Nanoparticle-neutrophils interactions for autoimmune regulation.

Neutrophils play an essential role as 'first responders' in the immune response, necessitating many immune-modulating capabilities. Chronic, unresolved inflammation is heavily implicated in the progression and tissue-degrading effects of autoimmune disease. Neutrophils modulate disease pathogenesis by interacting with the inflammatory and autoreactive cells through effector functions, including signaling, degranulation, and neutrophil extracellular traps (NETs) release. Since the current gold standard systemic glucocorticoid administration has many drawbacks and side effects, targeting neutrophils in autoimmunity provides a new approach to developing therapeutics. Nanoparticles enable targeting of specific cell types and controlled release of a loaded drug cargo. Thus, leveraging nanoparticle properties and interactions with neutrophils provides an exciting new direction toward novel therapies for autoimmune diseases. Additionally, recent work has utilized neutrophil properties to design novel targeted particles for delivery into previously inaccessible areas. Here, we outline nanoparticle-based strategies to modulate neutrophil activity in autoimmunity, including various nanoparticle formulations and neutrophil-derived targeting.

Selective Blood Cell Hitchhiking in Whole Blood with Ionic Liquid-Coated PLGA Nanoparticles to Redirect Biodistribution After Intravenous Injection.

Less than 5% of intravenously-injected nanoparticles (NPs) reach destined sites in the body due to opsonization and immune-based clearance in vascular circulation. By hitchhiking in situ onto specific blood components post-injection, NPs can selectively target tissue sites for unprecedentedly high drug delivery rates. Choline carboxylate ionic liquids (ILs) are biocompatible liquid salts <100X composed of bulky asymmetric cations and anions. This class of ILs has been previously shown to significantly extend circulation time and redirect biodistribution in BALB/c mice post-IV injection via hitchhiking on red blood cell (RBC) membranes. Herein, we synthesized & screened 60 choline carboxylic acid-based ILs to coat PLGA NPs and present the impact of structurally engineering the coordinated anion identity to selectively interface and hitchhike lymphocytes, monocytes, granulocytes, platelets, and RBCs in whole mouse blood for in situ targeted drug delivery. Furthermore, we find this nanoparticle platform to be biocompatible (non-cytotoxic), translate to human whole blood by resisting serum uptake and maintaining modest hitchhiking, and also significantly extend circulation retention over 24 hours in BALB/c healthy adult mice after IV injection. Because of their altered circulation profiles, we additionally observe dramatically different organ accumulation profiles compared to bare PLGA NPs. This study establishes an initial breakthrough platform for a modular and transformative targeting technology to hitchhike onto blood components with high efficacy and safety in the bloodstream post-IV administration.

CAR-neutrophil mediated delivery of tumor-microenvironment responsive nanodrugs for glioblastoma chemo-immunotherapy.

Glioblastoma (GBM) is one of the most aggressive and lethal solid tumors in human. While efficacious therapeutics, such as emerging chimeric antigen receptor (CAR)-T cells and chemotherapeutics, have been developed to treat various cancers, their effectiveness in GBM treatment has been hindered largely by the blood-brain barrier and blood-brain-tumor barriers. Human neutrophils effectively cross physiological barriers and display effector immunity against pathogens but the short lifespan and resistance to genome editing of primary neutrophils have limited their broad application in immunotherapy. Here we genetically engineer human pluripotent stem cells with CRISPR/Cas9-mediated gene knock-in to express various anti-GBM CAR constructs with T-specific CD3ζ or neutrophil-specific γ-signaling domains. CAR-neutrophils with the best anti-tumor activity are produced to specifically and noninvasively deliver and release tumor microenvironment-responsive nanodrugs to target GBM without the need to induce additional inflammation at the tumor sites. This combinatory chemo-immunotherapy exhibits superior and specific anti-GBM activities, reduces off-target drug delivery and prolongs lifespan in female tumor-bearing mice. Together, this biomimetic CAR-neutrophil drug delivery system is a safe, potent and versatile platform for treating GBM and possibly other devastating diseases.

Cargo-free particles divert neutrophil-platelet aggregates to reduce thromboinflammation.

The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.

Intravascularly infused extracellular matrix as a biomaterial for targeting and treating inflamed tissues.

Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Polymeric particle-based therapies for acute inflammatory diseases.

