Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

Abstract

Mass transport of drug delivery vehicles is guided by particle properties, such as shape, composition and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light chain variable region, fibrinogen, and complement component 1 compared to their anionic counterparts. The anionic-surface favored equal accumulation of microparticles in the liver and spleen, while cationic-surfaces favored preferential accumulation in the spleen. Immunohistochemistry supported macrophage internalization of both anionic and cationic silicon microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.

Introduction

For successful drug delivery, nanocarriers must vector their cargo to the appropriate cellular targets. Crucial to the success of these nanocarrier systems is their ability to navigate through the bloodstream and across biological barriers as they journey from the site of administration to the site of action.^1-2 Particle attributes, such as size, shape, density, elemental composition, and surface chemistry greatly affect convective transport in the blood stream, margination, cell adhesion, selective cellular uptake, and sub-cellular trafficking.^3-4 Particle composition and surface chemistry influence interactions with plasma constituents, which further impact effective transport and desired cellular interactions.⁵ As stated by Walczyk and colleagues, what a cell sees when it encounters a particle is the plasma-derived corona rather than the bare particle surface.⁶ A greater understanding of the impact of particle surface chemistry on biomolecular interactions will aid in creating particles with superior biodistributions.

The use of intrinsic particle properties as the driving force for cellular interactions and bio-barrier transport may prove superior to ligation of particles with antibody-based targeting ligands. The presence of antibodies drives uptake by macrophages of the reticuloendothelial system (RES) based on their high surface density of Fc gamma receptors. Opsonization of particles with plasma constituents can similarly drive sequestering of particles by the RES, with the liver and spleen comprising the endpoint organs for the majority of particle-based therapeutics. It is well known that bio-transport, cellular associations, and intracellular trafficking of drug delivery vehicles, as well as phagocytic engulfment of particles are driven in part by surface charge and the repertoire of macromolecules and plasma proteins that bind to the particle's surface. We recently reported that, under serum-free conditions, both anionic and cationic porous silicon microparticles are phagocytosed by human umbilical vein endothelial cells (HUVECs).¹ However, in the presence of serum, only microparticles with an originally cationic surface are internalized. One goal of this study is to define the impact of particle surface chemistry on protein adsorption and the resulting cellular associations and biodistribution.

In this study, using quasi-hemispherical and discoidal silicon microparticles with either an oxidized, anionic surface, or an aminosilylated, cationic surface, we sought to define plasma proteins adsorbing discriminately to the particle surface using two-dimensional gel electrophoresis (2DGE) and mass spectroscopy (MS). We hypothesized that different protein coronas would result in microparticle association with unique cellular populations based on affinities for different cell surface receptors, leading to disparate in vivo biodistributions. The impact of particle surface charge on in vivo cellular associations and biodistribution was studied in an animal model of breast cancer. Endothelial and RES targeting based on particle surface chemistry-driven opsonization was explored by defining cellular interactions in the liver and spleen. Cellular associations of particles with the intraluminal surface of blood vessels were imaged by scanning electron microscopy, while transmission electron microscopy was used to image intracellularly localized particles. Our broad goal is to enhance our understanding of the mechanics that drive in vivo trafficking of drug delivery vehicles.

Materials and Methods

Porous silicon microparticle fabrication

Nanoporous quasi-hemispherical and discoidal silicon microparticles were fabricated in the Microelectronics Research Center at The University of Texas at Austin. The mean particle diameter, 3.2 ± 0.2 μm, was determined using scanning electron microscopy and the average pore size, 26.3 ± 13.6 nm (hemispherical) and 51.3 ± 28.7 nm (discoidal) was based on BET measurements. Briefly, heavily doped p++ type (100) silicon wafers (Silicon Quest, Inc, Santa Clara, CA) were used as the silicon source. For discoidal microparticles, a SiO₂ layer was thermally grown on the wafer, followed by a layer of silicon nitride (SiN) deposited by Low Pressure Chemical Vapor Deposition. Standard photolithography, dry etch of SiN in CF4 plasma, and wet SiO2 etch in 5% HF were used to transfer a pattern of 2 μm circular arrays on the silicon wafer. A three-step electrochemical etch was used to make silicon particles consisting of a low porosity mechanical stabilization layer, a large pore device layer, and high porosity release layer. The masking SiN and SiO2 layers were removed in aqueous HF solution, and the silicon particles were released from the substrate by sonication in isopropanol. Details for creating hemispherical microparticles were recently published.⁷

