Bioengineering & Translational Medicine

Background: Tumor necrosis factor- inhibitors such as infliximab and adalimumab have transformed autoimmune disease treatment; however, infliximab is a mouse-human chimeric antibody that remains immunogenic, is associated with self-association/aggregation liability, and requires prolonged intravenous administration. We humanized infliximab and engineered infliximab-derived candidates with improved potency and developability. Methods: Infliximab complementarity-determining regions were grafted onto human germline frameworks to generate humanized infliximab. STage-Enhanced Maturation (STEM) technology produced an affinity-matured clone (hInBG4), followed by targeted amino-acid substitutions in the complementarity-determining regions to generate LW2Y, LW2YR2S, and LW2YHR1K. Variants were evaluated by a cell-based tumor necrosis factor alpha neutralization assay, affinity-capture self-interaction nanoparticle spectroscopy, a baculovirus particle enzyme-linked immunosorbent assay, size-exclusion high-performance liquid chromatography, transient expression in human embryonic kidney 293 cells, and tumor necrosis factor alpha binding kinetics by biolayer interferometry, including dissociation at pH 7.4 and 5.8. Results: All three variants showed two- to three-fold higher neutralization potency than chimeric infliximab and outperformed adalimumab. Affinity-capture self-interaction nanoparticle spectroscopy shifts decreased from double-digit parental values to low single digits, while baculovirus particle binding ratios remained acceptable. Size-exclusion chromatography showed cleaner monomer peaks with reduced tailing, and expression increased relative to humanized infliximab. LW2Y combined very high affinity at pH 7.4 with markedly faster dissociation at pH 5.8, consistent with pH-dependent antigen release. Conclusions: Humanization, affinity maturation, and targeted complementarity-determining region re-engineering generated infliximab-derived candidates with improved potency and developability and identified LW2Y as a lead for further preclinical evaluation.

Chronic, mechanically resilient thrombi remain difficult to remove rapidly and safely using existing therapies, which are limited by slow treatment speeds, reduced efficacy against aged clots and risks associated with embolic debris. Here we introduce Constrained Laser-Induced Cavitation (CLIC), a novel approach that confines laser-induced cavitation bubble generation and collapse within a miniaturized waveguide to enhance thrombolysis. Optimized CLIC removed retracted clots at a mass-loss rate of 393.5 mg/min, [~]40-fold higher than reported state-of-the-art sonothrombolysis under similar conditions. Systematic variation of channel length and laser parameters showed that CLIC efficacy depends strongly on treatment geometry and cavitation dynamics. Post-treatment analysis revealed cylindrical channels consistent with clot removal dominated by fluid jetting and suction-driven evacuation, with cavitation shockwaves likely contributing a secondary role. Debris fragment measurements remained predominantly below a 1 mm embolic-risk threshold, consistent with a promising embolic safety profile. These findings establish CLIC as a viable strategy for rapid thrombolysis of chronic, mechanically resistant thrombi.

Endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) are increasingly used to model vascular diseases and test therapeutic strategies. However, the efficiency and reproducibility of differentiation can vary depending on the culture medium and its supplemented factors and stages. Here, we directly compared two defined media, APEL and BPEL, for iPSC-to-ECs differentiation. iPSCs were differentiated over 10 days with sequential growth factor induction, followed by magnetic-activated cell sorting or flow cytometry for CD31+ cells. Both media produced ECs with similar morphology and marker expression, including CD31 and VE-cadherin. Functional assays demonstrated comparable tube formation, indicating equivalent endothelial functionality. Cost analysis indicated that APEL had a higher total reagent cost but generated a higher total cell yield, resulting in a comparable cost per 10 total cells, whereas BPEL was more cost-efficient for producing CD31/VE-cadherin endothelial-specific cells. Our results suggest that APEL and BPEL media are equally effective for generating iPSC-derived ECs, providing flexibility in method selection for vascular disease modeling and drug discovery applications.

Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, affect millions of individuals worldwide who have limited access to curative therapies. Autologous hematopoietic stem cell transplant following ex vivo CRISPR editing of the BCL11A erythroid enhancer reactivates fetal hemoglobin (HbF) and achieves an effective cure, but the resource constraints of clinically approved procedures for editing by electroporation (EP) severely limit widespread implementation. We directly compared the functional outcomes of EP delivery of Cas9 ribonucleoprotein with lipid nanoparticle (LNP) delivery of Cas9 mRNA in primary human HSPCs obtained from healthy HbAA donors and from patients with SCD. While higher editing rates are achieved with EP, LNP-treated HSPCs exhibited greater viability and cell yields that persisted throughout a multi-stage in vitro erythroid differentiation protocol. By day 20, the yield of mature red blood cells (CD71lowCD235ahigh) was lowest in the EP cohorts. Across treatment groups, we observed HbF induction proportional to indel frequency. LNP editing of SCD patient-derived HSPCs as low as 25% modified alleles still caused HbF production and reduced the propensity for sickling of in vitro differentiated RBCs. These findings highlight the critical trade-offs among manufacturing ease, delivery-associated toxicity, and functional performance across two modalities of therapeutic genome editing for hemoglobinopathies.

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-{gamma} and TNF-. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

Pulmonary hypertension (PH) is characterized by progressive structural and mechanical remodeling of the pulmonary vasculature, yet few computational frameworks directly link disease mechanisms to longitudinal progression and therapeutic response. In this study, we utilized a multiscale pulmonary arterial growth and remodeling (G&R) framework to capture evolving functional metrics from rat models of PH. This framework couples morphometric tree hemodynamics, constrained mixture theory-based wall mechanics, and maladaptive cellular remodeling. Disease progression was driven by three mechanistically interpretable parameters governing excess smooth muscle production, remodeling activation, and passive stiffening. These parameters were calibrated to longitudinal monocrotaline (MCT) measurements of pressure, wall thickness, and stiffness from prior work using a multiobjective optimization. To show the predictive value of this model, we simulated therapeutic intervention within the same disease-specific framework by using functional cell-level responses to therapy to inform changes in parameter values. Calibration to the study-specific MCT dataset reproduced the temporal increases in pressure, wall thickness, and stiffness, demonstrating that the model could capture multiple features of vascular remodeling simultaneously, with R2 values of 0.81, 0.83, and 0.95, respectively. Simulated treatment reduced pressure, wall thickness, and stiffness. Predicted pressure and wall-thickness responses agreed closely with the corresponding experimental treatment effects, whereas stiffness recovery was overpredicted, suggesting that additional mechanisms may contribute to persistent vascular stiffening after intervention. The framework also captured the overall progression of pulmonary pressure increases across both aggregated MCT and Sugen-hypoxia datasets, suggesting utility across studies and animal models. This work outlines a physics-based, multiscale framework that simulated quantities of direct clinical interest in a mechanistically interpretable platform for linking pulmonary vascular remodeling and treatment response. It supports comparisons across experimental phenotypes and interventions while identifying where constitutive refinements are needed to improve predictive capability across phenotypes.

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Sensory and motor neurons differ significantly in their morphology, structural organization, functional properties, and mode of action. However, despite this heterogeneity, many in vitro studies focus only on a single neuronal subtype, mainly being sensory neurons, limiting the translational potential and relevance of these studies for the evaluation of therapeutic options for spinal cord injury. In this study, we investigate the differentiation, maturation, and neuronal outgrowth of human induced pluripotent stem cell (iPSC)-derived motor and sensory neurospheres using polyethylene glycol (PEG)-microgels with various bioactive coatings. Our results show subtype-specific responses to the PEG-microgel scaffolds, with respect to motor and sensory neurosphere morphology and size. Furthermore, we compare the formation of the PEG-microgel/scaffolds when starting from iPSCs-derived precursor neuron spheres versus undifferentiated iPSCs. We observe notable differences in structural organization, maturity, and neuronal outgrowth between the two approaches, as well as between motor and sensory neurospheres. Together, these results underline the importance of studying motor and sensory neurons separately and highlight the need for a controlled, tunable culture platform to assess the impact of the microenvironment and to improve the physiological relevance of in vitro platforms for neuron-based research.

