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Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a “molecular grammar” for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or "membraneless organelles" such as nucleoli, stress granules, and P-bodies. Because of their propensity to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of a novel enzymatic activity in protein coacervates with the light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of Caenorhabditis elegans LAF-1, we functionalized condensates with enzymatic activity in vitro and show that enzyme localization to coacervates enhances assembly and activity of split enzymes. To build condensates that function as light-emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and displayed significantly increased reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. Because our model condensates have low viscosities, we predict NanoLuc diffusion-limited behavior in most other condensates and thus propose the condensate-Nanoluc system as a potential strategy for high-throughput screening of condensate targeting drugs. By splitting the NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. In addition, we demonstrate control of the spatial localization of the enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity and that this activity can be spatially and temporally controlled via biochemical reconstitution and design of protein surfactants.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to ninety percent of a targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division, and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

Cells contain membraneless compartments that assemble due to liquid-liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bio-inspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non-phase separating domain. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core, and the amphiphilic protein forming the surface film layer. Importantly, we found that MBP-based amphiphiles are surfactants and control droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations co-assemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. The resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

The material properties of biomolecular condensates play pivotal roles in many biological and pathological processes. Despite the rapid increase in the number of biomolecules identified that undergo liquid-liquid phase separation (LLPS), quantitative studies of the resulting condensates have been severely lagging behind. Here, we develop a micropipette-based technique, which uniquely allows quantifications of both the surface tension and viscosity of biomolecular condensates, independent of labeling and surface wetting effects. We demonstrate the accuracy and versatility of this technique by measuring condensates of LAF-1 RGG domains and a polymer-based aqueous two-phase system (ATPS). We anticipate this technique will be widely applicable to biomolecular condensates and will resolve several limitations regarding current approaches.

This perspective article highlights recent progress and emerging challenges in understanding the formation and function of membraneless organelles (MLOs). A long-term goal in the MLO field is to identify the sequence-encoded rules that dictate the formation of compositionally controlled biomolecular condensates, which cells utilize to perform a wide variety of functions. The molecular organization of the different components within a condensate can vary significantly, ranging from a homogeneous mixture to core-shell droplet structures. We provide many examples to highlight the richness of the observed behavior and potential research directions for improving our mechanistic understanding. The tunable environment within condensates can, in principle, alter enzymatic activity significantly. We examine recent examples where this was demonstrated, including applications in synthetic biology. An important question about MLOs is the role of liquid-like material properties in biological function. We discuss the need for improved quantitative characterization tools and the development of sequence-structure-dynamics relationships.

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

Diffusion through biological gels is crucial for effective drug delivery using nanoparticles. Here, we demonstrate a new method to measure diffusivity over a large range of length scales – from tens of nanometers to tens of micrometers – using photoactivatable fluorescent nanoparticle probes. We have applied this method to investigate the length-scale dependent mobility of nanoparticles in fibrin gels and in sputum from patients with cystic fibrosis (CF). Nanoparticles composed of poly(lactic-co-glycolic acid), with polyethylene glycol coatings to resist bioadhesion, were internally labeled with caged rhodamine to make the particles photoactivatable. We activated particles within a region of sample using brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles in that region to become fluorescent. We imaged the subsequent spatiotemporal evolution in fluorescence intensity and observed the collective particle diffusion over tens of minutes and tens of micrometers. We also performed complementary multiple particle tracking experiments on the same particles, extending significantly the range over which particle motion and its heterogeneity can be observed. In fibrin gels, both methods showed an immobile fraction of particles and a mobile fraction that diffused over all measured length scales. In the CF sputum, particle diffusion was spatially heterogeneous and locally anisotropic but nevertheless typically led to unbounded transport extending tens of micrometers within tens of minutes. These findings provide insight into the mesoscale architecture of these gels and its role in setting their permeability on physiologically relevant length scales, pointing toward strategies for improving nanoparticle drug delivery.

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membrane-less organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membrane-less organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bio-inspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. By altering the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation, we designed sequences with significantly increased phase separation propensity. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that in spite of these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance a predictive framework and identify key biophysical principles of sequence features important to phase separation.Significance StatementMembraneless organelles are assemblies of highly concentrated biomolecules that form through a liquid-liquid phase separation process. These assemblies are often enriched in intrinsically disordered proteins, which play an important role in driving phase separation. Understanding the sequence-to-phase behavior relationship of these disordered proteins is important for understanding the biochemistry of membraneless organelles, as well as for designing synthetic organelles and biomaterials. In this work, we explore a model protein, the disordered N-terminal domain of LAF-1, and highlight how three key features of the sequence control the protein’s propensity to phase separate. Combining predictive simulations with experiments, we find that phase behavior of this model IDP is dictated by the presence of a short conserved domain, charge patterning, and arginine-tyrosine interactions.

