Differential uptake of non-fouling particles by primary human neutrophils

The advent of targeted drug carriers has opened many new avenues for the delivery of therapeutics directly to the site of disease, reducing systemic side effects and enhancing the efficacy of therapeutic molecules. However, the packaging of therapeutics into particulate carriers for delivery comes with its own set of challenges and barriers. Among these, a great deal of research effort has focused on protecting carriers from clearance by phagocytes by altering carrier surface chemistry. Many groups have explored the use of polyethylene glycol (PEG) chain coatings to mitigate unwanted phagocytosis, as PEG is highly hydrophilic and is well-known for its anti-fouling propertiesNotably, very few papers have explored the effects of PEG on uptake by freshly obtained primary human phagocytes in physiological conditions, creating a disconnect between the prevailing literature and ultimate applications. In this work, we investigate the effect of PEGylation on uptake by primary human neutrophils in vitro, and compare these effects to several cell lines and other model phagocytic cells systems in evaluating the effects of surface chemistry on phagocytosis. We find that primary human neutrophils preferentially phagocytose PEGylated drug carriers, and that this effect is linked to factors present in human plasma. These findings have major implications for the efficacy of PEGylation in designing long-circulating drug carriers, as well as the need for thorough characterization of drug carrier platforms in a wide array of in vitro and in vivo assays. Please click Additional Files below to see the full abstract

Vascularâ targeted nanocarriers: design considerations and strategies for successful treatment of atherosclerosis and other vascular diseases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134422/1/wnan1414_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134422/2/wnan1414.pd

Experimental evaluation of receptor-ligand interactions of dual-targeted particles to inflamed endothelium

Vascular-targeted carriers (VTCs) are often designed as leukocyte mimics, conjugated with ligands that target leukocyte adhesion molecules (LAMs) to facilitate specific adhesion to diseased endothelium. VTCs must adhere in regions with dynamic blood flow, frequently requiring multiple ligand-receptor (LR) pairs to provide particle adhesion and high disease specificity. To study LR kinetics under flow, multiple research groups have used protein-coated plates to study the adhesion and rolling of dual-targeted particles in vitro.1-4 While important knowledge is contributed by these studies, they lack the complexity of a diseased physiologic endothelium, as spatiotemporal LAM expression varies widely. Despite decades of research with the ambition of mimicking leukocytes, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments. More specifically, there is a lack of mechanistic understanding of how multiple ligands interact with biologically complex endothelial surfaces under dynamic in vivo environments. Please click Additional Files below to see the full abstract

Evaluation of receptorâ ligand mechanisms of dualâ targeted particles to an inflamed endothelium

Vascularâ targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligandâ receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAMâ targeted VTCs remains poorly understood, especially in physiological environments. Here, we investigate particle binding to an inflamed mesentery via intravital microscopy using a series of particles with wellâ controlled ligand properties. We find that the total number of sites of a single ligand can drive particle adhesion to the endothelium, however, combining ligands that target multiple LR pairs provides a more effective approach. Combining sites of sialyl Lewis A (sLeA) and antiâ intercellular adhesion moleculeâ 1 (aICAM), two adhesive molecules, resulted in â ¼3â 7â fold increase of adherent particles at the endothelium over singleâ ligand particles. At a constant total ligand density, a particle with a ratio of 75% sLeA: 25% aICAM resulted in more than 3â fold increase over all over other ligand ratios tested in our in vivo model. Combined with in vivo and in silico data, we find the best dualâ ligand design of a particle is heavily dependent on the surface expression of the endothelial cells, producing superior adhesion with more particle ligand for the lesserâ expressed receptor. These results establish the importance of considering LRâ kinetics in intelligent VTC ligand design for future therapeutics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/1/btm210008-sup-0007-suppinfo07.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/2/btm210008_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/3/btm210008.pd

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by β2 Integrin Macâ 1

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153125/1/art41057.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153125/2/art41057_am.pd

Design of Vascular‐Targeted Carriers for Optimal Performance in Humans: Bringing Blood Cells and Plasma Proteins into the Game

Presented on April 15, 2015 from 4-5 pm in room G011 of the Molecular Science and Engineering Building on the Georgia Tech campus.Runtime: 62:01 minutesOverall, localized delivery of therapeutics offer the possibility of increased drug effectiveness while minimizing side effects often associated with systemic drug administration. Factors that affect the possibility of targeting therapeutics to reach the vascular wall include the ability to 1) identify disease‐specific target epitope expressed by the vascular cells; 2) identify optimum drug carrier type, shape and size for efficient interaction with the vascular wall; and 3) identify a drug‐carrier combination that allow for the effective release of therapeutics at the intended site. Existing literature has focused mainly on identifying target epitopes and the degradation/drug release characteristics of a wide range of drug‐carrier formulations. Our work focuses on elucidating the potential roles of particle shape and size and blood rheology/dynamics on the ability of vascular‐targeted drug carriers to interact with the blood vessel wall – an important consideration that will control the effectiveness of drug targeting regardless of the targeted disease or delivered therapeutic. This presentation will highlight key carrier‐blood cell and carrier‐plasma protein interactions that affect drug carrier binding to the vascular wall and suggest the optimum drug carrier

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells.

BackgroundEndothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.MethodologyWe investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.Results and conclusionNaïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8-12 hr of continuous shear-cytokine activation contrary to the commonly observed 4-6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation

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

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

Cycloheximide (CHX) inhibition of E-selectin synthesis and neutrophil binding assays for static- and shear-cytokine activated monolayers.

<p>(A) E-selectin expression on cycloheximide-treated monolayers. CHX at a non-lethal concentration of 1 µg mL⁻¹ was added to HUVEC monolayers at 4 hr activation time in both static (“SA-CHX,” diagonal stripes) and shear-cytokine (10 dyn cm⁻², “FA-CHX,” horizontal stripes) conditions. Static (“SA,” solid) and shear-cytokine (“FA,” white) controls were not treated with CHX. (B) Neutrophil binding density over HUVEC monolayers activated with 0.1 ng ml⁻¹ IL-1ß under static culture or 10 dyn cm⁻² shear for 4, 8, 12, and 24 hr periods. “NS” indicates no significance, “***,” <i>p</i><0.001, and “**,” <i>p</i><0.05.</p