Neurodegenerative diseases are increasingly associated with vascular dysfunction beyond progressive neuronal degeneration, yet how vascular pathology contributes to disease progression remains poorly understood, largely due to the lack of a neurodegenerative disease model capable of capturing neuronal pathology alongside associated vascular dysfunction. Here, we developed a microengineered 3D vascularized brain tissue model that integrates neurospheroids with a self-assembled, perfusable vascular network to recapitulate key features of the neurovascular interface. Using this model, we investigated the vascular contribution to Parkinson's disease pathology by introducing -synuclein preformed fibrils into the engineered vasculature. Intravascular -syn fibril exposure induced endothelial barrier disruption, vascular leakage, inflammation, and vascular regression. Notably, this vascular insult was accompanied by intraneuronal -synuclein aggregation within neurospheroids, suggesting that vascular dysfunction may facilitate the exposure of neural tissue to pathogenic -synuclein. Our neurodegenerative disease modeling approach establishes a versatile and tractable platform for investigating vascular contributions to neurodegenerative disease progression.

Current tuberculosis (TB) treatment suffers from drawbacks such as long regimens, high pill burden and side effects leading to non-adherence and poor treatment outcomes. Dissolution-controlled drug depot formulation with high drug loading is a clinically successful drug delivery strategy. Such depots reduce the dosing frequency for treatments requiring daily administration, thereby improving treatment adherence and compliance. However, dissolution-controlled depots for first-line TB drugs have not been demonstrated due to their high solubility and high dose requirements. In this study, we overcame this challenge by developing injectable, extended-release, dissolution-controlled depots of nanocrystalline rifapentine (NCRPT), microcrystalline rifapentine (MCRPT) and amorphous rifapentine microparticles (ARPT) with more than 75% loading. Crystalline formulations resulted in much slower depot dissolution compared to amorphous formulations. A single intramuscular (IM) injection of NCRPT in mice resulted in therapeutic serum concentrations for over a week. We then demonstrated the efficacy of NCRPT in both pre-exposure prophylaxis and therapeutic models of mice TB. NCRPT administered at 60 mg/kg once every two weeks demonstrated excellent efficacy in a mouse model of TB infection. In each case, a [~] 4-log-fold reduction in lung bacterial load compared to untreated mice was observed. These results open new avenues for developing LAI formulations of TB drugs and could improve patient compliance and TB management.

We present an active mixing toolhead for extrusion bioprinting. The tool enables the programmable fabrication of tumor microenvironment gradient arrays, through controlled deposition of mixed hydrogel precursor formulations into 384-well plates, pre-seeded with tumor cells. It operates on an open-source bioprinter and can actively mix arbitrary ratios of two hydrogel precursors prior to extrusion. These concentration gradient arrays are compatible with quantitative image analysis of cell viability and morphological responses to hydrogels conditioned with drug or extracellular matrix (ECM) proteins. The tools capacity to mix and print hydrogel precursor gradients was demonstrated using alginate and highly concentrated mCherry-conjugated mini-spidroin solutions. Hydrogel precursor stocks contained fluorescent reporters to facilitate quantifications of mixing efficiency, and as proxies for drug and ECM protein concentrations. The tool was applied to generate hydrogel-based gradients of the apoptosis-inducer staurosporine, from which concentration-dependent MDA-MB-231 breast cancer cell death responses were quantified. Gradient arrays of the ECM protein laminin-511, implicated in breast cancer tumorigenesis, were generated and revealed that increasing laminin-511 concentrations potentiated staurosporine-induced cell death. The study demonstrates the utility of this active mixing toolhead for producing hydrogel gradient arrays, and demonstrates the relevance of studying drug-responses in tumor microenvironment models that account for disease-specific ECM components.

