Particle-Blood Dynamics: Adhesion of Vascular-Targeted Pegylated Particles and Blood Cells in Flow.

Vascular targeting is a viable strategy for the therapeutic intervention of inflammatory diseases such as cardiovascular disease. The multiple components of blood (red blood cell, leukocytes, platelets, and plasma) create a complex transport process for optimizing tissue specific targeting. Attaching poly(ethylene glycol) (PEG) spacers to a drug carrier’s surface (PEGylation) is often proposed as a strategy for avoiding systemic clearance by minimizing plasma protein adsorption onto the drug carrier. However, it is unclear as to whether PEGylation influences ligand-based tissue targeting in blood flow. Additionally, both platelets and leukocytes localize and interact with the vessel wall during inflammation. Little is known whether the presence of inflammation targeting drug carriers in blood or at the vessel wall influences the homeostatic interaction of leukocytes and platelets with the same targeted tissue. Here, I investigate the dynamics between the non-red cell components of blood and vascular targeted carriers; specifically, how PEGylation influences carrier adhesion in blood and whether inflammation targeting drug carriers affect leukocyte and platelet adhesion to the same targeted tissue. The addition of a 5.5 kDa or 10 kDa PEG spacer to a model drug carrier improved targeting to a model vessel wall in laminar human blood flow, but only for an unfavorable targeting system (aICAM-1/ICAM-1). While PEGylation was unable to neutralize a negative plasma effect on the adhesion of an actual drug delivery system (poly(lactic-co-glycolic) acid spheres) in human blood, a high PEG surface density maintained the adhesiveness of a model drug carrier in pig blood. Also, high blood concentrations of 500 nm-5 µm targeted spheres were found to reduce the adhesion of leukocytes to the same targeted tissue under high shear conditions, with nearly a 100% reduction in leukocyte adhesion with 5 µm and 3 µm targeted spheres. This work contributes to the design and application of targeted drug carriers by recommending a 5.5 kDa or 10 kDa PEG spacer for targeting high shear areas associated with cardiovascular disease and identifying a new application for inflammation targeting particles through reducing leukocyte accumulation to inflammatory tissue.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111454/1/ponyskjr_1.pd

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Effect of PEGylation on Ligand-Based Targeting of Drug Carriers to the Vascular Wall in Blood Flow

The blood vessel wall plays a prominent role in the development of many life-threatening diseases and as such is an attractive target for treatment. To target diseased tissue, particulate drug carriers often have their surfaces modified with antibodies or epitopes specific to vascular wall-expressed molecules, along with poly­(ethylene glycol) (PEG) to improve carrier blood circulation time. However, little is known about the effect of poly­(ethylene glycol) on carrier adhesion dynamicsspecifically in blood flow. Here we examine the influence of different molecular weight PEG spacers on particle adhesion in blood flow. Anti-ICAM-1 or Sialyl Lewis^a were grafted onto polystyrene 2 μm and 500 nm spheres via PEG spacers and perfused in blood over activated endothelial cells at physiological shear conditions. PEG spacers were shown to improve, reduce, or have no effect on the binding density of targeted-carriers depending on the PEG surface conformation, shear rate, and targeting moiety

Differential Impact of Plasma Proteins on the Adhesion Efficiency of Vascular-Targeted Carriers (VTCs) in Blood of Common Laboratory Animals

Vascular-targeted carrier (VTC) interaction with human plasma is known to reduce targeted adhesion efficiency in vitro. However, the role of plasma proteins on the adhesion efficiency of VTCs in laboratory animals remains unknown. Here, in vitro blood flow assays are used to explore the effects of plasma from mouse, rabbit, and porcine on VTC adhesion. Porcine blood exhibited a strong negative plasma effect on VTC adhesion while no significant plasma effect was found with rabbit and mouse blood. A brush density poly­(ethylene glycol) (PEG) on VTCs was effective at improving adhesion of microsized, but not nanosized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as better models in preclinical research for predicting the in vivo functionality of VTCs for use in humans. These considerations hold great importance for the design of various pharmaceutical products and development of reliable drug delivery systems

Plasma protein corona modulates the vascular wall interaction of drug carriers in a material and donor specific manner.

