Elsevier

Biomaterials

Volume 30, Issue 4, February 2009, Pages 603-610
Biomaterials

The influence of protein adsorption on nanoparticle association with cultured endothelial cells

https://doi.org/10.1016/j.biomaterials.2008.09.050Get rights and content

Abstract

As materials are produced at smaller scales, the properties that make them especially useful for biological applications such as drug delivery, imaging or sensing applications also render them potentially harmful. There has been a reasonable amount of work addressing the interactions of biological fluids at material surfaces that demonstrates the high affinity of protein for particle surfaces and some looking at the role of particle surface chemistry in cellular associations, but mechanisms have been too little addressed outside the context of intended, specific interactions. Here, using cultured endothelium as a model for vascular transport, we demonstrate that the capacity of nanoparticle surfaces to adsorb protein is indicative of their tendency to associate with cells. Quantification of adsorbed protein shows that high binding nanoparticles are maximally coated in seconds to minutes, indicating that proteins on particle surfaces can mediate cell association over much longer time scales. We also remove many of the most abundant proteins from culture media which alters the profile of adsorbed proteins on nanoparticles but does not affect the level of cell association. We therefore conclude that cellular association is not dependent on the identity of adsorbed proteins and therefore unlikely to require specific binding to any particular cellular receptors.

Introduction

There is particulate matter in our environment from a wide range of sources; natural (smoke), unintended (pollution, workplace), and purposeful (diagnostic or drug delivery). Further, as technologies improve, nanoparticles are being produced at smaller scales, and with better control of properties. This vast landscape of possible interactions is beyond our resources to map point-by-point, so we seek to determine the properties of nanoparticles that underlie their biological fates by first understanding how particle interactions with cells depend on physical properties of particle surfaces. Along with the multiple sources of nanoparticles, there are also many routes of exposure. Inhalation is the most obvious, but there are also nanoparticles in sunscreen and many cosmetics applied to the skin, in some foods, and being used increasingly in novel applications like waterproofing of textiles and surfaces. As newer techniques render nanoparticles more suitable to in vivo applications [1], more types of particles are being used in medical procedures for diagnostic [2], [3] and therapeutic [4] purposes. Some effects of nanoparticle toxicity can occur at the point of exposure, but research has shown that particles also enter the circulation [5] and so our focus is on binding of proteins from blood plasma and subsequent interactions with endothelial cells, the cell type that lines blood vessels and mediates passage into surrounding tissues. This work is thus relevant to both therapeutic uses of and occupational and environmental exposures to nanoparticles.

Nanoparticles introduced to biological systems can follow many potential routes before reaching their eventual target [6]. A common aspect of each stage of their journey, however, is that surface interactions determine where a particle localizes, whether it remains attached and how it is handled by clearance, uptake and trafficking processes. Therefore, surface properties of particles, as they mediate protein binding are likely to be predictive of biological outcomes.

Protein adsorption at surfaces has been studied for decades in qualitative and quantitative work. Hydrophobic interactions tend to dominate the energy balance in most cases [7], [8], [9], but electrostatics can also play an important role [10], [11]. Early work on blood plasma adsorption identified complicated phenomena such as the Vroman effect [12], [13], where proteins that first adsorb are later replaced by other proteins with higher affinity for the surface. This phenomenon has since been attributed to differences in concentration and diffusion coefficients [14], as high affinity proteins such as fibrinogen are rapidly depleted near surfaces, allowing lower affinity proteins in higher abundance such as albumin to adsorb temporarily. However, details of mechanisms by which exchange occurs and the kinetics of this process are still being sorted. The character of a surface has been shown to affect affinities of individual proteins [10], [15] and the eventual balance of adsorbed protein [16], [17]. Other work has demonstrated protein mobility on surfaces by fluorescence recovery after photobleaching [18], also showing that there is an immobile fraction. More recent work suggests that this immobile fraction may result from conformational changes of protein [19] and aggregation [20] following adsorption. In previous work from this laboratory [21] and others [17], it has been shown that varying the material composition as well as the surface chemistry of particles [22] can affect the identity and amount of proteins adsorbed. However, there is little predictive understanding of the mechanisms involved and they are necessarily complicated by the broad ranges of protein and surface properties.