BackgroundContinuous glucose monitoring (CGM) systems are used not only for retrospective assessment of the glycemic profile but also for real-time decision-making, including automated insulin delivery. Accordingly, CGM performance characterization must capture not only the agreement of individual paired values but also the systems ability to reproduce the direction, rate, amplitude, and shape of glucose concentration change. Summary metrics, most notably MARD, cannot establish whether an observed deviation reflects an error in the formation of the test profile itself, a constant sensor offset, amplitude compression, a change in response rate, temporal misalignment, or hysteresis. ObjectiveTo adapt a programmable flow-based in vitro platform for the separate assessment of the experimentally delivered glucose profile and the dynamic response of CGM systems, and to propose a set of metrics that decomposes dynamic error into its components. MethodsGLU profiles were generated by programmable mixing of solutions at a constant total flow rate of 2 mL/min. Actual GLU concentration was independently measured with a SUPER GL2 glucose analyzer. Four static levels, three repeats of a 5.5[->]12.0[->]5.5 mmol/L profile, three repeats of a 6.0[->]3.0[->]6.0 mmol/L hypoglycemic profile, three 5.0[->]15.0[->]5.0 mmol/L profiles at different rates, one complex 4[->]18[->]3[->]12[->]5.5 mmol/L profile, and two proof-of-concept sensor experiments at 100- and 200-min transitions were investigated. Dynamic response was characterized by bias, MAE, RMSE, MARD, amplitude transfer coefficient K_A, rate transfer coefficients K_up and K_down, normalized shape RMSE, residual shift, and hysteresis loop area. ResultsAt the static levels, measured GLU exceeded the programmed value by 0.234-0.780 mmol/L. In the repeated 5.5[->]12.0[->]5.5 profiles, the ratio of actual to programmed rate was 0.978-1.083 on the rising phase and 0.987-1.157 on the falling phase, while the amplitude transfer coefficient was 0.967-1.066. In the hypoglycemic profile, minimum GLU was 2.55- 2.96 mmol/L, and time below 3.0 mmol/L was 15.2-72.6 min. The measured rates of 0.0519, 0.1045, and 0.2027 mmol/L/min preserved the intended ratio of approximately 1:2:4. In the complex profile, the programmed plateau of 18 mmol/L was not reached: mean measured GLU was 16.20 mmol/L. For CGM-A, K_A was 0.682 and 0.650, and K_up/K_down were 0.666/0.730 and 0.634/0.626; the corresponding values for CGM-B were 1.228 and 1.128, and 1.564/1.328 and 1.276/1.145. Hysteresis loop area differed 5- to 10-fold between the two sensor responses, exceeding an order of magnitude at the 100-min transition. ConclusionThe programmed concentration should be treated as a control setpoint, rather than as a reference measurement. The "programmed trajectory -- measured glucose -- CGM output" cascade first allows quantitative assessment of the agreement between the programmed and actually realized profile and only then separate characterization of sensor response. Decomposition of dynamic error into amplitude, rate, shape, and hysteresis components reveals differences that a single MARD value or correlation coefficient cannot capture.

