dependent on ECM concentration. Overall, our data supports the use of low molecular weight PEG coatings which provides an optimal balance of nanoparticle penetration through ECM and uptake in target cells. However, tissue-specific enhancements in ECM penetration and cellular uptake were observed for nanoparticles bearing a branched PEG coating. These studies provide new insight into tissue specific ECM barrier functions which can facilitate the design of nanoparticles that effectively transport through target tissues, improving their therapeutic efficacy.

bioengineering↗

bioRxiv2024

Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

The bone marrow is the organ responsible for blood production. Diverse non-hematopoietic cells contribute essentially to hematopoiesis. However, these cells and their spatial organization remain largely uncharacterized as they have been technically challenging to study in humans. Here, we used fresh femoral head samples and performed single-cell RNA sequencing (scRNA-Seq) to profile 29,325 enriched non-hematopoietic bone marrow cells and discover nine transcriptionally distinct subtypes. We next employed CO-detection by inDEXing (CODEX) multiplexed imaging of 18 individuals, including both healthy and acute myeloid leukemia (AML) samples, to spatially profile over one million single cells with a novel 53-antibody panel. We discovered a relatively hyperoxygenated arterio-endosteal niche for early myelopoiesis, and an adipocytic, but not endosteal or perivascular, niche for early hematopoietic stem and progenitor cells. We used our atlas to predict cell type labels in new bone marrow images and used these predictions to uncover mesenchymal stromal cell (MSC) expansion and leukemic blast/MSC-enriched spatial neighborhoods in AML patient samples. Our work represents the first comprehensive, spatially-resolved multiomic atlas of human bone marrow and will serve as a reference for future investigation of cellular interactions that drive hematopoiesis.

cell biology↗

bioRxiv2023

Mucus physically restricts influenza A viral particle access to the epithelium

Prior work suggests influenza A virus (IAV) crosses the airway mucus barrier in a sialic acid-dependent manner through the actions of the viral envelope glycoproteins, hemagglutinin and neuraminidase. However, host and viral factors that influence how efficiently mucus traps IAV remain poorly defined. In this work, we assessed how the physicochemical properties of mucus influence its ability to effectively capture IAV using fluorescence video microscopy and multiple particle tracking. We found an airway mucus gel layer must be produced with virus-sized pores to physically constrain IAV. While sialic acid binding by IAV may improve mucus trapping efficiency, sialic acid binding preference was found to have little impact on IAV mobility and the fraction of viral particles expected to penetrate the mucus barrier. Further, we demonstrate synthetic polymeric hydrogels engineered with mucus-like architecture are similarly protective against IAV infection despite their lack of sialic acid decoy receptors. Together, this work provides new insights on mucus barrier function toward IAV with important implications on innate host defense and interspecies transmission.

microbiology↗

bioRxiv2023

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel and native mucus viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

bioengineering↗

bioRxiv2023

Synthetic mucus biomaterials for antimicrobial peptide delivery

Despite the promise of antimicrobial peptides (AMPs) as treatments for antibiotic-resistant infections, their therapeutic efficacy is limited due to the rapid degradation and low bioavailability of AMPs. To address this, we have developed and characterized a synthetic mucus (SM) biomaterial capable of delivering AMPs and enhancing their therapeutic effect. LL37 loaded SM hydrogels demonstrated controlled release of LL37 over 8 hours as a result of charge-mediated interactions between mucins and LL37 AMPs. Compared to treatment with LL37 alone where antimicrobial activity was reduced after 3 hours, LL37-SM hydrogels inhibited Pseudomonas aeruginosa PAO1 growth over 12 hours. LL37-SM hydrogel treatment reduced PAO1 viability over 6 hours whereas a rebound in bacterial growth was observed when treated with LL37 only. These data demonstrate LL37-SM hydrogels enhance antimicrobial activity by preserving LL37 AMP activity and bioavailability. Overall, this work establishes SM biomaterials as a platform for enhanced AMP delivery for antimicrobial applications.

bioengineering↗