Nanoparticles: potential biomarker harvesters

David H Geho1,2, Clinton D Jones1,3, Emanuel F Petricoin1,2 and Lance A Liotta1,2
A previously untapped bank of information resides within the low molecular weight proteomic fraction of blood. Intensive efforts are underway to harness this information so that it can be used for early diagnosis of diseases such as cancer. The physicochemical malleability and high surface areas of nanoparticle surfaces make them ideal candidates for developing biomarker harvesting platforms. Given the variety of engineering strategies afforded through nanoparticle technologies, a significant goal is to tailor nanoparticle surfaces to selectively bind a subset of biomarkers, sequestering them for later study using high sensitivity proteomic tests. To date, applications of nanoparticles have largely focused on imaging systems and drug delivery vectors. As such, biomarker harvesting is an underutilized application of nanoparticle technology and is an area of nanotechnology research that will likely undergo substantial growth.
Addresses 1 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA 2 George Mason University,10900 University Blvd MS 4E3, Discovery Hall Room 182, Manassas, VA 20110, USA 3 Science Applications International Corporation (SAIC), GEO-CENTERS, R&D Center, 9460 Innovation Drive, Manassas, VA 20110, USA Corresponding author: Geho, David H (dgeho@gmu.edu)

major protein components in blood are high abundance, high molecular weight molecules (99% of the mass) that number fewer than 30 proteins [3]. It is in the LMW, low abundance population of blood proteins that the potential biomarker-rich population is currently thought to reside. Recent studies have demonstrated that these LMW species are associated non-covalently with the high abundance blood proteins [3–5] (Figure 1). Most proteomic studies have relied on the discarding of high abundance molecules in the blood such as albumin, to reduce the signal-suppressive effects of these proteins [6]. An alternative approach capitalizes on the carrier functions of these macromolecules through isolating proteins such as albumin, which then have their low abundance, LMW cargo eluted and measured by mass spectrometry [3–5]. This approach was recently used to identify potential diseaserelated biomarkers, such as proteolytic BRCA2 fragments, present in the blood of patients with ovarian cancer [7]. Detection of LMW cargo bound to albumin is changing the way that high abundance proteins are viewed from a proteomic perspective. Now, the goal is to find strategies that fully utilize this LMW information for the purposes of biomarker discovery and detection. One strategy for achieving this is to generate harvesting platforms out of nanoscale particles that bind and preserve low abundance, LMW biomarkers. In one scenario, the nanoparticle could be engineered to out-compete the native carrier proteins for the LMW biomarkers (Figure 2). Alternatively, the particles could be designed as functional biomolecule–nanoparticle conjugates, thereby creating selective affinity surfaces for the capture of LMW proteins (Figure 3). The LMW proteins could then be eluted from the biomolecule–nanoparticle conjugates. Because the application of nanoparticles to biomarker harvesting is in its infancy, examination of the previous uses of nanoparticles provides insight into the potential harvesting applicability of this technology. To date, these platforms have been used largely to release a therapeutic payload or as imaging agents (references described below). This review focuses on how these uses of nanoparticles, found in manuscripts published over the past 11 years, provide insight into the biomarker harvesting potential of nanoparticles.

Current Opinion in Chemical Biology 2006, 10:56–61 This review comes from a themed issue on Proteomics and genomics Edited by Garry P Nolan and Emanuel F Petricoin Available online 18th January 2006 1367-5931/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.01.003

Introduction
Recent application of highly sensitive mass spectrometry tools to study the proteomic content of blood has revealed a new subset of low abundance, low molecular weight (LMW) proteins that had been previously overlooked. Initial studies utilized mass spectrometry to survey the LMW serum proteome for the presence of m/z value patterns, or fingerprints, linked to disease states or even particular stages of a disease, such as ovarian cancer [1]. Since this early approach, a significant effort has been focused on the identity of LMW proteins and polypeptides in the blood, as reflected in a recent report of an atlas of 6929 distinct peptides identified in plasma [2]. The
Current Opinion in Chemical Biology 2006, 10:56–61

Nanoscale materials as potential biomarker harvesting platforms
Tools for reproducible isolation and preservation of low abundance, labile disease-related biomarkers will be increasingly required. Organic and inorganic materials
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Nanoparticles: potential biomarker harvesters Geho et al. 57