Surface modification of silicon microparticles

An isopropyl alcohol (IPA) suspension containing silicon microparticles was transferred to a glass petri dish and the IPA was evaporated using a hotplate set at 60°C overnight. The hotplate temperature was then raised to 120°C for 15 min to ensure evaporation of residual IPA from the porous matrix. Dried microparticles were then treated with piranha solution (1 volume H₂O₂ and 2 volumes of H₂SO₄) with heating to 110-120°C for 2 hour with intermittent sonication to disperse the microparticles. The microparticles were then washed three times and incubated in 10% nitric acid for 30 minutes. The microparticles were then washed in deionized water 3 times, followed by washing in IPA 3 times. For aminosilylation, oxidized microparticles were suspended in IPA containing 2% (v/v) 3-aminopropyltriethoxysilane (APTES; Sigma-Aldrich, St. Louis, MO) for 2 hours at 35°C, with mixing at 1300 rpm. APTES modified microparticles were washed in IPA, and the amine density was determined using a 4-nitrobenzaldehyde (NBA) colorimetric assay. Briefly, 2 × 10⁷ APTES-modified microparticles were washed in coupling solution [0.8% glacial acetic acid in 99.2% anhydrous ethanol (Sigma-Aldrich)] 3 times and resuspended in 1 ml of reaction solution [5 mM NBA in coupling solution] for 3 hours with mixing (900 RPM) at 50 °C. Particles were then washed with absolute ethanol 5 times and resuspended in 1 ml of hydrolysis solution [0.14% glacial acetic acid in 50% ethanol]. Standards were prepared with NBA and absorbance was determined at 268 nm. Microparticle concentration was determined using a Beckman Coulter Z Series Coulter Counter (Beckman Coulter, Inc; Brea, CA).

Zeta Potential Analysis

A ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Corp., Holtsville, NY) equipped with a 90Plus/BI-MAS Multi Angle Particle Sizing Option was used to measure the zeta potential of particles suspended in 0.01M phosphate buffer at a concentration 1-50 μg/ml silicon. The impact of IgG and serum protein binding on microparticle surface charge was studied by incubating the microparticles (1-3 × 10⁶) in serum or pure IgG for 30 minutes at 4, 25 or 37°C. Measurements were performed at pH 5.0 and pH 7.4. IgG binding was measured based on fluorescent intensity of a secondary antibody. The secondary antibody was incubated with particles at 10 μg/ml, both before and after exposure to bare IgG.

Fourier Transform Infra-Red Spectroscopy (FTIR)

Microparticle samples were applied to the diamond surface of a SMART ATR attachment on a Nicolet 6600 FTIR Spectrometer (Nicolet Instrument Tech., Middleton, WI). The applied sample was then dried with nitrogen and the FTIR spectra were read. A room temperature detector was used to collect all data, and all readings were made using a resolution of 4 and averaging 16 readings in absorbance mode. Analysis of the peaks was performed using Omnic peak identification software and Microsoft Excel.

Scanning Electron Microscopy (SEM)

J774 macrophages, purchased from American Type Culture Collection (Manassas, VA), were grown on 5 × 7 mm Silicon Chip Specimen Supports (Ted Pella, Inc., Redding, CA). Upon reaching 80% confluency, cells were incubated with silicon microparticles (1:10; cell:microparticle) for 15 min at 37°C. Samples were washed with PBS and fixed in 2.5% glutaraldehyde (Sigma-Aldrich; St. Louis, MO). After washing in PBS, cells were dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 90%, 95%, and 100%) for 10 min each. Cells were then incubated in 50% alcohol-hexamethyldisilazane (HMDS; Sigma-Aldrich) solution for 10 min followed by incubation in pure HMDS for 5 min to prepare for overnight incubation in a desiccator. Specimens were mounted on SEM stubs (Ted Pella, Inc.) using conductive adhesive tape (12mm OD PELCO Tabs, Ted Pella, Inc.), and then sputter-coated with a 10 nm layer of gold using a Plasma Sciences CrC-150 Sputtering System (Torr International, Inc., New Windsor, NY).

For SEM imaging of microparticles, samples were suspended in either IPA or water, then dried on ethanol washed SEM stubs (Ted Pella, Inc.) overnight in a desiccator. Micrographs were acquired under high vacuum, at 20-30 kV, spot size 3.0-5.0, using either a FEI Quanta 400 FEG ESEM or a Hitachi S-5500 high resolution SEM.

Tissue electron microscopy

For tissue SEM, at predetermined time points following intravascular injection of discoidal porous silicon microparticles (1 × 10⁸), animals were perfused by injection into the left ventricle with 30 ml of perfusion wash solution (Electron Microscopy Sciences, #1222SK) followed by 30 ml of perfusion fix solution (Electron Microscopy Sciences, #1223SK). Tissues were processed based on techniques adapted from Hashizume et al.⁸ Tissue was collected in fixative, followed by embedding in 3% agarose. Tissue sections of 100 μm thickness were cut using a Krumdieck MD-4000 Tissue Slicer (Alabama Research & Development, Munford, AL). Sections were rinsed with cacodylate buffer, immersed in cacodylate buffered 2% tannic acid for 24 hours, washed twice with 0.2 M sodium cacodylate, then incubated in cacodylate buffered 2% osmium tetroxide for two hours at 4°C, and washed again in 0.2 M sodium cacodylate and dehydrated in increasing concentrations of ethanol, followed by infiltration with 100% t-butanol. Samples were dried in a dessicator, and then mounted on SEM sample stubs using carbon adhesive tape. Following sputter coating with a 10 nm thick layer of gold, samples were imaged using an FEI Quanta 400 FEG SEM.

