Modulated Fibrosis and Mechanosensing of Fibroblasts by SB525334 in Pediatric Subglottic Stenosis.

OBJECTIVE: Subglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF-ß1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co-regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF-ß and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX). METHODS: Laryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by α-smooth muscle actin immunostaining and gene expression for α-SMA, FN, HGF, and CTGF markers. TGF-ß and Hippo signaling pathways were modulated during TGF-ß1-induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT-qPCR for differential gene expression and atomic force microscopy for ECM stiffness. RESULTS: SGS fibroblasts exhibited higher α-SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF-ß1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro-fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01). CONCLUSION: We demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF-ß1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF-ß and Hippo signaling pathways. LEVEL OF EVIDENCE: NA Laryngoscope, 134:287-296, 2024.

Amelioration of Subglottic Stenosis by Antimicrobial Peptide Eluting Endotracheal Tubes.

Introduction: Pediatric subglottic stenosis (SGS) results from prolonged intubation where scar tissue leads to airway narrowing that requires invasive surgery. We have recently discovered that modulating the laryngotracheal microbiome can prevent SGS. Herein, we show how our patent-pending antimicrobial peptide-eluting endotracheal tube (AMP-ET) effectively modulates the local airway microbiota resulting in reduced inflammation and stenosis resolution. Materials and Methods: We fabricated mouse-sized ETs coated with a polymeric AMP-eluting layer, quantified AMP release over 10 days, and validated bactericidal activity for both planktonic and biofilm-resident bacteria against Staphylococcus aureus and Pseudomonas aeruginosa. Ex vivo testing: we inserted AMP-ETs and ET controls into excised laryngotracheal complexes (LTCs) of C57BL/6 mice and assessed biofilm formation after 24 h. In vivo testing: AMP-ETs and ET controls were inserted in sham or SGS-induced LTCs, which were then implanted subcutaneously in receptor mice, and assessed for immune response and SGS severity after 7 days. Results: We achieved reproducible, linear AMP release at 1.16 µg/day resulting in strong bacterial inhibition in vitro and ex vivo. In vivo, SGS-induced LTCs exhibited a thickened scar tissue typical of stenosis, while the use of AMP-ETs abrogated stenosis. Notably, SGS airways exhibited high infiltration of T cells and macrophages, which was reversed with AMP-ET treatment. This suggests that by modulating the microbiome, AMP-ETs reduce macrophage activation and antigen specific T cell responses resolving stenosis progression. Conclusion: We developed an AMP-ET platform that reduces T cell and macrophage responses and reduces SGS in vivo via airway microbiome modulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00769-9.

Drug-Eluting Endotracheal Tubes for Preventing Bacterial Inflammation in Subglottic Stenosis.

OBJECTIVES/HYPOTHESIS: Subglottic stenosis (SGS) results from dysregulated extracellular matrix deposition by laryngotracheal fibroblasts causing scar tissue formation following intubation. Recent work has highlighted a relationship between this inflammatory state and imbalances in the upper airway microbiome. Herein, we engineer novel drug-eluting endotracheal (ET) tubes to deliver a model antimicrobial peptide Lasioglossin-III (Lasio) for the local modulation of the microbiome during intubation. STUDY DESIGN: Controlled in vitro study. METHODS: ET tubes were coated with a water-in-oil (w/o) emulsion of Lasio in poly(d,l-lactide-co-glycolide) (PLGA) by dipping thrice. Peptide release was quantified over 2 weeks via fluorometric peptide assays. The antibacterial activity was tested against airway microbes (Staphylococcus epidermidis, Streptococcus pneumoniae, and pooled human microbiome samples) by placing Lasio/PLGA-coated tubes and appropriate controls in 48 well plates with diluted bacteria. Bacterial inhibition and tube adhesion were tested by measuring optical density and colony formation after tube culture, respectively. Biocompatibility was tested against laryngotracheal fibroblasts and lung epithelial cells. RESULTS: We achieved a homogeneous coating of ET tubes with Lasio in a PLGA matrix that yields a prolonged, linear release over 1 week (typical timeframe before the ET tube is changed). We observed significant antibacterial activity against S. epidermidis, S. pneumoniae, and human microbiome samples, and prevention of bacterial adherence to the tube. Additionally, the released Lasio did not cause any cytotoxicity toward laryngotracheal fibroblasts or lung epithelial cells in vitro. CONCLUSION: Overall, we demonstrate the design of an effective-eluting ET tube to modulate upper-airway bacterial infections during intubation which could be deployed to help prevent SGS. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1356-1363, 2022.

Drug delivery to the pediatric upper airway.

