The Impact of the Plasma Protein Corona on the Adhesion Efficiency of Drug Carriers to the Blood Vessel Wall.

Upon injection of vascular-targeted drug carriers into the bloodstream, plasma proteins rapidly coat the carrier surface, forming a plasma protein corona. This corona is dependent on a host of parameters, including physicochemical particle properties and the plasma composition. Although several studies have identified key roles of the protein corona regarding circulation time, clearance, and biodistribution, the role of the plasma protein corona on the adhesion efficiency of vascular-targeted carriers (VTCs) to inflamed human umbilical vein endothelial cells (HUVECs) in human blood flow remains relatively unknown. In this dissertation, it is observed that the plasma protein corona exerts a negative effect on the adhesion of drug carriers in blood flow; however, the extent of these observations depend on a host of parameters including drug carrier material type, targeting ligand density, flow profile, plasma exposure time, and plasma anticoagulant. Furthermore, the magnitude of the corona-induced negative adhesion effects is shown to be heavily linked to the adsorption of immunoglobulin antibodies in plasma. This work has a variety of important implications for the intelligent design of VTCs. First, the fact that immunoglobulins heavily dictate adhesion reduction of drug carriers offers insight into specific directions to limit the effects of the protein corona. Specifically, tuning the corona to avoid adsorption of these proteins or coating with non-fouling dysopsonin proteins offers a method to maintain targeting efficiency in the presence of corona formation. Second, this work may explain why current targeted drug delivery systems often exhibit poor accumulation to the target site based on the large reduction of particles upon exposure to human plasma. Third, this work could be used to develop strategies to predict or diagnose a specific drug carrier based on its protein corona profile, hopefully leading to more successful translations of drug delivery systems to the market. Overall, this work shows that protein corona is a critical parameter when designing high-efficient targeted drug carriers and may be exploited or eliminated to achieve maximum adhesion specificity in vivo.PhDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133204/1/sobczdan_1.pd

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

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

A Supra-monolayer Nanopattern for Organic Nanoparticle Array Deposition

Nanopatterns have applications in many areas including sensors, optoelectronics, and crystallization screening. Particle lithography is a convenient method to manufacture nanoring nanopatterns based on organosilane surface chemistry. The pattern thickness is generally limited to the monolayer thickness. This work is focused on the chemical vapor deposition conditions that yield nanopatterns with multilayer thickness. The supra-monolayer n-octadecyltrichlorosilane (OTS) nanoring patterns are made using polystyrene particle lithography. The supra-monolayer nanopatterns are used as “nano-flasks” to deposit and nucleate nanoparticles of small organic molecules including <i>n</i>-docosane, aspirin, and clarithromycin. The supra-monolayer OTS nanopattern is an effective template for nanoparticle array deposition of all three chemicals with high degree of fidelity to the substrate pattern. The nanoparticle size is varied by solution concentration. The preferential deposition of the organic molecules inside the nanoring is attributed to the dewetting of the liquid film on the nanopattern. The dewetting process effectively distributes the liquid film among the “nano-flasks” so that millions of solution experiments can be carried out in isolated droplets with droplet volume as small as 1 × 10^–10 nL. The research demonstrates a method to manufacture “nano-flask” arrays for high-throughput nanoparticle deposition trials and manufacture of monodisperse organic/drug nanoparticles through self-assembly

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

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

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

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

Adhesion of PEGylated and non-PEGylated 1.4 µm sLe^a-targeted spheres to HUVEC in laminar buffer or whole blood flow at 200 s⁻¹ (5 min).

<p>A PEG density of 16,000 site/µm² estimated to be the brush conformation is used. sLe^a density  = 1,800+/−200 sites/µm² for both PEGylated and un-PEGylated particles. Particle concentration in flow  = 5e5 spheres/mL.</p