Figure 1

and biochemical substances offer a platform that will allow researchers to develop systems for biomarker harvesting from the vascular compartment [8,9]. Such new technologies will fill the need for ex vivo biomarker harvesting systems that could be used in the physician’s office. It can also be envisioned that particles designed for in vivo administration might be used to provide surveillance of disease processes underway in patients. A wide variety of materials and biomolecules can be used to fashion nanometer scale particles, including inorganic substances and aggregates of biomolecules [10,11,12]. These materials provide a rich palette for the creation of nanoporous, nanoscale substances, in the size range of macromolecular complexes, which have specific molecular interaction characteristics. On the basis of the surface topology, charge and chemical functional groups, a given particle would be expected to have selective molecular interactive properties. Hence, it is likely that nanoparticle preparations can be created with selective specificity to isolate certain fractions of the LMW proteome of the blood.

Prototypical carrier protein. A limited number of proteins in the blood represent the majority of its proteomic content. These high abundance proteins, such as albumin, have known binding properties for LMW proteins and fragments of proteins that have considerable potential as disease biomarkers. A critical need within clinical proteomics research is to develop harvesting platforms that retrieve the low abundance, low molecular weight proteomic content of the blood for testing using tools such as mass spectrometry.

Two fundamental classes of capturing particles can be envisioned: 1. Particles with surface properties for selective protein binding (Figure 2). 2. Particles conjugated with proteins or other chemical groups that function as bait molecules for LMW proteomic species (Figure 3).

Figure 2

Nanoparticle harvesting platform. Nanoparticle surfaces can be engineered with properties such as varying porosity, surface charges and functional groups. Further, the nanoparticle may contain an intrinsic label, such as a quantum dot, which functions as a tracer element. The surface properties of the particle provides a binding site for selected proteins or protein fragments that circulate in the blood bound to other carrier proteins, such as albumin. www.sciencedirect.com Current Opinion in Chemical Biology 2006, 10:56–61

58 Proteomics and genomics

Figure 3

Nanoparticle bioconjugate harvesting platform. Nanoparticle surfaces can be engineered with functional groups that enable affinity groups such as proteins (e.g. antibodies) to be added to their surfaces. The nanoparticle can contain an intrinsic label, such as a quantum dot, which functions as a tracer element. The affinity molecules on the surface of the particle provide binding sites for selected proteins or protein fragments that circulate in the blood bound to other carrier proteins, such as albumin.

Biomolecule harvesting nanoparticles
A simple configuration for a biomarker harvesting platform is to design a set of nanoparticles with distinct physicochemical properties. For example, the relative surface area, presence or absence of size-selective pores, and surface chemistries of nanoparticles can be tailored to provide a physicochemically based fractionation tool for blood proteins. One scenario for use of these particles is to apply them to a serum sample. Following association of molecules with the particles, the particles are washed, removing the subset of unbound molecules. The depleted serum may then be analyzed, or alternatively, the particles may be treated with an eluant to remove bound molecules for subsequent analysis. There are a wide variety of substrates that could be used for such a platform, including silica particles. As an introductory approach, nanoporous silica particles have been used to fractionate serum proteins before subsequent mass spectrometry (DH Geho et al., manuscript submitted for review). This approach can be extended in the future to include metal, polymeric and biomolecule-based particles as well.

that specifically target certain biomarkers for isolation from serum. For example, the biomarker associates with the affinity molecule attached to the nanoparticle. The complex is then isolated, followed by elution and detection of the targeted biomarker. This approach for biomarker harvesting remains to be validated; however, there is significant evidence that proteins can be attached to nanoparticles and still retain their native binding functions (see below).
Proteins conjugated to labeled nanoparticles

Functional biomolecule–nanoparticle conjugates
A more complicated, and perhaps more selective, harvesting approach is to conjugate affinity proteins onto the surface of nanoparticles to develop harvesting platforms
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Of particular interest are bioconjugated particles that contain a reporter system, which functions as an intrinsic tracer element. For example, quantum dots are sizetunable nanoparticles that have been widely used as labels for biological probes [11]. In one instance, they have been conjugated to streptavidin, creating a reporter complex for molecular studies such as protein microarrays [13]. Moreover, the quantum dot–streptavidin complexes were further modified with polyethylene glycol. This demonstrates the versatility of nanoparticle-based platforms in forming higher-order structures with specific biomolecular activities. Quantum dots are just one example of an inorganic nanoparticle that can be attached to functional proteins; the composite organic–inorganic nanoparticles (COINs) are an alternative nanoparticle class that have been used as labels for immunoassays
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Nanoparticles: potential biomarker harvesters Geho et al. 59

