Conditional Antimicrobial Peptide Therapeutics.

Antimicrobial peptides (AMPs) constitute a promising class of alternatives to antibiotics to curb antimicrobial resistance. Nonetheless, their utility as a systemic agent is hampered by short circulation time and toxicity. Infection sites, analogous to tumors, harbor an aberrant microenvironment that has the potential to be exploited to develop conditionally activated therapeutics with an improved therapeutic index. In particular, we identified strategies to prolong systemic circulation of small, cationic AMPs in a mouse model of bacterial pneumonia. Specifically, we report an albumin-binding domain (ABD)-AMP conjugate as a long-circulating conditional AMP therapeutic with a masked activity that can be liberated by proteases in the infected tissue microenvironment. Our systemically administered conjugate enhanced the pulmonary delivery of active AMP while also reducing AMP exposure to other off-target organs. Importantly, this reduction in off-target exposure improved the safety profile of the AMP. The framework we present can be generalized to quantify and optimize the performance of this emerging class of conditional therapeutics.

Host protease activity classifies pneumonia etiology.

Community-acquired pneumonia (CAP) has been brought to the forefront of global health priorities due to the COVID-19 pandemic. However, classification of viral versus bacterial pneumonia etiology remains a significant clinical challenge. To this end, we have engineered a panel of activity-based nanosensors that detect the dysregulated activity of pulmonary host proteases implicated in the response to pneumonia-causing pathogens and produce a urinary readout of disease. The nanosensor targets were selected based on a human protease transcriptomic signature for pneumonia etiology generated from 33 unique publicly available study cohorts. Five mouse models of bacterial or viral CAP were developed to assess the ability of the nanosensors to produce etiology-specific urinary signatures. Machine learning algorithms were used to train diagnostic classifiers that could distinguish infected mice from healthy controls and differentiate those with bacterial versus viral pneumonia with high accuracy. This proof-of-concept diagnostic approach demonstrates a way to distinguish pneumonia etiology based solely on the host proteolytic response to infection.

Advances in activity-based diagnostics for infectious disease and microbiome health.

In the human body, pathogens and the endogenous microbiome produce enzymes that aid in replication and survival. The activity from these enzymes as well as energy-dependent transport processes can be used as functional biomarkers for pathogen identification, antimicrobial treatment monitoring, and surveillance of microbiome health. To produce visual and/or quantifiable readouts from this activity, concepts from chemical biology and nanomedicine have been utilized to develop signal-producing probes for patient samples or for direct administration in vivo. In the context of infection, activity-based diagnostics offer several potential advantages over current diagnostics including the ability to differentiate between active infection and sterile inflammation, which is made possible by targeting microbial enzymes with orthogonal activity to that of the host. In this review, we discuss new developments in the making of activity-based infection diagnostics and the beginnings of microbiome activity-based diagnostics.

Selective Permeabilization of Gram-Negative Bacterial Membranes Using Multivalent Peptide Constructs for Antibiotic Sensitization.

The drug-impermeable bacterial membrane in Gram-negative pathogens limits antibiotic access to intracellular drug targets. To expand our rapidly waning antibiotic arsenal, one approach is to improve the intracellular delivery of drugs with historically poor accumulation in Gram-negative bacteria. To do so, we engineered macromolecular potentiators to permeabilize the Gram-negative membrane to facilitate drug influx. Potentiators, known as WD40, were synthesized by grafting multiple copies of a cationic α-helical antimicrobial peptide, WLBU2, onto a dextran polymer scaffold. WD40 enabled drug uptake in the model pathogen P. aeruginosa, a capability that was not observed with unmodified WLBU2 peptide. WD40 was able to reduce minimum inhibitory concentrations of a drug panel by up to 3 orders of magnitude. Hydrophobic and highly three-dimensional antibiotics exhibited the greatest potentiation. Antibiotic activity was potentiated in several clinical strains and resulted in sensitization of drug-resistant strains to rifampin, a drug not previously used for Gram-negative infections.

Fusogenic porous silicon nanoparticles as a broad-spectrum immunotherapy against bacterial infections.

Bacterial infections are re-emerging as substantial threats to global health due to the limited selection of antibiotics that are capable of overcoming antibiotic-resistant strains. By deterring such mutations whilst minimizing the need to develop new pathogen-specific antibiotics, immunotherapy offers a broad-spectrum therapeutic solution against bacterial infections. In particular, pathology resulting from excessive immune response (i.e. fibrosis, necrosis, exudation, breath impediment) contributes significantly to negative disease outcome. Herein, we present a nanoparticle that is targeted to activated macrophages and loaded with siRNA against the Irf5 gene. This formulation is able to induce >80% gene silencing in activated macrophages in vivo, and it inhibits the excessive inflammatory response, generating a significantly improved therapeutic outcome in mouse models of bacterial infection. The versatility of the approach is demonstrated using mice with antibiotic-resistant Gram-positive (methicillin-resistant Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) muscle and lung infections, respectively. Effective depletion of the Irf5 gene in macrophages is found to significantly improve the therapeutic outcome of infected mice, regardless of the bacteria strain and type.