Acute inflammation is essential for initiating and coordinating the body's response to injuries and infections. However, in acute inflammatory diseases, inflammation is not resolved but propagates further, which can ultimately lead to tissue damage such as in sepsis, acute respiratory distress syndrome and deep vein thrombosis. Currently, clinical protocols are limited to systemic steroidal treatments, fluids and antibiotics that focus on eradicating inflammation rather than modulating it. Strategies based on stem cell therapeutics and selective blocking of inflammatory molecules, despite showing great promise, still lack the scalability and specificity required to treat acute inflammation. By contrast, polymeric particle systems benefit from uniform manufacturing at large scales while preserving biocompatibility and versatility, thus providing an ideal platform for immune modulation. Here, we outline design aspects of polymeric particles including material, size, shape, deformability and surface modifications, providing a strategy for optimizing the targeting of acute inflammation.

Dual Coating of Chitosan and Albumin Negates the Protein Corona-Induced Reduced Vascular Adhesion of Targeted PLGA Microparticles in Human Blood.

Vascular-targeted carriers (VTCs) have the potential to localize therapeutics and imaging agents to inflamed, diseased sites. Poly (lactic-co-glycolic acid) (PLGA) is a negatively charged copolymer commonly used to construct VTCs due to its biodegradability and FDA approval. Unfortunately, PLGA VTCs experienced reduced adhesion to inflamed endothelium in the presence of human plasma proteins. In this study, PLGA microparticles were coated with chitosan (CS), human serum albumin (HSA), or both (HSA-CS) to improve adhesion. The binding of sialyl Lewis A (a ligand for E-selectin)-targeted PLGA, HSA-PLGA, CSPLGA, and HSA-CSPLGA to activated endothelial cells was evaluated in red blood cells in buffer or plasma flow conditions. PLGA VTCs with HSA-only coating showed improvement and experienced 35-52% adhesion in plasma compared to plasma-free buffer conditions across all shear rates. PLGA VTCs with dual coating-CS and HSA-maintained 80% of their adhesion after exposure to plasma at low and intermediate shears and ≈50% at high shear. Notably, the protein corona characterization showed increases at the 75 and 150 kDa band intensities for HSA-PLGA and HSA-CSPLGA, which could correlate to histidine-rich glycoprotein and immunoglobulin G. The changes in protein corona on HSA-coated particles seem to positively influence particle binding, emphasizing the importance of understanding plasma protein-particle interactions.

Effect of the Emulsion Solvent Evaporation Technique Cosolvent Choice on the Loading Efficiency and Release Profile of Anti-CD47 from PLGA Nanospheres.

INTRODUCTION: Side effects associated with using antibodies as therapeutics can limit systemic administration at the high concentrations often needed for therapeutic impact. Thus, therapeutic antibodies are usually considered for targeted delivery. Antibody encapsulation in polymeric nanoparticles via the emulsion-based nanofabrication methods typically yields low loading efficiencies. Therefore, the fabrication techniques need to be modified to maximize the loading efficiency of antibodies. In this work, we utilized various cosolvents with the emulsion solvent evaporation technique to improve the loading efficiency of anti-CD47, a therapeutic antibody used to block CD47 activity in atherosclerotic plaques and cancer lesions. METHODS AND RESULTS: The double emulsion solvent evaporation technique was used to fabricate anti-CD47-loaded polymeric nanoparticles. The primary oil phase solvent, chloroform, was doped with different cosolvents, including ethyl acetate, acetonitrile, ethanol, and methanol, to investigate the impact of cosolvents on the loading efficiency of anti-CD47. The release profile and loading efficiency were quantified by measuring the fluorescence signal of the released antibody. The activity of the antibody released from particles fabricated in the presence of the cosolvent was confirmed by quantifying its adherence to red blood cells. Ethyl acetate was the optimum cosolvent, improving the loading efficiency of anti-CD47 in poly(lactic-co-glycolic acid), PLGA, nanoparticles to 90% or higher, and the antibody was found to retain its activity after being released from nanoparticles. CONCLUSION: Our results demonstrate that a minimum amount of a cosolvent with minimal hydrophilicity can stabilize the antibody in the oil phase; thus, improving the antibody's loading efficiency significantly.

PLGA's Plight and the Role of Stealth Surface Modification Strategies in Its Use for Intravenous Particulate Drug Delivery.