The nanoscale plasma protein interaction with intravenously injected particulate carrier systems is known to modulate their organ distribution and clearance from the bloodstream. However, the role of this plasma protein interaction in prescribing the adhesion of carriers to the vascular wall remains relatively unknown. Here, we show that the adhesion of vascular-targeted poly(lactide-co-glycolic-acid) (PLGA) spheres to endothelial cells is significantly inhibited in human blood flow, with up to 90% reduction in adhesion observed relative to adhesion in simple buffer flow, depending on the particle size and the magnitude and pattern of blood flow. This reduced PLGA adhesion in blood flow is linked to the adsorption of certain high molecular weight plasma proteins on PLGA and is donor specific, where large reductions in particle adhesion in blood flow (>80% relative to buffer) is seen with ∼60% of unique donor bloods while others exhibit moderate to no reductions. The depletion of high molecular weight immunoglobulins from plasma is shown to successfully restore PLGA vascular wall adhesion. The observed plasma protein effect on PLGA is likely due to material characteristics since the effect is not replicated with polystyrene or silica spheres. These particles effectively adhere to the endothelium at a higher level in blood over buffer flow. Overall, understanding how distinct plasma proteins modulate the vascular wall interaction of vascular-targeted carriers of different material characteristics would allow for the design of highly functional delivery vehicles for the treatment of many serious human diseases

Adhesion of sLe^a–coated PS spheres or anti-ICAM-coated PLGA spheres to activated HUVEC under various flow conditions.

<p>(A) Adhesion of 5 µm sLe^a–coated PS spheres in laminar whole blood and buffer flows to activated HUVEC at 200 s⁻¹ for 7 human subjects. N = 2 (distinct trials) for each blood bar. (B) Average adhesion of 5 µm anti-ICAM-coated PLGA spheres to activated HUVEC from laminar buffer, plasma, or whole blood flow of three low PLGA binding donors at 200 s⁻¹. Laminar flow was run for 5 min. Particle concentration in flow  = 5e5 spheres/mL. sLe^a density  = 1,800+/−200 sites/µm² (SEM) and anti-ICAM-1 density  = 3500+/−500 sites/µm² (SEM). #  =  Not significant with respect to the whole blood trial. N = 3 distinct trials (donors) for the plasma and blood flow assays.</p

Phase and fluorescence images of small PLGA spheres.

<p>Phase image of 1.4 µm PLGA particles in PBS+/+ (A) and plasma (B), and fluorescence image of 330 nm PLGA in PBS+/+ (C) and plasma (D). Particles were added to the desired medium for 5 min in static after which a small amount of the particle solution is placed on a coverslip for imaging. All images shown were taken at a 40× magnification. Scale bar  = 10 µm.</p

Summary of the adhesion of PLGA spheres from flow of buffer, plasma and blood to an activated endothelial cell monolayer.

<p>(A) A depiction of particle margination in buffer, plasma and blood flow. Binding density after 5 min of flow for (B) 5 µm, (C) 1.4 µm and (D) 300 nm sLe^a-coated pLGA particles to activated HUVEC monolayer from laminar buffer, plasma, or whole blood at 200 s⁻¹. Particle concentration  = 5e5 particles/mL for 5 and 1.4 µm data and 1e6 particles/mL for the 330 nm particles. sLe^a density  = 1,700+/−100 sites/µm² (SEM) surface for 5 µm, 1500+/−100 sites/µm² (SEM) for 1.4 µm and 9,000+/−400 sites/µm² (SEM) for 330 nm particles. N = 3 distinct donors (donors A, B and C).</p

Sample images of activated HUVEC monolayers exposed to PLGA in different flow mediums.

<p>Phase image of 1.4 µm sLe^a-coated PLGA spheres bound to IL1-β-activated HUVEC monolayer after 5 min of flow of particles in (A) RBC-in-Buffer, (B) whole blood, and (C) plasma at 200 s⁻¹. Image taken at 20× magnification. sLe^a density  = 1500+/−100 sites/µm² (SEM). Particle concentration  = 5e5 particles/mL. Scale bar  = 20 µm</p

The average mean particle diameter (Z-average) and polydispersity indexes (PDI) for PLGA spheres in filtered phosphate buffer saline with calcium and magnesium ions (PBS+/+) are measured via dynamic light scattering (DLS) technique using a Malvern Zetasizer Nano ZSP equipped with a back scattering detector (173 degrees).

<p>The average mean particle diameter (Z-average) and polydispersity indexes (PDI) for PLGA spheres in filtered phosphate buffer saline with calcium and magnesium ions (PBS+/+) are measured via dynamic light scattering (DLS) technique using a Malvern Zetasizer Nano ZSP equipped with a back scattering detector (173 degrees).</p