Significance Light–oxygen–voltage (LOV) domain photoreceptors are found ubiquitously in nature and possess highly diverse signaling roles and mechanisms. Here, we show that a class of fungal LOV proteins dynamically associates with anionic plasma membrane phospholipids by a blue light-switched electrostatic interaction. This reversible association is rapidly triggered by blue light and ceases within seconds when illumination ceases. Within the native host, we predict that these proteins regulate G-protein signaling by the controlled recruitment of fused regulator of G-protein signaling (RGS) domains; in applied contexts, we anticipate that engineered chimeric versions of such proteins will be useful for rapid optogenetic membrane localization of fused proteins through direct interaction with the membrane itself, without requiring additional components to direct subcellular localization., We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells., Designer organelles with new biochemical functionalities are of great interest in synthetic biology and cellular engineering. Here the authors present a single-protein-based platform for generating synthetic membraneless compartments that is capable of enzymatically-triggered alterations to phase behavior and of recruiting and concentrating cargo proteins.

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Particle tracking is a powerful microscopy technique to quantify the motion of individual particles at high spatial and temporal resolution in complex fluids and biological specimens. Particle tracking's applications and impact in drug and gene delivery research have greatly increased during the last decade. Thanks to advances in hardware and software, this technique is now more accessible than ever, and can be reliably automated to enable rapid processing of large data sets, thereby further enhancing the role that particle tracking will play in drug and gene delivery studies in the future. We begin this review by discussing particle tracking-based advances in characterizing extracellular and cellular barriers to therapeutic nanoparticles and in characterizing nanoparticle size and stability. To facilitate wider adoption of the technique, we then present a user-friendly review of state-of-the-art automated particle tracking algorithms and methods of analysis. We conclude by reviewing technological developments for next-generation particle tracking methods, and we survey future research directions in drug and gene delivery where particle tracking may be useful.

Debunking the mucoadhesion myth: Nonsticky particles for enhanced pulmonary drug delivery., Mucoadhesive particles (MAP) have been widely explored for pulmonary drug delivery because of their perceived benefits in improving particle residence in the lungs. However, retention of particles adhesively trapped in airway mucus may be limited by physiologic mucus clearance mechanisms. In contrast, particles that avoid mucoadhesion and have diameters smaller than mucus mesh spacings rapidly penetrate mucus layers [mucus-penetrating particles (MPP)], which we hypothesized would provide prolonged lung retention compared to MAP. We compared in vivo behaviors of variously sized, polystyrene-based MAP and MPP in the lungs following inhalation. MAP, regardless of particle size, were aggregated and poorly distributed throughout the airways, leading to rapid clearance from the lungs. Conversely, MPP as large as 300 nm exhibited uniform distribution and markedly enhanced retention compared to size-matched MAP. On the basis of these findings, we formulated biodegradable MPP (b-MPP) with an average diameter of

Poor drug distribution and short drug half-life within tumors strongly limit efficacy of chemotherapies in most cancers, including primary brain tumors. Local or targeted drug delivery via controlled-release polymers is a promising strategy to treat infiltrative brain tumors, which cannot be completely removed surgically. However, drug penetration is limited with conventional local therapies since small-molecule drugs often enter the first cell they encounter and travel only short distances from the site of administration. Nanoparticles that avoid adhesive interactions with the tumor extracellular matrix may improve drug distribution and sustain drug release when applied to the tumor area. We have previously shown model polystyrene nanoparticles up to 114 nm in diameter were able to rapidly diffuse in normal brain tissue, but only if coated with an exceptionally dense layer of poly(ethylene glycol) (PEG) to reduce adhesive interactions. Here, we demonstrate that paclitaxel (PTX)-loaded, poly(lactic-co-glycolic acid) (PLGA)-co-PEG block copolymer nanoparticles with an average diameter of 70 nm were able to diffuse 100-fold faster than similarly sized PTX-loaded PLGA particles (without PEG coatings). Densely PEGylated PTX-loaded nanoparticles significantly delayed tumor growth following local administration to established brain tumors, as compared to PTX-loaded PLGA nanoparticles or unencapsulated PTX. Delayed tumor growth combined with enhanced distribution of drug-loaded PLGA-PEG nanoparticles to the tumor infiltrative front demonstrates that particle penetration within the brain tumor parenchyma improves therapeutic efficacy. The use of drug-loaded brain-penetrating nanoparticles is a promising approach to achieve sustained and more uniform drug delivery to treat aggressive gliomas and potentially other brain disorders.

Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.

A major role of respiratory mucus is to trap inhaled particles, including pathogens and environmental particulates, to limit body exposure. Despite the tremendous health implications, how particle size and surface chemistry affect mobility in respiratory mucus from humans without lung disease is not known. We prepared polymeric nanoparticles densely coated with low molecular weight polyethylene glycol (PEG) to minimize muco-adhesion, and compared their transport to that of uncoated particles in human respiratory mucus, which we collected from the endotracheal tubes of surgical patients with no respiratory comorbidities. We found that 100 and 200 nm diameter PEG-coated particles rapidly penetrated respiratory mucus, at rates exceeding their uncoated counterparts by approximately 15- and 35-fold, respectively. In contrast, PEG-coated particles ≥ 500 nm in diameter were sterically immobilized by the mucus mesh. Thus, even though respiratory mucus is a viscoelastic solid at the macroscopic level (as measured using a bulk rheometer), nanoparticles that are sufficiently small and muco-inert can penetrate the mucus as if it were primarily a viscous liquid. These findings help elucidate the barrier properties of respiratory mucus and provide design criteria for therapeutic nanoparticles capable of penetrating mucus to approach the underlying airway epithelium.

Clinical manifestations of cystic fibrosis (CF) result from an increase in the viscosity of the mucus secreted by epithelial cells that line the airways. Particle-tracking microrheology (PTM) is a widely accepted means of determining the viscoelastic properties of CF mucus, providing an improved understanding of this disease as well as an avenue to assess the efficacies of pharmacologic therapies aimed at decreasing mucus viscosity. Among its advantages, PTM allows the measurement of small volumes, which was recently utilized for an in situ study of CF mucus formed by airway cell cultures. Typically, particle tracks are obtained from fluorescence microscopy video images, although this limits one's ability to distinguish particles by depth in a heterogeneous environment. Here, by performing PTM with high-resolution micro-optical coherence tomography ( μ OCT), we were able to characterize the viscoelastic properties of mucus, which enables simultaneous measurement of rheology with mucociliary transport parameters that we previously determined using μ OCT. We obtained an accurate characterization of dextran solutions and observed a statistically significant difference in the viscosities of mucus secreted by normal and CF human airway cell cultures. We further characterized the effects of noise and imaging parameters on the sensitivity of μ OCT-PTM by performing theoretical and numerical analyses, which show that our system can accurately quantify viscosities over the range that is characteristic of CF mucus. As a sensitive rheometry technique that requires very small fluid quantities, μ OCT-PTM could also be generally applied to interrogate the viscosity of biological media such as blood or the vitreous humor of the eye in situ.

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.

Rationale: The mechanisms underlying cystic fibrosis (CF) lung disease pathogenesis are unknown. Objectives: To establish mechanisms linking anion transport with the functional microanatomy, we evaluated normal and CF piglet trachea as well as adult swine trachea in the presence of selective anion inhibitors. Methods: We investigated airway functional microanatomy using microoptical coherence tomography, a new imaging modality that concurrently quantifies multiple functional parameters of airway epithelium in a colocalized fashion. Measurements and Main Results: Tracheal explants from wild-type swine demonstrated a direct link between periciliary liquid (PCL) hydration and mucociliary transport (MCT) rates, a relationship frequently invoked but never experimentally confirmed. However, in CF airways this relationship was completely disrupted, with greater PCL depths associated with slowest transport rates. This disrupted relationship was recapitulated by selectively inhibiting bicarbonate transport in vitro and ex vivo. CF mucus exhibited increased viscosity in situ due to the absence of bicarbonate transport, explaining defective MCT that occurs even in the presence of adequate PCL hydration. Conclusions: An inherent defect in CF airway surface liquid contributes to delayed MCT beyond that caused by airway dehydration alone and identifies a fundamental mechanism underlying the pathogenesis of CF lung disease in the absence of antecedent infection or inflammation.