[14]. These have been used in a sandwich protein array for cytokine detection, demonstrating that conjugation of an antibody to the COIN platform can be achieved without destroying the immunoaffinity characteristics of the antibody. Silica nanoparticles, alternatively, can be loaded, or doped, with metallo-organic luminophores, such as tris(2,20 -bipyridyl)dichlororuthenium (II) hexahydrate, a type of photostable luminophore [15]. The surface of the luminophore-doped particles can then act as intermediate substrates for bioconjugate formation with biomolecules, such as antibodies. These three examples of reporter-containing particles are reflective of the diverse approaches enabled by nanoscale engineering. Other potential particle labels include metallic barcodes and DNA-based bar codes [16,17]. Labeled particles will be valuable components of biomarker fractionation, as distinct particles with distinct biomarker cargo can be given discriminating labels for high-throughput sample processing.
Biomolecular nanoparticles

using 3-aminopropyltriethoxysilane. The particles were in turn loaded with N-acetylhomocysteine thiolactone to attach a thiol reactive group. This provided a linkage group for the attachment of conformationally intact peptomers, or head-to-tail linked peptides, derived from an amino acid sequence from the HIV-1 gp120 protein. The particles had a mean maximum diameter of 355 nm and carried a load of 53 000 Æ 42 000 peptides. In animal models, this nanoparticle–immunogen complex generated a specific immune response to the peptide epitope of interest. This demonstrated that proteinaceous conjugates can be linked to nanoparticles and still retain their conformational integrity [19]. In summary, numerous nanoparticles have been conjugated with proteins, with the latter retaining their functional properties. This provides significant evidence that an affinity protein–nanoparticle conjugate system could be designed to harvest disease relevant markers from serum.

At the other end of the particle spectrum, nanoparticles have been made out of gelatin through higher order structures created through glutaraldehyde-based crosslinking, resulting in particles with an average size of 250–300 nm [12]. This nanoparticle platform has builtin functional groups for bioconjugate derivatization, such as lysine residues. In this particular study, these functional groups were thiolated to serve as linkage points for Neutravidin1, which served as a docking site for biotinylated antibodies. Roughly 200 antibody molecules could be attached to each particle, illustrating an advantage of the high-surface area of the nanoparticles. In the study, the particles were created for potential use as a drug delivery vehicle. The biomarker harvesting profiles of biomolecular nanoparticles remain to be determined, but the built-in selective protein-binding properties of proteins themselves potentially make them attractive building blocks for biomarker harvesting tools.
Rigorous analysis of the conformational integrity of biomolecule–nanoparticle conjugates

In vivo nanoparticle exposure
A long-term goal of research into nanoparticle-based biomarker harvesting is the administration of in vivo harvesting nanoparticles [8]. These particles would have access to numerous tissue microenvironments in the host, thereby enabling a thorough sampling of the proteins being expressed, and providing an overall portrait of the patient’s health (Figure 4). In this system, the particles would be periodically harvested from the vascular comFigure 4

As nanoparticle–biomolecule platforms are developed for biomarker harvesting, proteins will be chosen on the basis of their molecular binding affinities for targeted biomarkers. One question for each biomolecule–nanoparticle pairing is whether the desired binding function of the immobilized protein will be retained on the particle surface. The immune system is exquisitely sensitive to conformational changes in protein structure. Therefore, a rigorous test system for the conformational state of nanoparticle conjugated proteins is the development of antibodies that recognize a natively folded protein after immunization with a nanoparticle–polypeptide antigen complex. With an eye towards the development of a new type of immunization platform, one group performed such a test using calcinated aluminum oxide nanoparticles [18]. These particles were coated with amine groups
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Nanoparticle distribution in tissue. In the future, a potential utilization of harvesting nanoparticles is to infuse them into patients to provide sustained surveillance of the overall state of health of an individual. Studies have shown that infused nanoparticles are found within the blood, in endothelial cells, in the interstitium, and within tumor cells. The kinetics of nanoparticle movement from the blood to the tumor (path illustrated with black arrows) and then from the tumor back to the blood stream (path illustrated with green arrows) remain to be thoroughly evaluated. ECM, extracellular matrix. Current Opinion in Chemical Biology 2006, 10:56–61

60 Proteomics and genomics

partment for analysis. Issues for study as this approach is developed include the tissue distribution of particles, the likelihood of retrieving the particles from the patient’s blood, and the potential toxicity profiles of the particles.
Tissue distribution of infused nanoparticles