Tissue TEM imaging was done on 100 nm sections of resin-embedded tissue using a JEOL 1200 transmission electron microscope at 60 kV with digital images collected using a 1 k × 1 k Gatan BioScan camera Model 792.

In vitro microparticle association studies

HUVEC, purchased from Lonza Walkersville, Inc. (Walkersville, MD), were cultured in EBM®-2 medium (Clonetics®, CC-3156; Lonza). Serum-free experiments used EBM®-2 medium (Clonetics®) supplemented with only hydrocortisone and GA-1000, plus 0.2% BSA. HUVECs were discarded after 7-8 passages. For flow cytometry studies, HUVECs (1.5 × 10⁵ cells/well) were seeded into 6-well plates and 24 hours later the cells were incubated with silicon microparticles (10-20 microparticles/cell) in the indicated media for 60 minutes. For opsonization experiments, microparticles were preincubated with 100 μl of serum or plasma on ice for 30 minutes. Stimulated cells were treated with TNF-α (10 ng/ml) and IFN-γ (100 U/ml) for 48 hrs prior to incubation with microparticles. Following cellular internalization, cells were washed with PBS, harvested by trypsinization and resuspended in PBS containing 1.0% BSA and 0.1% sodium azide (FACS wash buffer). Microparticle association with cells was determined by measuring side scatter using a Becton Dickinson FACSCalibur Flow Cytometer (Becton Dickinson; San Jose, CA) equipped with a 488-nm argon laser and CellQuest software. Data is presented as the percentage of cells associating with microparticles (percent of cells with high side scatter). Side scatter due to cells in the absence of particles was subtracted from the presented data. Sample populations were compared using a two-tailed distribution, two-sample equal variance t-test.

Animal Studies

Breast cancer xenografts were established in Crl:NU-Foxn1^nu mice (Charles Rivers Laboratories International, Inc., Wilmington, MA) by intramammary injection of 1 × 10⁵ 4T1 mouse mammary tumor cells (ATCC, Manassas, VA). After approximately 3 weeks, when the tumors reached 1 cm in diameter, mice were injected by tail vein with vehicle control (saline) or 1 × 10⁸ 3.2 μm oxidized or APTES-modified silicon microparticles in 100 μl saline (n=3 mice per group). Twenty four hours after injection, the mice were sacrificed and tissues were excised and either frozen for immunohistochemistry or weighed, then moved to 5 mL of 80% 1M NaOH/20% EtOH for analysis of tissue biodistribution using inductively coupled plasma atomic emission spectrometry (ICP-AES).

Frozen tissues were sectioned and stained with antibodies specific for endothelia (anti-mouse CD34; Beckman Coulter, Fullerton, CA) or macrophages (anti-mouse CD204, scavenger receptor Type I/II; AbD Serotec, Raleigh, North Carolina). CD34 was visualized using the STAT-Q Peroxidase (HRP) system and DAB, purchased from Innovex Biosciences (Richmond, CA). The secondary antibody used for the CD204 antibody was goat-anti-rat IgG:HRP (1:50 dilution; STAR72), purchased from AbD Serotec. Nuclei were stained with hematoxylin.

Tissue samples suspended in NaOH for ICP-AES were homogenized, placed on a tube rotator overnight, and then centrifuged at 4200 RPM (Beckman Coulter centrifuge). A 0.5 mL aliquot of the tissue slurry was moved to a conical tube with 4.5 mL of distilled water. Samples were analyzed for silicon using a Varian Vista AX at a power of 1 kW, with plasma flow set to 15 L/min, auxiliary flow of 1.5 L/min, and a nebulizer flow of 0.75 L/min, with 5 replicate readings at 15 seconds between each reading. A standard curve was generated using Fisher Scientific (Pittsburgh, PA) Silicon Reference Standard Solution.

Differential Protein Estimation by Two-dimensional Gel Electrophoresis (2DGE)

Plasma was collected from 10 mice by orbital bleeding and placed in tubes containing ethylenediamine tetraacetic acid as the anti-coagulant. Following centrifugation at 1500 × g for 10 min, the upper plasma layers were pooled and frozen. Prior to use, plasma was thawed on ice. Oxidized and APTES modified discoidal microparticles (1 × 10⁸; 0.5 mg each) were each incubated with 0.5 ml of plasma on ice for 30 minutes after vortexing. Microparticles were then washed 3 times with PBS, washed once in 0.1x PBS, and resuspended in 100 μl of Destreak Rehydration Solution (Fisher Scientific). Samples were vortexed, incubated at room temperature for 10 minutes, then centrifuged and the protein solution frozen at -80°C.