Numerous human disorders can benefit from targeted, intravenous (IV) drug delivery. Polymeric nanoparticles have been designed to undergo systemic circulation and deliver their therapeutic cargo to target sites in a controlled manner. Poly(lactic-co-glycolic) acid (PLGA) is a particularly promising biomaterial for designing intravenous drug carriers due to its biocompatibility, biodegradability, and history of clinical success across other routes of administration. Despite these merits, PLGA remains markedly absent in clinically approved IV drug delivery formulations. A prominent factor in PLGA particles' inability to succeed intravenously may lie in the hydrophobic character of the polyester, leading to the adsorption of serum proteins (i.e., opsonization) and a cascade of events that end in their premature clearance from the bloodstream. PEGylation, or surface-attached polyethylene glycol chains, is a common strategy for shielding particles from opsonization. Polyethylene glycol (PEG) continues to be regarded as the ultimate "stealth" solution despite the lack of clinical progress of PEGylated PLGA carriers. This review reflects on some of the reasons for the clinical failure of PLGA, particularly the drawbacks of PEGylation, and highlights alternative surface coatings on PLGA particles. Ultimately, a new approach will be needed to harness the potential of PLGA nanoparticles and allow their widespread clinical adoption.

Polysalicylic Acid Polymer Microparticle Decoys Therapeutically Treat Acute Respiratory Distress Syndrome.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain problematic due to high mortality rates and lack of effective treatments. Neutrophilic injury contributes to mortality in ALI/ARDS. Here, technology for rapid ARDS intervention is developed and evaluated, where intravenous salicylic acid-based polymer microparticles, i.e., Poly-Aspirin (Poly-A), interfere with neutrophils in blood, reducing lung neutrophil infiltration and injury in vivo in mouse models of ALI/ARDS. Importantly, Poly-A particles reduce multiple inflammatory cytokines in the airway and bacterial load in the bloodstream in a live bacteria lung infection model of ARDS, drastically improving survival. It is observed that phagocytosis of the Poly-A microparticles, with salicylic acid in the polymer backbone, alters the neutrophil surface expression of adhesion molecules, potentially contributing to their added therapeutic benefits. Given the proven safety profile of the microparticle degradation products-salicylic acid and adipic acid-it is anticipated that the Poly-A particles represent a therapeutic strategy in ARDS with a rare opportunity for rapid clinical translation.

Method article: an in vitro blood flow model to advance the study of platelet adhesion utilizing a damaged endothelium.

In vitro flow assays utilizing microfluidic devices are often used to study human platelets as an alternative to the costly animal models of hemostasis and thrombosis that may not accurately represent human platelet behavior in vivo. Here, we present a tunable in vitro model to study platelet behavior in human whole blood flow that includes both an inflamed, damaged endothelium and exposed extracellular matrix. We demonstrate that the model is adaptable across various anticoagulants, shear rates, and proteins for endothelial cell culture without the need for a complicated, custom-designed device. Furthermore, we verified the ability of this 'damaged endothelium' model as a screening method for potential anti-platelet or anti-thrombotic compounds using a P2Y12 receptor antagonist (ticagrelor), a pan-selectin inhibitor (Bimosiamose), and a histamine receptor antagonist (Cimetidine). These compounds significantly decreased platelet adhesion to the damaged endothelium, highlighting that this model can successfully screen anti-platelet compounds that target platelets directly or the endothelium indirectly.

Deformable microparticles for shuttling nanoparticles to the vascular wall.

Vascular-targeted drug carriers must localize to the wall (i.e., marginate) and adhere to a diseased endothelium to achieve clinical utility. The particle size has been reported as a critical physical property prescribing particle margination in vitro and in vivo blood flows. Different transport process steps yield conflicting requirements-microparticles are optimal for margination, but nanoparticles are better for intracellular or tissue delivery. Here, we evaluate deformable hydrogel microparticles as carriers for transporting nanoparticles to a diseased vascular wall. Depending on microparticle modulus, nanoparticle-loaded poly(ethylene glycol)-based hydrogel microparticles delivered significantly more 50-nm nanoparticles to the vessel wall than freely injected nanoparticles alone, resulting in >3000% delivery increase. This work demonstrates the benefit of optimizing microparticles' efficient margination to enhance nanocarriers' transport to the vascular wall.

Characterizing bulk rigidity of rigid red blood cell populations in sickle-cell disease patients.

In this work, we utilized a parameterization model of ektacytometry to quantify the bulk rigidity of the rigid red blood cell (RBC) population in sickle cell disease (SCD) patients. Current ektacytometry techniques implement laser diffraction viscometry to estimate the RBC deformability in a whole blood sample. However, the diffraction measurement is an average of all cells present in the measured sample. By coupling an existing parameterization model of ektacytometry to an artificially rigid RBC model, we formulated an innovative system for estimating the average rigidity of the rigid RBC population in SCD blood. We demonstrated that this method could more accurately determine the bulk stiffness of the rigid RBC populations. This information could potentially help develop the ektacytometry technique as a tool for assessing disease severity in SCD patients, offering novel insights into the disease pathology and treatment.

