Targeted Nanoparticle Binding to Hydroxyapatite in a High Serum Environment for Early Detection of Heart Disease

Abstract

The impact of the protein-rich in vivo environment on targeted binding of functionalized nanoparticles has been an active field of research over the past several years. Current research aims at better understanding the nature of the protein corona and how it may be possible for targeted binding to occur even in the presence of serum. Much of the current research focuses on nanoparticles targeted to particular cell receptors or features with the aim of cellular uptake. However, similar research has not been performed on nanoparticles that are targeted to non-protein disease features, such as hydroxyapatite (HA). HA is a crystalline calcium-phosphate mineral that is present in large quantities in bone, and in smaller quantities in diseased cardiovascular tissue in cases of atherosclerosis or various stenoses. Our work aims to gain a better understanding of the behavior of PEGylated, peptide-coated superparamagnetic iron oxide nanoparticles (SPIONs) in a biologically-relevant high-protein environment (50% serum). We first determined that specific binding to HA occurs at significantly higher rates than non-specific binding in the absence of serum protein. We then examined nanoparticle interactions with serum proteins, including determination of the relative quantities of protein in the hard vs. soft protein corona. Finally, we examined specific and non-specific binding of targeted SPIONs in 50% serum, and determined that targeted binding may still occur with significant (p < 0.05) selectivity. We hypothesize that this may be because the nature of the binding interactions between the peptides and the HA are, by definition, less specific than the protein-protein interactions required for nanoparticles to bind to specific cells or cell features. These results suggest that these targeted SPIONs may be further developed for use in early detection of heart diseases such as atherosclerosis and aortic stenosis.

Keywords: iron oxide nanoparticles; non-specific binding; protein corona; serum; targeted nanoparticles.

Figure 1:

(A) Schematic of particle fabrication and functionalization. A ligand exchange is performed on magnetic SPION cores coated in oleic acid to produce SPIONs coated in citric acid (CA-SPIONs), and citric acid coated SPIONs are PEGylated using EDC/NHS chemistry. Targeting peptides HABP and OPN are added via an iodoacetyl/thiol interaction between iodoacetyl-PEG and peptides with cysteine residues.TEM images of ~10nm SPION cores. SPION core sizes were quantified in ImageJ, which yielded an average core size of 9.39 ± 0.98nm (mean ± standard deviation). (C) Results from a LIVE/DEAD assay indicating that HABP-, OPN-, and PEG-SPIONs do not show higher rates of cell death after incubation than a control sample incubated without nanoparticles.

Figure 2:

(A) T2-weighted MRI (3 Tesla) of saline solutions containing HABP-, OPN -, and PEG-SPIONs at different concentrations (ppm) exhibits decreasing signal intensity with increasing concentration for all 3 types of SPION (i.e., the color of each tube gets darker with increasing ppm). (B) The loss in signal intensity with increasing concentration is a consequence of the high R2 relaxivities of HABP-, OPN-, and PEG-SPIONs determined from the relaxation rate of each sample measured from MR images like those in (A) but acquired at multiple echo times. Error bars shown are standard error in the calculated slope.

Figure 3:

(A) Binding of HABP SPIONs to HA. From left to right, tubes contain 0, 5, 10, 25, 50, 100, and 200mg of HA. After filtration of the HA, the concentration of SPIONs remaining in the tubes is visibly different depending on the amount of HA in each tube – higher quantities of HA result in fewer particles remaining unbound. (B) Quantification of SPION binding from samples bound in the same setup as that illustrated in (A). Generally, 50mg of HA was sufficient to fully bind 9.5 × 10¹³ HABP-SPIONs, and 7.7 × 10¹³ OPN-SPIONs. (C) Quantification of HABP- and OPN-SPION binding to various biologically-relevant surfaces. HA-targeted SPIONs bind significantly more to HA than to other surfaces tested, although some degree of off-target binding did occur. Error bars are standard deviation; n=3.

Figure 4:

Fold change in nanoparticle size over time in a 50% serum solution. Effective nanoparticle diameter would include the nanoparticles in addition to any tightly associated and loosely associated proteins (i.e. both the hard and the soft protein corona). (A) HABP-SPIONs, (B) OPN-SPIONs, and (C) PEG-SPIONs were all tested under identical conditions. Both targeted (HABP- and OPN-SPIONs) and non-targeted (PEG-SPIONs) bind to proteins in serum, as evidenced by changes in effective particle diameter upon exposure to 50% serum. The protein corona that develops around all 3 functionalized SPIONs tested does not appear to alter significantly over the course of a 24-hour incubation. HABP-SPIONs appear to accumulate the largest protein corona, while OPN-SPIONs have the smallest. Despite the intended anti-biofouling function of PEG, PEG-SPIONs do still appear to accumulate a protein corona in 50% serum. Error bars are standard deviation; n = 3.

Figure 5:

Soft and hard protein corona measured as mg of associated protein per particle after incubation in a 50% serum solution for (A) 0 minutes, (B) 30 minutes, and (C) 2 hours, followed by 3 wash steps via centrifugation and resuspension in DI water. Protein associated with the particles with no wash steps is considered to be part of the soft corona; protein associated with the particles after 3 washes is considered to be part of the hard corona. Error bars are standard deviation; n=3.

Figure 6:

SDS-PAGE gels stained with silver nitrate. (A) Samples (HABP-, OPN-, and PEG-SPIONs, respectively) that were not incubated with serum. Wells in (B), (C), and (D) are samples (HABP-, OPN-, and PEG-SPIONs in each well, respectively) that were incubated with serum, washed 3x with water by centrifugation, and then mixed with an SDS-based running buffer and boiled to denature any protein remaining attached to the particles. Differences between wells reflect differences in which proteins may have preferentially adsorbed to different surface functionalizations; differences between gels reflect potential differences in the hard protein corona with longer incubations times. Samples were incubated with serum for: (B) 0 minutes, (C) 30 minutes, and (D) 2 hours prior to beginning the wash steps.

Figure 7:

(A) Images taken at 20x of Prussian-Blue stained SPIONs bound to different surfaces to observe specific vs. non-specific binding. (B) Quantification of multiple runs of binding assays (n = 3) demonstrate that both HABP- and OPN-SPIONs bind at significantly higher rates to HA than other biologically relevant surfaces, while PEG-SPIONs and CA-SPIONs do not bind significantly to HA. PEG-SPIONs and CA-SPIONs exhibit significant off-target binding to cholesterol, potentially due to the hydrophobic nature of the cholesterol surface. All surfaces were fabricated such that ~25mg of each material is present for testing against each type of nanoparticle.