2DGE was conducted using Pharmacia's (GE Healthcare, Pittsburgh, PA) IPGphor multiple sample IEF device for the first dimension, and Bio-Rad's multiple gel SDS-PAGE systems (Protean Plus and Criterion Dodeca cells) for the second as first described by O’Farrell.⁹ Isoelectric focusing (IEF) was performed with 11 cm precast IPG strips (Bio-Rad, Hercules, CA). Sample aliquots were loaded onto dehydrated immobilized pH gradient (IPG) strips, and rehydrated overnight. IEF was performed at 20°C with the following parameters: 50 Volts (V), 11 hours; 250 V, 1 hours; 500 V, 1 hours; 1000 V, 1 hours; 8000 V, 2 hours; 8000 V, 6 hours. The IPG strips were then incubated in 4 ml of Equilibration Buffer (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8.8, 20% glycerol) containing 10 μl/ml tri-2 (2-carboxyethyl) phosphine (Geno Technology, Inc., St. Louis, MO) for 15 minutes at 22°C with shaking. The samples were incubated in another 4 ml of Equilibration Buffer with 25 mg/ml iodoacetamide for 15 min at 22°C with shaking in order to ensure protein S-alkylation. Electrophoresis was performed at 150 V for 2.25 h, 4°C with precast 8-16% polyacrylamide gels in Tris-glycine buffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS, pH 8.3). After electrophoresis, the gels were fixed (10% methanol, 7% acetic acid in ddH20), stained with SYPRO-Ruby ¹⁰ (Bio-Rad), and destained in fixative. The destained gels were scanned at a 100 μm resolution using the Perkin-Elmer ProXPRESS 2D™ Proteomic Imaging System (Boston, MA) using 460/80 nm excitation and 620 nm (long pass) emission filters.

Measurement of relative spot intensities

The 2D gel images were analyzed using Progenesis/ SameSpots software (Nonlinear Dynamics, Ltd. Newcastle Upon Tyne, UK). Analysis relies on geometric correction of the scans and projecting them all into the same reference space, performing pixel-to-pixel matching, and then spot detection. Subsequent to automatic spot detection, spot filtering was manually performed and spots with an area of less than 250 pixels were filtered out, and spots with a volume (intensity)/area ratio of less than 150 pixels were also filtered. The matching of spots between the gels is manually reviewed and adjusted as necessary. In our previous work, we found that the log-transformed normalized spot volumes were normally distributed, indicating that nonparametric statistical comparisons such as t-tests could be applied to identify those proteins whose expression was significantly changed by treatment. A change in normalized spot volumes greater than ± 25-fold was used to select spot that were subsequently robotically picked, trypsin-digested, and peptide masses identified by MALDI TOF. Protein identification was performed using a Bayesian algorithm,¹¹ where high probability matches are indicated by expectation score, an estimate of the number of matches that would be expected in that database if the matches were completely random. To determine gel-to-gel reproducibility, hierarchical clustering was performed by the hclust algorithm in the Splus statistical package (S-PLUS 6, Insightful Inc.), using Euclidian distance. ¹²

Protein identification

Protein gel spots were excised and prepared for MALDI TOF/TOF analysis using Genomic Solutions’ ProPic II and ProPrep robotic instruments following the manufacturer's protocols. Briefly, gel pieces were incubated with trypsin (20 μg/mL in 25 mM ammonium bicarbonate, pH 8.0, Promega Corp.) at 37°C for 4 h. The 4800 MALDI TOF/TOF was used for peptide mass fingerprinting and sequence analysis and database interrogation. Data were acquired with an Applied Biosystems (AB) 4800 MALDI TOF/TOF and AB software package 4000 Series Explorer (v. 3.0 RC1) with Oracle Database Schema (v. 3.19.0) and data version (3.80.0) to acquire both MS and MS/MS spectral data. The parameters used are included as supplemental material.

Results

Silicon microparticles and cellular associations

Porous silicon microparticles were fabricated from silicon wafers using procedures that resulted in quasi-hemispherical (Figure 1a-b) or discoidal shapes (Figure 1c). Previous in vitro studies show that both microparticle shape variants are candidates for cellular uptake by phagocytic cells.¹³ SEM images in Figure 1b-c show microparticles on or near the surface of J774 macrophages. The mechanism of internalization has previously been shown to be actin-dependent phagocytosis and macropinocytosis.¹⁴ In Figure 1d, a discoidal silicon microparticle is being internalized by a macrophage, and the formation of cellular filopodia can be seen both adhering to the microparticle surface and as a larger extension approaching the microparticle. We have previously shown that murine macrophages internalize both cationic and anionic silicon microparticles in the presence and absence of serum.¹⁴

Figure 1.

In vitro cellular associations with porous silicon microparticles. A-C) Two shapes of porous silicon microparticles, quasi-hemispherical (a & b) and discoidal (c), are shown in scanning electron micrographs (bars 1 μm). D) Scanning electron micrographs of a cell internalizing a discoidal silicon microparticle at increasing magnification. E) Left: impact of serum protein binding and pro-inflammatory cytokines (stimulated) on endothelial association with anionic silicon microparticles (adapted from Serda et al. ¹⁴). Right: endothelial association with anionic silicon microparticles in media containing plasma or FCS (fetal calf serum), with and without pre-opsonization of microparticles.