Mucus barriers lining mucosal epithelia reduce the effectiveness of nanocarrier-based mucosal drug delivery and imaging (“theranostics”). Here, we describe liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, e.g., the diaCEST MRI contrast agent barbituric acid (BA). We observed that polyethylene glycol (PEG)-coated liposomes containing ≥ 7 mol% PEG diffused only ~ 10-fold slower in human cervicovaginal mucus (CVM) compared to their theoretical speeds in water. 7 mol%-PEG liposomes contained sufficient BA loading for diaCEST contrast, and provided improved vaginal distribution compared to 0 and 3 mol%-PEG liposomes. However, increasing PEG content to ~ 12 mol% compromised BA loading and vaginal distribution, suggesting that PEG content must be optimized to maintain drug loading and stability. Non-invasive diaCEST MRI illustrated uniform vaginal coverage and longer retention of BA-loaded 7 mol%-PEG liposomes compared to unencapsulated BA. Liposomal MPP with optimized PEG content hold promise for drug delivery and imaging at mucosal surfaces. From the Clinical Editor This team of authors characterized liposome-based mucus-penetrating particles (MPP) capable of loading hydrophilic agents, such as barbituric acid (a diaCEST MRI contrast agent) and concluded that liposomal MPP with optimized PEG coating enables drug delivery and imaging at mucosal surfaces.

Paradigm-shifting research has revealed that certain intrinsically disordered proteins (IDPs) phase separate into spherical liquid droplets that act as membrane-less organelles in living cells. Here, we investigated protease activity as a strategy for triggering controlled droplet disassembly without changing temperature, salt concentration, or other variables that are difficult to manipulate in vivo. We selected a naturally occurring phase-separating IDP, the RGG domain from LAF-1, and produced a variant consisting of two RGG domains in tandem. This RGG tandem protein exhibits enhanced phase behavior - that is, it self-assembles into liquid droplets at lower protein concentration and higher temperature as compared to a single RGG domain. We incorporated a thrombin cleavage site between the two RGG domains of this construct and discovered that upon treatment of the RGG tandem droplets with thrombin, the droplet diameter shrank at a rate of 0.5 micrometers per hour, as measured by quantitative optical microscopy. This occurred because the single RGG domains that result upon cleavage of RGG tandem with thrombin have weaker phase behavior and do not phase separate under the same conditions as does RGG tandem. Through additional protein engineering, we generated a variant that releases a fluorescent reporter when the protein droplet disassembles upon treatment with protease. This is a modular platform for enzymatically-triggered disassembly and release of proteins from droplets. We envision applications of this concept in protein drug delivery, such as in release of antimicrobial peptides for wound healing, and in controlling the behavior of membrane-less organelles within cells in an inducible manner.

Protective mucus layers serve as the body’s first line of defense at exposed surfaces of the eyes and respiratory, gastrointestinal, and cervicovaginal tracts. These highly viscoelastic and adhesive mucus gels trap most foreign pathogens and environmental ultrafine particles, which are then removed by mucus clearance mechanisms[1] (on the order of seconds to a few hours, depending on anatomical site). However, mucus also immobilizes and rapidly clears therapeutic nanoparticles, including synthetic drug carriers,[2] and clinically tested viral[3] and nonviral gene vectors[4] and, therefore, represents a critical obstacle to localized drug and gene delivery at mucosal surfaces for the treatment of a variety of diseases.[1b]

Diffusion through biological gels is crucial for effective nanoparticle drug delivery. In this work, we develop a method to measure diffusivity over a large range of length scales -- from tens of nanometers to tens of microns -- using photoactivatable nanoparticle probes. Nanoparticles composed of block copolymers of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) were synthesized and confirmed to possess dense PEG coatings that resist bioadhesion, and these particles were conjugated with caged rhodamine to make them photoactivatable. Using confocal microscopy, we activated a region of these particles with a brief, targeted exposure to UV light, uncaging the rhodamine and causing the particles to become fluorescent. We observed their collective diffusion over tens of minutes and tens of microns, thus obtaining a measure of their diffusivity. This technique is complementary to traditional multiple particle tracking (MPT); it extends the range over which particle motion can be directly observed. We confirmed the accuracy of this technique with reference to MPT measurements and the known diffusivity of particles in water. We applied the method to a model fibrin gel system, and found that both our method and MPT measurements show an immobile fraction of particles and mobile fraction that diffuses over long scales. Finally, we examined nanoparticle diffusion in sputum collected from cystic fibrosis patients, and we measured particle diffusion over distances relevant to drug delivery in the lungs. Coupled with traditional MPT, this technique enables multiscale characterization of particle mobility in complex biological fluids