Decoupling Fluorous Protein Coatings Yield Heat-Stable and Intrinsically Sterile Bioformulations.

Thermal inactivation is a major bottleneck to the scalable production, storage, and transportation of protein-based reagents and therapies. Failures in temperature control both compromise protein bioactivity and increase the risk of microorganismal contamination. Herein, we report the rational design of fluorochemical additives that promiscuously bind to and coat the surfaces of proteins to enable their stable dispersion within fluorous solvents. By replacing traditional aqueous liquids with fluorinated media, this strategy conformationally rigidifies proteins to preserve their structure and function at extreme temperatures (≥90 °C). We show that fluorous protein formulations resist contamination by bacterial, fungal, and viral pathogens, which require aqueous environments for survival, and display equivalent serum bioavailability to standard saline samples in animal models. Importantly, by designing dispersants that decouple from the protein surface in physiologic solutions, we deliver a fluorochemical formulation that does not alter the pharmacologic function or safety profile of the functionalized protein in vivo. As a result, this nonaqueous protein storage paradigm is poised to open technological opportunities in the design of shelf-stable protein reagents and biopharmaceuticals.

Real-Time, In Situ Imaging of Macrophages via Phase-Change Peptide Nanoemulsions.

Macrophages are specialized phagocytes that play central roles in immunity and tissue repair. Their diverse functionalities have led to an evolution of new allogenic and autologous macrophage products. However, realizing the full therapeutic potential of these cell-based therapies requires development of imaging technologies that can track immune cell migration within tissues in real-time. Such innovations will not only inform treatment regimens and empower interpretation of therapeutic outcomes but also enable prediction and early intervention during adverse events. Here, phase-changing nanoemulsion contrast agents are reported that permit real-time, continuous, and high-fidelity ultrasound imaging of macrophages in situ. Using a de novo designed peptide emulsifier, liquid perfluorocarbon nanoemulsions are prepared and show that rational control over interfacial peptide assembly affords formulations with tunable acoustic sensitivity, macrophage internalization, and in cellulo stability. Imaging experiments demonstrate that emulsion-loaded macrophages can be readily visualized using standard diagnostic B-mode and Doppler ultrasound modalities. This allows on-demand and long-term tracking of macrophages within porcine coronary arteries, as an exemplary model. The results demonstrate that this platform is poised to open new opportunities for non-invasive, contrast-enhanced imaging of cell-based immunotherapies in tissues, while leveraging the low-cost, portable, and safe nature of diagnostic ultrasound.

Deciphering the Mechanistic Basis for Perfluoroalkyl-Protein Interactions.

Although rarely used in nature, fluorine has emerged as an important elemental ingredient in the design of proteins with altered folding, stability, oligomerization propensities, and bioactivity. Adding to the molecular modification toolbox, here we report the ability of privileged perfluorinated amphiphiles to noncovalently decorate proteins to alter their conformational plasticity and potentiate their dispersion into fluorous phases. Employing a complementary suite of biophysical, in-silico and in-vitro approaches, we establish structure-activity relationships defining these phenomena and investigate their impact on protein structural dynamics and intracellular trafficking. Notably, we show that the lead compound, perfluorononanoic acid, is 106 times more potent in inducing non-native protein secondary structure in select proteins than is the well-known helix inducer trifluoroethanol, and also significantly enhances the cellular uptake of complexed proteins. These findings could advance the rational design of fluorinated proteins, inform on potential modes of toxicity for perfluoroalkyl substances, and guide the development of fluorine-modified biologics with desirable functional properties for drug discovery and delivery applications.

Heparin-Peptide Nanogranules for Thrombosis-Actuated Anticoagulation.

Despite nearly a century of clinical use as a blood thinner, heparin's rapid serum clearance and potential to induce severe bleeding events continue to urge the development of more effective controlled delivery strategies. Subcutaneous depots that steadily release the anticoagulant into circulation represent a promising approach to reducing overdose frequency, sustaining therapeutic concentrations of heparin in plasma, and prolonging anticoagulant activity in a safe and effective manner. Subcutaneously deliverable heparin-peptide nanogranules that allow for long-lasting heparin bioavailability in the circulatory system, while enabling on-demand activation of heparin's anticoagulant effects in the thrombus microenvironment, are reported. Biophysical studies demonstrate this responsive behavior is due to the sequestration of heparin within self-assembling peptide nanofibrils and its mechanically actuated decoupling to elicit antithrombotic effects at the clotting site. In vivo studies show these unique properties converge to allow subcutaneous nanogranule depots to extend heparin serum concentrations for an order of magnitude longer than standard dosing regimens while enabling prolonged and controlled anticoagulant activity. This biohybrid delivery system demonstrates a potentially scalable platform for the development of safer, easier to administer, and more effective antithrombotic nanotechnologies.