We also previously demonstrated that cellular uptake of porous silicon microparticles by human endothelial cells is altered by serum opsonins binding to the surface of microparticles in a charge dependent fashion.¹⁴ Cationic, aminosilylated silicon microparticles are internalized similarly in both serum-free and serum containing media, however, internalization of anionic, oxidized silicon microparticles is reduced in the presence of serum proteins.¹⁴ The results in Figure 1e summarize previously published findings in which serum proteins and pure IgG inhibit uptake of anionic silicon microparticles by endothelial cells, in the presence or absence of the inflammatory cytokines TNF-α and IFN-γ.¹⁴ The negative impact of serum biomolecules on microparticle uptake was greater in the presence of plasma compared to serum, and pre-opsonization of microparticles before addition to the cell culture did not alter uptake compared to adding non-opsonized microparticles to cells in the presence of media containing serum.

Microparticle characterization and serum opsonization

Verification of microparticle surface modification was obtained using Fourier Transform Infra-Red (FTIR) spectroscopy (Figure 2a). Oxidized microparticles exhibited a broad peak at 3350 cm^-1, indicating the presence of hydroxyl units. Aminosilylation of the microparticles introduced a broad peak near 3400 cm^-1 due to –NH stretching, and thereby supporting surface modification with amine groups. The presence of surface amines on aminosilylated microparticles was also demonstrated by detection of amines using a 4-nitrobenzaldehyde colorimetric assay (Figure 2b). One milliliter containing 2 × 10⁷ APTES-modified microparticles had an amine density of 0.35 ± 0.004 mM.

Figure 2.

Characterization of silicon microparticles. A) FTIR analysis of positive (APTES) and negative (oxidized) silicon microparticles. B) Amine density on discoidal microparticles, before and after APTES modification. C) Zeta potential analysis of silicon microparticles before (control) and after incubation in serum at different temperatures for 30 minutes.

The zeta potential, an indicator of surface charge, of silicon microparticles in the oxidized and aminosilylated state was -19.45 ± 0.45 and 27.86 ± 0.45 mV, respectively, in phosphate buffer, pH 5.0 (Figure 2c). Incubation of microparticles with fetal bovine serum (FBS) across 3 temperatures for 30 minutes resulted in near neutral surface charges for both oxidized and aminosilylated silicon microparticle, with no major alterations caused by incubation at different temperatures.

At physiological pH (7.4) oxidized and aminosilylated silicon microparticles had surface potentials of -25.7 and 6.3 mV, respectively. Exposure of particles to pure IgG resulted in both particle subsets having similar, negatively charged surface (Figure 2d). Unlabeled IgG displayed a greater affinity for anionic (oxidized) compared to cationic (APTES) microparticles, based on flow cytometry measurements that detected binding of a fluorochrome-labeled secondary antibody on the particle surface (Figure 2e). Binding of the secondary, labeled antibody to particles in the absence of unlabeled primary antibody was relatively low, especially for oxidized microparticles with a net negative surface charge.

As reported previously, at physiological pH, the surface charge of serum opsonized microparticles is negative for both oxidized and aminosilylated silicon microparticles despite the original surface charge.¹⁴ In contradiction to the belief that cationic particles enhance interactions with cell surfaces based on affinity to negative gylcocalx moieties, these data argue against surface charge as the sole explanation for selective endothelial binding and uptake of originally cationic microparticles.

Biodistribution of cationic and anionic microparticles

A murine 4T1 model of breast carcinoma was used to study the biodistribution of silicon microparticles 24 hours after tail vein injection with 2 × 10⁸ discoidal silicon microparticles in 100 μl saline (Figure 3a). The study included 3 groups: vehicle control, cationic silicon microparticles, and anionic silicon microparticles (n=3 per group). ICP-AES analysis of silicon content (ng silicon/mg tissue) in excised tissue showed low levels of microparticles in tumor, kidney, and lungs. Anionic silicon microparticles were found in liver and spleen at similar concentrations (9.8 and 10.6, respectively), while cationic silicon microparticles showed preferential accumulation in the spleen compared to the liver (35.5 and 17.8, respectively; p<0.02; n=3). The mass of cationic microparticles in the spleen was significantly greater than that of anionic microparticles when the two groups were compared (p<0.002). These findings are supported by Choi et al. who demonstrated that intravenous administration of a cationic lipid/emulsion DNA complex favors splenic accumulatior over liver. The trend was reversed for cationic lipid emulsion complexes delivered intra-arterially or intraportally.

Figure 3.

Tissue biodistribution and cellular associations with silicon microparticles. A) ICP-AES analysis of silicon content in tissues following intravenous injection of saline or microparticles, either positive or negative. B) Histological evaluation of in vivo microparticle association with macrophages and endothelial in liver and spleen based on surface charge (bars 10 μm).