Pediatric upper airway disorders are frequently life-threatening and require precise assessment and intervention. Targeting these pathologies remains a challenge for clinicians due to the high complexity of pediatric upper airway anatomy and numerous potential etiologies; the most common treatments include systemic delivery of high dose steroids and antibiotics or complex and invasive surgeries. Furthermore, the majority of innovative airway management technologies are only designed and tested for adults, limiting their widespread implementation in the pediatric population. Here, we provide a comprehensive review of the most recent challenges of managing common pediatric upper airway disorders, describe the limitations of current clinical treatments, and elaborate on how to circumvent those limitations via local controlled drug delivery. Furthermore, we propose future advancements in the field of drug-eluting technologies to improve pediatric upper airway management outcomes.

Peptide functionalized liposomes for receptor targeted cancer therapy.

Most clinically approved cancer therapies are potent and toxic small molecules that are limited by severe off-target toxicities and poor tumor-specific localization. Over the past few decades, attempts have been made to load chemotherapies into liposomes, which act to deliver the therapeutic agent directly to the tumor. Although liposomal encapsulation has been shown to decrease toxicity in human patients, reliance on passive targeting via the enhanced permeability and retention (EPR) effect has left some of these issues unresolved. Recently, investigations into modifying the surface of liposomes via covalent and/or electrostatic functionalization have offered mechanisms for tumor homing and subsequently controlled chemotherapeutic delivery. A wide variety of biomolecules can be utilized to functionalize liposomes such as proteins, carbohydrates, and nucleic acids, which enable multiple directions for cancer cell localization. Importantly, when nanoparticles are modified with such molecules, care must be taken as not to inactivate or denature the ligand. Peptides, which are small proteins with <30 amino acids, have demonstrated the exceptional ability to act as ligands for transmembrane protein receptors overexpressed in many tumor phenotypes. Exploring this strategy offers a method in tumor targeting for cancers such as glioblastoma multiforme, pancreatic, lung, and breast based on the manifold of receptors overexpressed on various tumor cell populations. In this review, we offer a comprehensive summary of peptide-functionalized liposomes for receptor-targeted cancer therapy.

Pathogen-specific antimicrobials engineered de novo through membrane-protein biomimicry.

Precision antimicrobials aim to kill pathogens without damaging commensal bacteria in the host, and thereby cure disease without antibiotic-associated dysbiosis. Here we report the de novo design of a synthetic host defence peptide that targets a specific pathogen by mimicking key molecular features of the pathogen's channel-forming membrane proteins. By exploiting physical and structural vulnerabilities within the pathogen's cellular envelope, we designed a peptide sequence that undergoes instructed tryptophan-zippered assembly within the mycolic acid-rich outer membrane of Mycobacterium tuberculosis to specifically kill the pathogen without collateral toxicity towards lung commensal bacteria or host tissue. These mycomembrane-templated assemblies elicit rapid mycobactericidal activity and enhance the potency of antibiotics by improving their otherwise poor diffusion across the rigid M. tuberculosis envelope with respect to agents that exploit transmembrane protein channels for antimycobacterial activity. This biomimetic strategy may aid the design of other narrow-spectrum antimicrobial peptides.

Re-engineering Antimicrobial Peptides into Oncolytics Targeting Drug-Resistant Ovarian Cancers.

INTRODUCTION: Bacteria and cancer cells share a common trait-both possess an electronegative surface that distinguishes them from healthy mammalian counterparts. This opens opportunities to repurpose antimicrobial peptides (AMPs), which are cationic amphiphiles that kill bacteria by disrupting their anionic cell envelope, into anticancer peptides (ACPs). To test this assertion, we investigate the mechanisms by which a pathogen-specific AMP, originally designed to kill bacterial Tuberculosis, potentiates the lytic destruction of drug-resistant cancers and synergistically enhances chemotherapeutic potency. MATERIALS AND METHODS: De novo peptide design, paired with cellular assays, elucidate structure-activity relationships (SAR) important to ACP potency and specificity. Using the sequence MAD1, microscopy, spectrophotometry and flow cytometry identify the peptide's anticancer mechanisms, while parallel combinatorial screens define chemotherapeutic synergy in drug-resistant cell lines and patient derived ex vivo tumors. RESULTS: SAR investigations reveal spatial sequestration of amphiphilic regions increases ACP potency, but at the cost of specificity. Selecting MAD1 as a lead sequence, mechanistic studies identify that the peptide forms pore-like supramolecular assemblies within the plasma and nuclear membranes of cancer cells to potentiate death through lytic and apoptotic mechanisms. This diverse activity enables MAD1 to synergize broadly with chemotherapeutics, displaying remarkable combinatorial efficacy against drug-resistant ovarian carcinoma cells and patient-derived tumor spheroids. CONCLUSIONS: We show that cancer-specific ACPs can be rationally engineered using nature's AMP toolbox as templates. Selecting the antimicrobial peptide MAD1, we demonstrate the potential of this strategy to open a wealth of synthetic biotherapies that offer new, combinatorial opportunities against drug resistant tumors.