Engineering synthetic breath biomarkers for respiratory disease.

Human breath contains many volatile metabolites. However, few breath tests are currently used in the clinic to monitor disease due to bottlenecks in biomarker identification. Here we engineered breath biomarkers for respiratory disease by local delivery of protease-sensing nanoparticles to the lungs. The nanosensors shed volatile reporters upon cleavage by neutrophil elastase, an inflammation-associated protease with elevated activity in lung diseases such as bacterial infection and alpha-1 antitrypsin deficiency. After intrapulmonary delivery into mouse models with acute lung inflammation, the volatile reporters are released and expelled in breath at levels detectable by mass spectrometry. These breath signals can identify diseased mice with high sensitivity as early as 10 min after nanosensor administration. Using these nanosensors, we performed serial breath tests to monitor dynamic changes in neutrophil elastase activity during lung infection and to assess the efficacy of a protease inhibitor therapy targeting neutrophil elastase for the treatment of alpha-1 antitrypsin deficiency.

PolySTAT-modified chitosan gauzes for improved hemostasis in external hemorrhage.

Positively-charged chitosan gauzes stop bleeding from wounds by electrostatically interacting with negatively-charged cell membranes of erythrocytes to cause erythrocyte agglutination and by sealing wounds through tissue adhesion. In the following work, nonwoven chitosan gauze was impregnated with PolySTAT, a synthetic polymer that enhances coagulation by cross-linking fibrin, to generate PolySTAT/chitosan gauzes with improved hemostatic efficacy. When comparing nonwoven chitosan and PolySTAT/chitosan to a commercially-available chitosan-containing gauze (Celox® Rapid), no appreciable differences were observed in fiber size, morphology, and pore size. However, PolySTAT/chitosan demonstrated more rapid blood absorption compared to Celox® Rapid. In a rat model of femoral artery injury, PolySTAT/chitosan gauzes reduced blood loss and improved survival rate compared to non-hemostatic controls and Celox® Rapid. While Celox® Rapid had stronger adherence to tissues compared to PolySTAT/chitosan gauzes, blood loss was greater due to hematoma formation under the Celox® dressing. Animals treated with PolySTAT/chitosan gauzes required less saline infusion to restore and maintain blood pressure above the target blood pressure (60mmHg) while other treatment groups required more saline due to continued bleeding from the wound. These results suggest that PolySTAT/chitosan gauzes are able to improve blood clotting and withstand increasing arterial pressure with the addition of a fibrin cross-linking hemostatic mechanism. STATEMENT OF SIGNIFICANCE: Blood loss remains one of the leading causes of death after traumatic injury in civilian populations and on the battlefield. Advanced biomaterials that interact with blood components and/or accelerate the clotting process to form a hemostatic plug are necessary to staunch bleeding after injury. Chitosan-based gauzes, which stop bleeding by causing red blood cell aggregation, are currently used on the battlefield and have shown variable performance under high pressure arterial blood flow in animal studies, suggesting that red blood cell aggregates require further mechanical stabilization for more reliable performance. In this work, we investigate the binding and cross-linking of fibrin, a major component in blood clots, on chitosan gauze fiber surfaces to structurally reinforce red blood cell aggregates.

A Fibrin Cross-linking Polymer Enhances Clot Formation Similar to Factor Concentrates and Tranexamic Acid in an in vitro Model of Coagulopathy.

Transfusion of blood components and factor concentrates is clinically used to replenish clotting factors and treat coagulopathy after injury when bleeding is severe. Alternatively, direct manipulation of fibrin polymerization via synthetic cross-linking agents may also improve clot formation during coagulopathic conditions as a novel way to treat coagulopathy. We recently developed a synthetic hemostatic polymer, PolySTAT, that promotes clot formation and stabilizes fibrin network structure by cross-linking fibrin monomers. In this study, we used rotational thromboelastometry (ROTEM) to monitor the effect of PolySTAT on the mechanical strength of clots during clot formation and breakdown in comparison to replacement clotting factors and antifibrinolytics under conditions of simulated trauma-induced coagulopathy (sTIC). Human recombinant activated Factor VII (rFVIIa) shortened clotting onset time and accelerated clotting rate, while tranexamic acid (TXA) eliminated clot lysis and restored maximal clot firmness.In contrast, fibrinogen and PolySTAT were both able to speed up clot formation, increase maximal firmness, and inhibit clot lysis. Furthermore, PolySTAT acted synergistically with TXA and fibrinogen, enhancing their individual effects on clot formation. Thus, manipulating fibrin clot structure by physical cross-linking with a synthetic polymer has beneficial effects on clot formation and may be a viable transfusion strategy for treatment of coagulopathy.

Synthetic Strategies for Engineering Intravenous Hemostats.