While the reported lung accumulation of both cationic and anionic microparticles is low in this study, we have previously found that microparticle manipulations that cause aggregation increase lung accumulation drastically, making the lung the major organ for microparticle accumulation (unpublished data). Our presented data is also consistent with findings reported by Liu et al¹⁵ in which hydrogel microparticles (1-5 microns) rapidly clear from the lung while nanoparticles (20-40 nm) have prolonged lung retention.”

To determine if in vitro cell-type specific interactions also occurr in vivo, we examined the impact of particle surface presentation on in vivo associations with endothelial cells and macrophages (Figure 3b). Liver and spleen tissues, harvested 24 hours after intravenous microparticle injection, were labeled with antibodies specific for macrophage scavenger receptor (CD204) and for endothelial CD34. In the spleen, silicon microparticles were predominately located in the splenic cords and did not appear to associate with either endothelia or macrophages. However, in the liver, silicon microparticles were associated with macrophages, regardless of original surface chemistry. Cationic microparticles were also found in association with hepatic vascular endothelial cells.

As stated previously, accumulation of silicon microparticles was low in the lungs and tumor (Figure 3a), despite high macrophage content. Immunohistochemistry analysis of tissues from mice injected with FITC-labeled silicon microparticles (anionic) also supported heavy uptake by liver macrophages, with low macrophage uptake in the spleen and lungs (Figure 4a). Thus it appears that tissue-specific macrophages differ in their binding affinities for opsonized microparticles. This specificity may be driven in part by the unique repertoire of cell surface receptors expressed by each macrophage tissue subtype. Tissue TEM images show both cationic and anionic silicon microparticles inside liver macrophages (Figure 4b).

Figure 4.

Tissue specific uptake of silicon microparticles by macrophages. A) Histological evaluation of in vivo microparticle association with macrophages from liver, lung, and spleen (100x oil immersion lens; bars 10 μm). B) Tissue TEM images showing silicon microparticles internalized by hepatic macrophages (Kupffer cells). Each tissue is presented at 10 and 60 k magnification [bars 10 μm (left) and 1 μm (right)].

Cellular associations in hepatic blood vessels and sinusoids were examined by SEM 1-2 hours after injection of anionic microparticles. Figure 5 (top left) shows a scanning electron micrograph of an open blood vessel (pseudo-colored in red) surrounded by hepatocytes (green). The upper right image shows a red blood cell in the opening of a sinusoid. In the lower micrographs, anionic silicon microparticles (blue) can be seen attached to the vascular wall (shown at two magnifications). Cellular pseudopodia can be seen engaging the microparticles.

Figure 5.

In vivo imaging of hepatic blood vessels and sinusoids. Scanning electron micrographs of murine hepatic tissue slices following injection of silicon microparticles. A) An open blood vessel surrounded by hepatocytes pseudo-colored in green. B) An RBC in the opening of a sinusoid. C-D) Two silicon particles (pseudo-colored in blue) engaged by cellular constituents lining the vessel wall.

Hepatic tissue from mice injected with cationic silicon microparticles is shown in Figure 6. The boxed region of the blood vessel (top left image) is progressively magnified in the subsequent images. Among white blood cells is a cationic silicon microparticle. Cellular competition for the microparticle is seen between an endothelial cell, shown below the microparticle, and cellular constituents lining the vessel wall (seen above the microparticle). In the pseudo-colored image, endothelial cells are shown in red, lymphocytes in green, and the silicon microparticle in blue. Higher magnification images, showing detailed cellular interactions with both cationic (left) and anionic (right) silicon microparticles, are presented in Figure 7.

Figure 6.

In vivo cellular interactions with discoidal porous silicon microparticles. Microparticles were injected intravascularly into nude mice, followed 2 hrs post injection by vascular perfusion and tissue collection. The boxed region of the hepatic blood vessel in the upper left image is show at increasing magnification in the succeeding scanning electron micrographs (bars 400, 30, 10, 5, 2 μm). A silicon microparticle is shown near the center of each image and is displayed in blue in the final image.

Figure 7.

In vivo cellular interactions with silicon microparticles. High magnification scanning electron micrographs show diverse cellular constituents competing for a cationic silicon microparticle (left) and engagement of an anionic (oxidized) silicon microparticle by cells lining the vascular wall (right).

Identification of serum components selectively binding to silicon microparticles

Silicon microparticles with opposable surface charges were incubated with murine plasma for 30 minutes, followed by washing with PBS, and removal of bound proteins using a protocol adapted ffrom the National Cancer Institute's National Characterization Laboratory (NCL; www.ncl.cancer.gov). Proteins were separated by 2DGE and following automated spot detection proteins with normalized spot volumes greater than ± 25-fold different between the two populations (Figure 8) were subsequently robotically picked, trypsin-digested, and peptide masses identified by MALDI TOF. Using this criteria, 11 proteins were identified, all displaying higher selectivity for cationic microparticles (Table 1). The proteins fell into three categories: (1) Ig light chain variable region; (2) fibrinogen alpha polypeptide; and (3) complement component 1. Spots having the same protein ID are a common occurrence and may represent post-translational modifications, including proteolysis.