Because of their potential use as imaging agents and drug delivery vectors, nanoparticles have been studied in in vivo contexts previously. For example, monocrystalline iron oxide nanoparticles, which can be used as MRI contrast agents, have been injected into the cortex of rat brain and the local distribution of the particles was evaluated. These particles were roughly 5 nm in diameter with an outer core of dextran, creating an overall particle size of roughly 20 nm. These were dispersed within the brain cortex over a span of roughly 7.93 mm, a distance many times greater than the size of the particle [20], demonstrating the ability of the particles to permeate complex tissue architectures. As another example of in vivo studies, long circulating dextran coated iron oxide (LCDIO) nanoparticles have been used in imaging studies, including in clinical trials [21]. These particles can cross the capillary endothelium that surrounds tumors [22,23]. In addition, uptake by tumor cells has been demonstrated [22,24]. In one animal study, LCDIO particles were shown to have the following biodistribution after 24 h (top five locations): lymph node 25%, spleen 9.8%, liver 1.9%, kidney 0.8% and blood 0.5% when 125I labeled particles were studied [25]. With regard to the tumor microenvironment in particular, histological studies showed the particles to be in both the extracellular and intracellular compartments. In the extracellular space, particles were observed in both the vascular (4.5%) and interstitial (19%) compartments. Tumor cells contained 49% of the observed particles whereas endothelial cells contained 6.5% and tumor associated macrophages contained 21%. Thus, particles were found in blood, within endothelial cells, in the interstitium and in tumor cells (Figure 4). These regions are important sites for the generation and dissemination of potential biomarkers, and all of these contained significant populations of nanoparticles. Of particular interest is the ability of nanoparticles to infiltrate tumor environment, a rich bioreactor for the generation of disease related biomarkers. Of note, a significant number of particles were present in the vascular compartment, which is essential for particle retrieval in a clinical setting. However, the feasibility of the retrieval of infused nanoparticles from peripheral blood vessels, as might be envisioned in a clinical scenario, remains to be thoroughly evaluated.
Inflammatory properties of nanoparticles

study investigated the interaction of cultured endothelial cells with a panel of nanoparticles: polyvinyl chloride (PVC) (130 nm mean particle size), TiO2 (70 nm mean particle size), SiO2 (14 nm mean particle size), Co (120 nm mean particle size), and Ni (50 nm mean particle size) [26]. Of these, the PVC-, TiO2-, SiO2- and Co particles were internalized by the endothelial cells, whereas the Ni particles were not. Of note, the Co-, SiO2-, and TiO2 particles caused the release of the proinflammatory cytokine IL-8, demonstrating a potential side effect of nanoparticle infusion. Previous work has also demonstrated that SiO2 particles can cause pulmonary inflammatory disease [27], a finding compatible with the endothelial cell experiments. In another system, the inflammatory characteristics of biodegradable polylactide particles have been studied in the eye. These particles have been used in delivery studies, having been loaded with proteins, oligonucleotides and even plasmids [28–31]. These particles can be synthesized in a controlled manner, allowing tunable degradation properties to be engineered into the particle [32,33]. In animal ocular studies, intravitreous injection of dye-loaded polylactide particles resulted in the migration of the particles into the retinal pigment epithelium [34]. The nanoparticles were present at the site for 4 months, even after a single injection. There was a mild inflammatory cell infiltrate in the ciliary body 6 hr after injection and the posterior vitreous of the retina had an inflammatory infiltrate after 18 to 24 h. The inflammation markedly decreased after 48 h. The characteristics of the infused particles will undoubtedly impact on the host response to the particle. Sophisticated, standardized validation systems will be required to test host–particle interaction, if nanoparticle infusion systems are to be fully developed.

Conclusions
Nanoparticles are a diverse class of nanoscale material surfaces that can be modified to create selective surfaces for targeted molecular interactions. As the biomarker populations present in blood are more fully characterized, nanoparticle harvesting platforms will have significant potential as the first line for specimen preparation and preservation. As with any new field, diverse strategies and approaches must be tested to develop reproducible particle-based systems for biomarker harvesting. However, there is significant innovative potential for the development of in vitro and in vivo harvesting systems to improve the detection of diseases at an early, more treatable stage.

References and recommended reading
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For infused particles, endothelial cells function as the primary gatekeeper, mediating access of the particles into extravascular spaces, and are therefore key sites of potential early nanoparticle-induced toxicity. To this end, one
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Nanoparticles: potential biomarker harvesters Geho et al. 61

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