Relative to protein adsorption, there has been little work investigating the mechanisms of particle binding to cell surfaces. The earliest studies identified opsonins, plasma proteins including immunoglobulins and complement proteins that adsorb to particle surfaces. Opsonization targets foreign matter for clearance by the reticulo-endothelial system and mononuclear phagocytic system, primarily via the liver and spleen [23]. Most recent studies have aimed to bypass this process to allow for targeted drug delivery [24], [25], most often using poly-ethylene glycol (PEG) to inhibit opsonization and allow other surface groups to bind specifically to receptors at cell surfaces [26]. With adsorbed proteins, the picture is more complicated than opposite surface charges mediating electrostatic interactions. The charge of the protein coat rather than the native particle surface will determine electrostatic binding. Only a few groups have investigated the role of particle properties in interactions with cell layers, showing phenomena such as a peak in cell association for 50 nm gold particles with spherical rather than rod shaped geometry [27] and a role for surface charge [28].

Given the ubiquity of protein rich fluids in organisms, we seek a bridge between detailed knowledge of protein–surface interactions and the fate of particles within organisms. Toward this end, we investigate protein adsorption to polystyrene nanoparticles with defined surface characteristics and size and measure nanoparticle-cell binding to cultured endothelial cells derived from human veins. Our main finding is that in contexts without detailed control of proteins on particle surfaces, the association between nanoparticles and cells most likely depends on non-specific interactions rather than the presence of specific mediators of cellular binding on nanoparticle surfaces.

Section snippets

Particle and reagent sources

Sources and properties for all the particles used are listed in Table 1. 5 kDa PEG was from Shearwater (Shearwater Polymers – Huntsville, AL). All other reagents were from Sigma if the source is not explicitly stated.

Particle modification and protein binding assay

Covalent modifications were performed by a two-step carbodiimide reaction linking free amines to COOH groups on particle surface as described previously [21]. Particles were sonicated at ∼4 V rms for a total of ∼20 s at a 50% duty cycle in an ice bath once each during the activation

Modification of nanoparticle surfaces by serum proteins

To investigate cellular interactions between cells and nanoparticles, we used a monolayer culture of endothelial cells. The growth medium for these cells includes ten percent fetal bovine serum (FBS) and thus there is plentiful protein in the cellular environment, as is the case in vivo. Therefore, the first question we address here is the rate at which nanoparticle surfaces adsorb protein to determine whether nanoparticle surfaces will be modified before they reach cells.

The time course of

Discussion

The experimental results presented here show first: that nanoparticle surface chemistry, as it mediates the protein adsorbing capacity of particles determines the cellular binding of nanoparticles, and second: that non-specific interactions account for a large portion of nanoparticle binding to endothelial cell surfaces. The main data supporting the first conclusion is rapid coating of particles by serum proteins and a positive correlation observed between protein adsorption to particle

Conclusions

This work demonstrates the importance of protein adsorption to nanoparticle surfaces in mediating binding to the endothelial cell surface. This relationship does not depend on the identity of proteins adsorbed to particle surfaces, but rather on the capacity of the surfaces to bind protein. Thus, there does not appear to be unique mechanism by which general nanoparticles will interact with endothelial cells after exposure to serum. An assessment of adsorptive capacity should be useful to

Acknowledgments

The authors would like to thank Christina Reed, Arshad Rahman, and members of his lab for help with cell culture, as well as Jessica Snyder, Michael Springer, Hsin Peng and Tom Gaborski for help in and out of the lab. This work was supported by a Department of Defense Multi-University Research Initiative (MURI) grant, award number: FA9550-04-1-0430.

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