Figure 8.

2D-Gel analysis of particle binding proteome. A. Eluted proteins from OX-treated beads. B. Eluted proteins from APTES-treated beads. Spot key: 1 – Ig light chain V region (clone 17s.2); 2 – complement component 1q, C chain; 3 – Ig light chain V region (clone 17s.2); 4, 5 – alpha fibrinogen, polypeptide isoform 2; 6 – apolipoprotein E; 7 – apolipoprotein A; 8 – anti-ds-DNA immunoglobulin light chain V region; 9 – alpha fibrinogen, polypeptide isoform 2; 10 – γ - fibrinogen; 11, 12 – alpha fibrinogen, polypeptide isoform 2; 13 - Ig light chain V region (clone 17s.2); 14 – complement component 1q, B chain; 15 – Ig light chain V region (clone 17s.2); 16 – alpha fibrinogen.

Table 1.

Summary of selected protein spot performance. Spot numbers refer to numbered spots in Figure 6. Only spots exhibiting greater than 25-fold abundance in the APTES treated samples (Fig. 7B) are included. “q-value” represents the false discovery rate.²⁷

Protein name	Accession No.	Spot Number	MS ID Expectation Value	Protein Abundance	Statistics
				Fold	Anova (p)	q Value	Power
Ig light chain V region (clone 17s.2) - mouse (fragment)	gi|284919	1	1.25594E-05	26.06	3.66E-04	9.60E-03	1.00
complement component 1, q subcomponent, C chain	gi|399145	2	7.92447E-20	36.71	3.58E-04	9.60E-03	1.00
Ig light chain V region (clone 17s.2) - mouse (fragment)	gi|284919	3	1.58114E-14	30.04	1.01E-03	1.16E-02	1.00
fibrinogen, alpha polypeptide isoform 2 [Mus musculus]	gi|33563252	4	3.15479E-24	69.26	1.13E-05	3.91E-03	1.00
Ig light chain V region (clone 17s.2) - mouse (fragment)	gi|284919	5	7.92447E-12	34.40	1.22E-04	8.43E-03	1.00
anti-ds-DNA immunoglobulin light chain V region [Mus musculus]	gi|736420	8	3.97164E-14	27.19	2.70E-04	9.35E-03	1.00
fibrinogen, alpha polypeptide, isoform CRA_b [Mus musculus]	gi|148683477	9	1.25594E-05	9.51	1.23E-02	2.63E-02	0.89
fibrinogen, alpha polypeptide isoform 2 [Mus musculus]	gi|33563252	10	3.97164E-68	25.14	1.10E-03	1.16E-02	1.00
fibrinogen, alpha polypeptide isoform 2 [Mus musculus]	gi|33563252	11	1.58114E-65	9.51	1.23E-02	2.63E-02	0.89
fibrinogen, alpha polypeptide isoform 2 [Mus musculus]	gi|33563252	12	1.58114E-75	25.52	1.05E-03	1.16E-02	1.00
Ig light chain V region (clone 17s.2) - mouse (fragment)	gi|284919	13	0.000250594	27.87	4.07E-03	1.71E-02	0.99
complement component 1q, B chain	gi|115202	14	3.97164E-14	23.94	1.65E-03	1.30E-02	1.00
Ig light chain V region (clone 17s.2) - mouse (fragment)	gi|284919	15	7.92447E-11	25.24	3.03E-04	9.54E-03	1.00

Anionic microparticles were more selective in protein binding. None of the proteins eluted from the microparticles had spot volumes that differed by greater than 9-fold. Although not strikingly greater, there was selective binding of apolipoproteins A and E on anionic microparticles (Figure 6, spots 6 and 7), with 1.8 and 3.9-fold greater binding (ANOVA 0.962 and 0.218, respectively).

Discussion

Particle surface charge influences the extent and specificity of binding to serum proteins, which further regulates cellular associations and biodistribution. In vitro endothelial associations of anionic silicon microparticles are significantly reduced in the presence of plasma proteins or pure IgG. Conversely, cationic silicon microparticles continue to adhere to and be internalized by endothelial cells in the presence of serum. This is consistent with findings by Donald McDonald and colleagues¹⁶ who have demonstrated that cationic, but not anionic, neutral, or sterically stabilized liposomes associate with the luminal surface of tumor endothelial cells and are subsequently internalized. Marc Dellian et al. have also reported selective accumulation of cationic liposomes on vascular endothelium.¹⁷

We identified three categories of proteins absorbed to cationic silicon microparticles: fibrinogen alpha polypeptide, Ig light chain variable region, and complement component 1. Fibrinogen alpha polypeptide, a component of fibrinogen, is a blood-borne glycoprotein composed of three pairs of polypeptide chains. It is 644 amino acids and approximately 79 kDa. Various cleavage products of fibrinogen regulate cell adhesion and spreading, making fibrinogen-coated components likely candidates to regulate adhesion of particles to specific cell populations. Fibrinogen nanoparticles are reported to be internalized by L929 (murine fibroblast) cells,¹⁸ supporting a role for fibrinogen components in cellular adhesion and uptake.