Supramolecular Peptide Assemblies as Antimicrobial Scaffolds.

Antimicrobial discovery in the age of antibiotic resistance has demanded the prioritization of non-conventional therapies that act on new targets or employ novel mechanisms. Among these, supramolecular antimicrobial peptide assemblies have emerged as attractive therapeutic platforms, operating as both the bactericidal agent and delivery vector for combinatorial antibiotics. Leveraging their programmable inter- and intra-molecular interactions, peptides can be engineered to form higher ordered monolithic or co-assembled structures, including nano-fibers, -nets, and -tubes, where their unique bifunctionalities often emerge from the supramolecular state. Further advancements have included the formation of macroscopic hydrogels that act as bioresponsive, bactericidal materials. This systematic review covers recent advances in the development of supramolecular antimicrobial peptide technologies and discusses their potential impact on future drug discovery efforts.

Discovery of antitumor lectins from rainforest tree root transcriptomes.

Glycans are multi-branched sugars that are displayed from lipids and proteins. Through their diverse polysaccharide structures they can potentiate a myriad of cellular signaling pathways involved in development, growth, immuno-communication and survival. Not surprisingly, disruption of glycan synthesis is fundamental to various human diseases; including cancer, where aberrant glycosylation drives malignancy. Here, we report the discovery of a novel mannose-binding lectin, ML6, which selectively recognizes and binds to these irregular tumor-specific glycans to elicit potent and rapid cancer cell death. This lectin was engineered from gene models identified in a tropical rainforest tree root transcriptome and is unusual in its six canonical mannose binding domains (QxDxNxVxY), each with a unique amino acid sequence. Remarkably, ML6 displays antitumor activity that is >105 times more potent than standard chemotherapeutics, while being almost completely inactive towards non-transformed, healthy cells. This activity, in combination with results from glycan binding studies, suggests ML6 differentiates healthy and malignant cells by exploiting divergent glycosylation pathways that yield naïve and incomplete cell surface glycans in tumors. Thus, ML6 and other high-valence lectins may serve as novel biochemical tools to elucidate the glycomic signature of different human tumors and aid in the rational design of carbohydrate-directed therapies. Further, understanding how nature evolves proteins, like ML6, to combat the changing defenses of competing microorganisms may allow for fundamental advances in the way we approach combinatorial therapies to fight therapeutic resistance in cancer.

The Ca2+ export pump PMCA clears near-membrane Ca2+ to facilitate store-operated Ca2+ entry and NFAT activation.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+-extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

Lipopeptisomes: Anticancer peptide-assembled particles for fusolytic oncotherapy.

Anticancer peptides (ACPs) are cationic amphiphiles that preferentially kill cancer cells through folding-dependent membrane disruption. Although ACPs represent attractive therapeutic candidates, particularly against drug-resistant cancers, their successful translation into clinical practice has gone unrealized due to their poor bioavailability, serum instability and, most importantly, severe hemolytic toxicity. Here, we exploit the membrane-specific interactions of ACPs to prepare a new class of peptide-lipid particle, we term a lipopeptisome (LP). This design sequesters loaded ACPs within a lipid lamellar corona to avoid contact with red blood cells and healthy tissues, while affording potent lytic destruction of cancer cells following LP-membrane fusion. Biophysical studies show ACPs rapidly fold at, and integrate into, liposomal membranes to form stable LPs with high loading efficiencies (>80%). Rational design of the particles to possess lipid combinations mimicking that of the aberrant cancer cell outer leaflet allows LPs to rapidly fuse with tumor cell membranes and afford localized assembly of loaded ACPs within the bilayer. This leads to preferential fusolytic killing of cancer cells with minimal collateral toxicity towards non-cancerous cells and erythrocytes, thereby imparting clinically relevant therapeutic indices to otherwise toxic ACPs. Thus, integration of ACPs into self-assembled LPs represents a new delivery strategy to improve the therapeutic utility of oncolytic agents, and suggests this technology may be added to targeted combinatorial approaches in precision medicine. STATEMENT OF SIGNIFICANCE: Despite their significant clinical potential, the therapeutic utility of many ACPs has been limited by their collateral hemolysis during administration. Leveraging the membrane-specific interactions of ACPs, here we prepare self-assembled peptide-lipid nanoparticles, or 'lipopeptisomes' (LPs), capable of preferentially fusing with and lysing cancer cell membranes. Key to this fusolytic action is the construction of LPs from lipids simulating the cancer cell outer leaflet. This design recruits the oncolytic peptide payload into the carrier lamella and allows for selective destruction of cancer cells without disrupting healthy cells. Consequently, LPs impart clinically relevant therapeutic indexes to previously toxic ACPs, and thus open new opportunities to improve the clinical translation of oncolytics challenged by narrow therapeutic windows.