While there are currently many well-established topical hemostatic agents for field administration, there are still limited tools to staunch bleeding at less accessible injury sites. Current clinical methods to restore hemostasis after large volume blood loss include platelet and clotting factor transfusion, which have respective drawbacks of short shelf life and risk of viral transmission. Therefore, synthetic hemostatic agents that can be delivered intravenously and encourage stable clot formation after localizing to sites of vascular injury are particularly appealing. In the past three decades, platelet substitutes have been prepared using drug delivery vehicles such as liposomes and PLGA nanoparticles that have been modified to mimic platelet properties. Additionally, structural considerations such as particle size, shape, and flexibility have been addressed in a number of reports. Since platelets are the first responders after vascular injury, platelet substitutes represent an important class of intravenous hemostats under development. More recently, materials affecting fibrin formation have been introduced to induce faster or more stable blood clot formation through fibrin cross-linking. Fibrin represents a major structural component in the final blood clot, and a fibrin-based hemostatic mechanism acting downstream of initial platelet plug formation may be a safer alternative to platelets to avoid undesired thrombotic activity. This Review explores intravenous hemostats under development and strategies to optimize their clotting activity.

A synthetic fibrin cross-linking polymer for modulating clot properties and inducing hemostasis.

Clotting factor replacement is the standard management of acute bleeding in congenital and acquired bleeding disorders. We present a synthetic approach to hemostasis using an engineered hemostatic polymer (PolySTAT) that circulates innocuously in the blood, identifies sites of vascular injury, and promotes clot formation to stop bleeding. PolySTAT induces hemostasis by cross-linking the fibrin matrix within clots, mimicking the function of the transglutaminase factor XIII. Furthermore, synthetic PolySTAT binds specifically to fibrin monomers and is uniformly integrated into fibrin fibers during fibrin polymerization, resulting in a fortified, hybrid polymer network with enhanced resistance to enzymatic degradation. In vivo hemostatic activity was confirmed in a rat model of trauma and fluid resuscitation in which intravenous administration of PolySTAT improved survival by reducing blood loss and resuscitation fluid requirements. PolySTAT-induced fibrin cross-linking is a novel approach to hemostasis using synthetic polymers for noninvasive modulation of clot architecture with potentially wide-ranging therapeutic applications.

Synthesis and characterization of anti-EGFR fluorescent nanoparticles for optical molecular imaging.

Molecular imaging, the visualization of molecular and cellular markers, is a promising method for detection of dysplasia and early cancer in the esophagus and can potentially be used to identify regions of interest for biopsy or tumor margins for resection. EGFR is a previously reported cell surface receptor with stepwise increases in expression during the progression from Barrett's metaplasia to adenocarcinoma. In this work, a 200 nm fluorescent nanoparticle contrast agent was synthesized for targeted imaging of EGFR through a series of surface modifications to dye-encapsulated polystyrene particles. Amino-functionalized polystyrene particles were PEGylated using a heterobifunctional PEG linker. Subsequently, thiolated M225 antibodies were conjugated to maleimide functional groups on attached PEGs for EGFR targeting. In vitro binding studies using flow cytometry demonstrated specific binding of M225-PEG-NP to EGFR-expressing cells with minimal nonspecific binding in EGFR(-) cells. Binding was shown to increase proportionally with the number of conjugated M225 antibodies. Adsorbed formulations with unmodified M225 antibodies, M225 + PEG-NP, were synthesized using the same antibody feeds used in M225-PEG-NP synthesis to determine the contribution of adsorbed antibodies to EGFR targeting. Adsorbed antibodies were less efficient at mediated nanoparticle targeting to EGFR than conjugated antibodies. Finally, M225-PEG-NP demonstrated binding to EGFR-expressing regions in human esophageal tissue sections.

The transduction of Coxsackie and Adenovirus Receptor-negative cells and protection against neutralizing antibodies by HPMA-co-oligolysine copolymer-coated adenovirus.

Adenoviral (AdV) gene vectors offer efficient nucleic acid transfer into both dividing and non-dividing cells. However issues such as vector immunogenicity, toxicity and restricted transduction to receptor-expressing cells have prevented broad clinical translation of these constructs. To address this issue, engineered AdV have been prepared by both genetic and chemical manipulation. In this work, a polymer-coated Ad5 formulation is optimized by evaluating a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-co-oligolysine copolymers synthesized by living polymerization techniques. This synthesis approach was used to generate highly controlled and well-defined polymers with varying peptide length (K(5), K(10) and K(15)), polymer molecular weight, and degradability to coat the viral capsid. The optimal formulation was not affected by the presence of serum during transduction and significantly increased Ad5 transduction of several cell types that lack the Coxsackie and Adenovirus Receptor (CAR) by up to 6-fold compared to unmodified AdV. Polymer-coated Ad5 also retained high transduction capability in the presence of Ad5 neutralizing antibodies. The critical role of heparan sulfate proteoglycans (HSPGs) in mediating cell binding and internalization of polymer-coated AdV was also demonstrated by evaluating transduction in HSPG-defective recombinant CHO cells. The formulations developed here are attractive vectors for ex vivo gene transfer in applications such as cell therapy. In addition, this platform for adenoviral modification allows for facile introduction of alternative targeting ligands.