Complement fragments bound to microorganisms promote leukocyte chemoattraction and phagocytosis. Various cell types have specific receptors for complement, with CR1 being the best characterized. C1 is a complex of three molecules, C1q, C1r, and C1s. C1r and C1s are proteases while C1q (460 kDa) binds to antigen-bound antibody via the Fc region, promoting binding to phagocytes.¹⁹ C1q is the heterotrimeric C terminal globlular region of C1. Different allotypes differ in their net surface charge and areas of charged clusters mediate interactions. ²⁰ Association of antibody and complement components to cationic silicon microparticles may thus favor interaction with professional phagocytes (i.e. macrophages), while binding to non-professional phagocytes (i.e. endothelial cells) may be mediated by the diverse combination of opsonins bound to the particle surface. The abundance of Ig and fibrinogen components adsorbed to cationic microparticles may also reflect non-specific entrapment of abundant serum components accompanying particle aggregation, which was observed during the PBS wash steps following incubation of cationic silicon microparticles with plasma.

It has been reported that “neutral” particles in serum have an outer protein corona consisting of fibrinogen, IgG, and albumin.²¹ As just stated, both fibrinogen and Ig variable light chain were abundantly (>25-fold) present on cationic silicon microparticles compared to anionic silicon microparticles, however albumin was identified as binding to both functionalities, with selectivity never exceeding 8.5-fold for any of its putative forms. The zeta potential of unbound cationic silicon microparticles at pH 7.4 is approximately 6 mV. The low charge density may reflect the porous nature of the microparticles and may render the APTES-modified microparticles more similar to neutrally-charged particles than their nonporous counterparts. However, the slight positive charge of the APTES-modified particles may also explain adsorption by fibrinogen, based on the negative surface charge of this molecule. ²²

Contrary to these findings, we also showed by flow cytometry that IgG is preferentially adsorbed to anionic microparticles. Atomic force microscopy analysis of porous silicon chips incubated with pure IgG, demonstrates binding of IgG to both cationic and anionic porous silicon (Supplemental Figure 1&2). However, the presentation of immunoglobulin on the silicon chips is dissimilar and supports unique antibody binding orientations. Presentation of the Fc portion of the antibody on microparticles would be expected to inhibit uptake by endothelial cells, which express little or no Fc gamma receptors, ¹⁴ and simultaneously increase uptake by macrophages.

Proteins bound to anionic microparticles include apolipoproteins A and E. Apolipoproteins bind to lipids to aid in their transport in the bloodstream and to provide an anchor for LDL receptors on target cells. Apolipoprotein A ranges in size from 419-838 kDa ²³, with diameters of 3.8- 4.6 nm, and isoelectric points of 5.36-5.62.²⁴ On the other hand, Apolipoprotein E has 200 amino acids and is approximately 35 kDa.²⁵ Apolipoprotein E (Apo E) consists of two domains, a lipid associating domain and a globular domain containing the LDL receptor binding site.²⁶ The latter Apo E domain is arginine-rich and may aid in binding to silicon microparticles that have a net negative surface charge density. The presence of Apo E on the surface of anionic silicon microparticles may aid in delivery of oxidized microparticles to the liver.

Immunohistochemistry images of liver and spleen following intravenous administration of microparticles supported phagocytic uptake of both microparticle formulations by liver macrophages, as well as selective association of cationic microparticles with hepatic endothelial cells. We demonstrated a greater uptake of cationic microparticles in the spleen, compared to the liver. This may reflect lower levels of uptake of cationic, compared to anionic, microparticles by Kupffer cells in the liver and thereby greater interaction with other classes of cells, including vascular endothelial cells. Scanning electron micrographs support competition of different classes of phagocytic cells for cationic silicon microparticles. We have also demonstrated that, despite high macrophage content in the lungs, microparticles predominately exist independent of lung macrophages and are present in low abundance. Based on these results, as well as the low tumor accumulation of microparticles, it appears that macrophage uptake of both cationic and anionic silicon microparticles is predominately restricted to liver Kupffer cells. Mechanisms that lower Kupffer cell uptake of microparticles may thus result in strategic localization of microparticles extracellularly, permitting sustained release of secondary nanoparticles, and ultimately therapeutic and/or imaging agents.

In summary, microparticle surface charge and serum opsonization of particles plays a major role in cellular associations, internalization, and biodistribution. Engineering drug delivery vectors for selective binding to serum components represents a mechanism to potentially direct cellular interactions and thus mass transport of therapeutic agents, as well as imaging agents and theranostics.

Non-standard abbreviations

APTES

3-aminopropyltriethoxysilane

HMDS

hexamethyldisilazane

IPA

isopropyl alcohol

SEM

scanning electron microscopy

4-NBA

4-nitrobenzaldehyde