Activation of the STING pathway can induce potent antitumor immunity, but effective delivery of STING agonists to the tumor while limiting systemic exposure remains challenging. We previously developed MUSIC, an ultrasound-guided platform that uses microbubbles (MBs) to deliver the STING agonist 2'3'-cGAMP and locally activate antitumor immunity. However, the vascular confinement of MBs and the need for intratumoral administration limit the potential for systemic tumor targeting. To overcome these limitations, we developed SONATA (Systemic Oncotherapy using Nanobubbles for Acoustically-guided Tumor Activation), which employs nanobubbles (NBs) that are approximately 10-fold smaller than conventional MBs, enabling systemic administration and tumor extravasation. Following NB accumulation within tumors, ultrasound exposure triggers localized cGAMP release, facilitating delivery to targeted CD11b+ antigen-presenting cells (APCs) and STING activation with spatial and temporal control. NBs are composed of the same components as MBs, including phospholipid shells and a perfluorobutane core and are functionalized with anti-CD11b antibodies to target CD11b+ APCs and spermine-modified dextran to stably load cGAMP through nanocomplex formation. Upon ultrasound activation, SONATA induced phosphorylation of STING, TBK1, and IRF3 and increased IFN-{beta} production in bone marrow-derived macrophages. In an orthotopic breast cancer model, intravenously administered SONATA combined with tumor-localized ultrasound significantly inhibited tumor growth compared with controls. Furthermore, SONATA synergized with immune checkpoint blockade prolonged the median survival of tumor-bearing mice. Collectively, these findings establish SONATA as a systemically administered immunotherapy platform that enables ultrasound-guided, spatially controlled STING activation.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Chronic inflammation drives persistent systemic cytokine signaling that contributes to vascular dysfunction and secondary vasculitis, yet mechanistic studies are limited by models that fail to capture the multicellular architecture and dynamics of human arteries. In contrast, perfusing intact vessels ex vivo has limited tractability because of material availability and difficulty of genetic or biochemical manipulation. We developed a modular, perfused artery-on-a-chip platform by tri-axially bioprinting primary human vascular cells to recapitulate the concentric organization of the intimal, medial, and adventitial layers. The engineered vessels are viable longer than 21 days, with functional endothelial barriers, contractile smooth muscle behavior, and actively remodeled extracellular matrices bearing hallmarks of native vascular tissue. Addition of tumor necrosis factor alpha (TNF) induces altered transcript levels of proinflammatory mediators and secretion of cytokines and matrix-remodeling enzymes without compromising vessel viability. Importantly, this secretory response is effectively attenuated by both a small-molecule JAK1 inhibitor (ABT-317) and anti-TNF antibody (Infliximab), demonstrating the models utility for therapeutic evaluation.

Immune-epithelial interactions govern the initiation and progression of airway diseases, yet their heterogeneity is difficult to capture using existing in vitro models. Although conventional Transwell and lung-on-chip systems reproduce airway compartmentalisation and permit epithelial-immune interactions, they lack the spatial and analytical resolution needed to visualise dynamic immune behaviour during infection. Here, we present the "Single Cell resolved Airway-Immune Recruitment" (scAIR) platform designed to interrogate immune-epithelial interactions during airway infection. The scAIR device features a modular central chamber accommodating a Transwell insert with primary airway epithelial cells (AECs) pre-differentiated under air-liquid interface (ALI), flanked by immune compartments connected through a precision-patterned microchannel array. This architecture enables real-time single-cell imaging of immune cell migration while preserving epithelial physiology. The scAIR device coupled with a machine learning analysis (MLA) pipeline enables automated tracking and quantification of individual immune cell speed, direction, and behavioural heterogeneity. Using this platform, respiratory syncytial virus (RSV) infection is modelled to generate a type 1 inflammatory airway epithelium that drives neutrophil recruitment. TNF-alpha neutralisation with adalimumab reveals distinct migratory behaviours that are obscured by population-averaged measurements. This integrated platform quantifies airway immune responses during infection and therapeutic modulation, enabling mechanistic studies, drug evaluation, and precision modelling of airway inflammation.

Nonunion fractures remain a costly and persistent challenge in regenerative medicine, with current treatments limited by donor-site morbidity, restricted graft availability, and severe adverse effects associated with supraphysiological bone morphogenetic protein 2 (BMP-2) delivery, including ectopic ossification and inflammation. Endogenous BMP-2 signaling is tightly regulated in adult tissues, constraining the precision and scalability of approaches based on transcriptional upregulation or bolus growth factor administration. To address these limitations, we developed a two-phase integrated computational-experimental pipeline for the de novo design of protein binders targeting the BMP-2 knuckle epitope, a receptor-binding surface corresponding to BMPR-II engagement, enabling affinity-tuned modulation of BMP-2 activity rather than uncontrolled pathway activation. Phase I employed PyRosetta-based {beta}-strand motif grafting and physics-based docking protocols to generate 264 candidate binders, followed by deep-learning-driven refinement in Phase II using partial RFDiffusion and ProteinMPNN with AlphaFold2 validation, yielding 22 candidates with stable {beta}-sheet architectures consistent with knuckle-epitope targeting. Experimental validation demonstrated dose-dependent BMP-2 binding, with the lead construct exhibiting an apparent KD of 2.07 nM toward BMP-2. Targeted alanine substitutions revealed differential residue contributions, with mutation of T42 significantly disrupting binding, while other substitutions had more modest effects, indicating a partially hotspot-driven interface supported by other interactions.