Fund Black scientists.

Our nationwide network of BME women faculty collectively argue that racial funding disparity by the National Institutes of Health (NIH) remains the most insidious barrier to success of Black faculty in our profession. We thus refocus attention on this critical barrier and suggest solutions on how it can be dismantled.

Biodegradable, bile salt microparticles for localized fat dissolution.

Bile acids are proposed as therapeutic agents for various diseases, including liver diseases and obesity. However, oral or subcutaneous administration of a solubilized version of these drugs has limited efficacy and imposes unwanted side effects. Here, we describe a gold-templating method for fabricating stable, bile salt-cholate or deoxycholate-microparticles. The gold ions' reduction at the oil-water interface in a double emulsion solvent evaporation process enables a gold-bile salt interaction and the formation of bile salt particles. We demonstrate that composite microparticles release cholate/deoxycholate into solution via a surface erosion process. We illustrate these particles' capability to lyse adipocytes, both in vitro and in vivo, with minimal side effects, contrary to the Food and Drug Administration-approved salt solution that leads to severe inflammation and ulceration. Overall, particle-based cholate/deoxycholate opens opportunities for localized delivery of these salts, improving efficacy while minimizing side effects associated with oral and subcutaneous use.

Neutrophils preferentially phagocytose elongated particles-An opportunity for selective targeting in acute inflammatory diseases.

Polymeric particles have recently been used to modulate the behavior of immune cells in the treatment of various inflammatory conditions. However, there is little understanding of how physical particle parameters affect their specific interaction with different leukocyte subtypes. While particle shape is known to be a crucial factor in their phagocytosis by macrophages, where elongated particles are reported to experience reduced uptake, it remains unclear how shape influences phagocytosis by circulating phagocytes, including neutrophils that are the most abundant leukocyte in human blood. In this study, we investigated the phagocytosis of rod-shaped polymeric particles by human neutrophils relative to other leukocytes. In contrast to macrophages and other mononuclear phagocytes, neutrophils were found to exhibit increased internalization of rods in ex vivo and in vivo experimentation. This result suggests that alteration of particle shape can be used to selectively target neutrophils in inflammatory pathologies where these cells play a substantial role.

The influence of red blood cell deformability on hematocrit profiles and platelet margination.

The influence of red blood cell (RBC) deformability in whole blood on platelet margination is investigated using confocal microscopy measurements of flowing human blood and cell resolved blood flow simulations. Fluorescent platelet concentrations at the wall of a glass chamber are measured using confocal microscopy with flowing human blood containing varying healthy-to-stiff RBC fractions. A decrease is observed in the fluorescent platelet signal at the wall due to the increase of stiffened RBCs in flow, suggesting a decrease of platelet margination due to an increased fraction of stiffened RBCs present in the flow. In order to resolve the influence of stiffened RBCs on platelet concentration at the channel wall, cell-pair and bulk flow simulations are performed. For homogeneous collisions between RBC pairs, a decrease in final displacement after a collision with increasing membrane stiffness is observed. In heterogeneous collisions between healthy and stiff RBC pairs, it is found that the stiffened RBC is displaced most. The influence of RBC deformability on collisions between RBCs and platelets was found to be negligible due to their size and mass difference. For a straight vessel geometry with varying healthy-to-stiff RBC ratios, a decrease was observed in the red blood cell-free layer and platelet margination due to an increase in stiffened RBCs present in flow.

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by ß2 Integrin Mac-1.

OBJECTIVE: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion. METHODS: Patients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures. RESULTS: Upon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, ß2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01). CONCLUSION: APS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.

Model Particulate Drug Carriers Modulate Leukocyte Adhesion in Human Blood Flows.

Drug carriers have been widely explored as a method of improving the efficacy of therapeutic drugs for a variety of diseases, including those involving inflammation. However, few of these formulations have advanced past clinical trials. There are still major gaps in our understanding of how drug carriers impact leukocytes, particularly in inflammatory conditions. In this work, we investigated how targeted and nontargeted drug carriers affect the function of leukocytes in blood flow. We explored three primary mechanisms: (1) collisions in blood flow disrupt leukocyte adhesion, (2) specific binding to the endothelium competes with leukocytes for binding sites, and (3) particle phagocytosis alters leukocyte phenotype, resulting in reduced adhesion. We find that each of these mechanisms contributes to significantly reduced leukocyte adhesion to an inflamed endothelium, and that particle phagocytosis may be the most significant driver of this effect. These results are crucial for understanding the totality of the impact of drug carriers on leukocyte behavior and response to inflammation and should inform the future design of any such drug carriers.