Inhalable SARS-CoV-2 Mimetic Particles Induce Pleiotropic Antigen Presentation.

Coronavirus disease 2019 (Covid-19) has caused over 5.5 million deaths worldwide, and viral mutants continue to ravage communities with limited access to injectable vaccines or high rates of vaccine hesitancy. Inhalable vaccines have the potential to address these distribution and compliance issues as they are less likely to require cold storage, avoid the use of needles, and can elicit localized immune responses with only a single dose. Alveolar macrophages represent attractive targets for inhalable vaccines as they are abundant within the lung mucosa (up to 95% of all immune cells) and are important mediators of mucosal immunity, and evidence suggests that they may be key cellular players in early Covid-19 pathogenesis. Here, we report inhalable coronavirus mimetic particles (CoMiP) designed to rapidly bind to, and be internalized by, alveolar macrophages to deliver nucleic acid-encoded viral antigens. Inspired by the SARS-CoV-2 virion structure, CoMiP carriers package nucleic acid cargo within an endosomolytic peptide envelope that is wrapped in a macrophage-targeting glycosaminoglycan coating. Through this design, CoMiP mimic several important features of the SARS-CoV-2 virion, particularly surface topography and macromolecular chemistry. As a result, CoMiP effect pleiotropic transfection of macrophages and lung epithelial cells in vitro with multiple antigen-encoding plasmids. In vivo immunization yields increased mucosal IgA levels within the respiratory tract of CoMiP vaccinated mice.

Extracellular matrix-inspired inhalable aerogels for rapid clearance of pulmonary tuberculosis.

Tuberculosis (TB) remains a leading cause of death from a single infectious agent, and limiting the spread of multidrug-resistant TB (MDR-TB) is now an urgent global health priority. Essential to the persistence of this disease is the ability of Mycobacterium tuberculosis (Mtb) to circumvent host defenses by infecting lung macrophages to create a cellular niche for its survival and proliferation. This has urged the development of new therapeutic strategies that act through mechanisms distinct from conventional antibiotics, and thus are effective against MDR bacteria, while being able to efficiently kill persister Mtb cells in infected host macrophages. Here, we report a new class of gel-like microparticle aerosols, or 'aerogels', designed to exploit metabolic vulnerabilities of Mtb pathogens and TB-infected macrophages to enable preferential delivery of synergistic peptide-antibiotic combinations for potent and rapid antitubercular therapy. This is achieved by formulating aerogels through the supramolecular assembly of a de novo designed anti-TB peptide and the extracellular matrix (ECM)-derived polysaccharide, hyaluronic acid (HA). Importantly, HA serves as a nutrient source for Mtb cells during tissue invasion and proliferation, and is recognized by CD44 receptors highly expressed on lung macrophages during TB infection. By exploiting this metabolic substrate for pathogen targeting, HA aerogels are shown to avidly bind and kill both drug-sensitive and drug-resistant mycobacteria, while being efficiently internalized into macrophage host cells in vitro and in vivo to clear Mtb persisters. This multifaceted bioactivity suggests aerogels may serve as a versatile inhalable platform upon which novel biomaterials-enabled therapeutics can be developed to rapidly clear pulmonary MDR-TB.

Tuning the Interfacial Properties of Fluorous Colloids Toward Ultrasound Programmable Bioactivity.

Liquid-in-liquid emulsions are kinetically stable colloids that undergo liquid-to-gas phase transitions in response to thermal or acoustic stimuli. Perfluorocarbons (PFCs) are preferred species as their highly fluorinated nature imparts unique properties that are unparalleled by nonfluorinated counterparts. However, traditional methods to prepare PFC emulsions lack the ability to precisely tune the thermodynamic stability of the fluorous-water interphase and consequently control their vaporization behavior. Here, we report a privileged fluoroalkanoic acid that undergoes concentration-dependent assembly on the surfaces of fluorous droplets to modulate interfacial tension. This allows for the rational formulation of orthogonal PFC droplets that can be programmed to vaporize at specified ultrasound powers. We exploit this behavior in two exemplary biomedical settings by developing emulsions that aid ultrasound-mediated hemostasis and enable bioorthogonal delivery of molecular sensors to mammalian cells. Mechanistic insights gained from these studies can be generalized to tune the thermodynamic interfacial equilibria of PFC emulsions toward designing controllable tools for precision medicine.

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.

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.

Bioresponsive peptide-polysaccharide nanogels - A versatile delivery system to augment the utility of bioactive cargo.

We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.