Lung-targeted delivery of mRNA with lipid nanoparticles (LNPs) demonstrates high potential for therapeutic applications in pulmonary disorders. However, progress in pulmonary mRNA therapeutics is constrained by the challenges of engineering lipids that are both safe and highly effective at targeting the lungs. To meet these critical needs, we designed triazine-based (TZ) ionizable lipids with cyanuric chloride as the linker between the cationic head and the lipophilic tail, which allows for easy derivatization capable of systemic mRNA delivery. Three TZ-based lipids were synthesized using the same ionizable headgroups while differing in the carbon tail length and evaluated for their in vitro and in vivo protein expression. Notably, all three lipids result in pulmonary expression after intravenous administration, but the TZ lipid containing a C14 tail does so without any indication of thrombosis, both in vitro and in vivo as compared to other formulations. Our findings highlight the effect of minor chemical modifications driving altered in vivo activity, thus enabling new opportunities for safe pulmonary delivery of mRNA for lung-related diseases.

Ulcerative colitis (UC) is a global health challenge driven by immune dysregulation and gut microbiota imbalance.1 Current treatments, limited by insufficient efficacy and systemic toxicity during prolonged use, fail to resolve the intertwined immune-microbial pathology.2 Here, we report an orally administered self-assembled hydrogel C2-(IIRR)2I-NH2 (CIR), engineered from host defense peptides, which disrupts the immune-microbiota entanglement. The CIR hydrogel exhibits structural transformation at the inflamed sites rich in liposaccharide (LPS), a pro-inflammatory molecule derived from pathogenic bacteria. Stable {beta}-sheet nanofibers can transfer to bioactive -helix conformations, enabling localized therapeutic action with minimal off-target toxicity. In murine colitis models, CIR restores mucosal integrity and suppresses disease severity, outperforming the first-line drug 5-aminosalicylic acid (5-ASA). Microbiome profiling reveals its capacity to rebalance gut microbiota, depleting LPS produced pathogenic bacteria like Prevotellaceae. Transcriptomic analyses further indicate that CIR silences TLR4-mediated signaling pathway. By synergistically targeting immune dysregulation and microbial dysbiosis, this self-assembled peptide hydrogel establishes a paradigm-shifting strategy for UC, offering clinically translatable potential for multifactorial gastrointestinal disorders.

In 1992, inhaled NO (iNO) at low doses entered the practice of medicine for cardiopulmonary indications. Recently, iNO at higher doses has been tested in diverse pulmonary infections. However, nothing is known about the ability of exogenous NO gas to kill Mycobacterium tuberculosis (Mtb), the leading cause of death from infection between major viral pandemics. Here we mimicked exposure conditions used in recent human studies of high-dose iNO to explore the effects of NO gas against Mtb in vitro and in mice. We saw a profound bactericidal effect of NO gas in vitro against Mtb incubated in shallow, mildly acidic fluid. Mtb-infected mice tolerated inhaled NO well, except for developing more methemoglobinemia than humans at the same level of exposure. In Mtb-infected mice with poorly aerated pulmonary infiltrates, inhaled NO had an anti-inflammatory effect but did not reduce the bacterial burden. These results may help inform the decision whether to test inhaled NO as an adjunctive treatment for tuberculosis, and if so, in what settings and with what goals.