Core-shell hydrogel particles harvest, concentrate and preserve labile low abundance biomarkers

Abstract

Background: The blood proteome is thought to represent a rich source of biomarkers for early stage disease detection. Nevertheless, three major challenges have hindered biomarker discovery: a) candidate biomarkers exist at extremely low concentrations in blood; b) high abundance resident proteins such as albumin mask the rare biomarkers; c) biomarkers are rapidly degraded by endogenous and exogenous proteinases.

Methodology and principal findings: Hydrogel nanoparticles created with a N-isopropylacrylamide based core (365 nm)-shell (167 nm) and functionalized with a charged based bait (acrylic acid) were studied as a technology for addressing all these biomarker discovery problems, in one step, in solution. These harvesting core-shell nanoparticles are designed to simultaneously conduct size exclusion and affinity chromatography in solution. Platelet derived growth factor (PDGF), a clinically relevant, highly labile, and very low abundance biomarker, was chosen as a model. PDGF, spiked in human serum, was completely sequestered from its carrier protein albumin, concentrated, and fully preserved, within minutes by the particles. Particle sequestered PDGF was fully protected from exogenously added tryptic degradation. When the nanoparticles were added to a 1 mL dilute solution of PDGF at non detectable levels (less than 20 picograms per mL) the concentration of the PDGF released from the polymeric matrix of the particles increased within the detection range of ELISA and mass spectrometry. Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.

Conclusions and significance: We envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

Conflict of interest statement

Competing Interests: The technology described herein is licensed (patent pending) to the authors institutions (George Mason University, USA and Istituto Superiore di Sanita', Italy). Under faculty guidelines the University authors can received a share of the patent royalties owned by the university.

Figures

Figure 1. Bait chemistry.

Figure 2. Schematic illustration of core shell particle.

The nanoparticle consists of a NIPAm-AAc core that functions as a bait. After adding particles to the protein solution, biomarkers are attracted and entrapped in this bait. A NIPAm shell increases the sieving properties of nanoparticles.

Figure 3. Light scattering and atomic force microscopy characterization of nanoparticles.

(A) At room temperature, core is approximately 360 nm in size whereas adding core-shell particles have a diameter of 700 nm at pH 4.5. Core and core shell particles follow a typical temperature dependent behavior. (B) Particle suspension in MilliQ water (pH 5.5, 1 µg/mL) was deposited on freshly cleaved mica under humid atmosphere at room temperature for 15 minutes and dried under nitrogen. Atomic force microscopy (AFM) image of nanoparticles was acquired. Particles have a diameter of approximately 800 nm and exhibit a homogeneous size distribution. The scale bar for particle height shows a maximum height of 168 nm. The AFM picture was acquired under dry status therefore the particles are distorted (flattened) from their spherical shape due to drying on the mica surface.

Figure 4. SDS-PAGE Analysis of PDGF incubated particles.

(A) Lane 1) Starting solution containing BSA and PDGF (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles remove PDGF from carrier albumin with a total exclusion of albumin itself. (B) Lane 1) Starting solution containing PDGF, BSA, aprotinin (MW 6,500 Da), lysozyme (MW 14,400 Da), trypsin inhibitor (MW 21,500 Da), carbonic anhydrase (MW 31,000 Da), and ovalbumin (MW 45,000 Da) (Control), 2) Supernatant (OUT); 3) Particle content (IN). Particles harvest PDGF together with low molecular weight proteins and exclude proteins above ca 20,000 Da.

Figure 5. Core shell particles raise the concentration of undetectable PDGF into the detection range of ELISA assay.

(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 18.92+/−4.313 pg/mL and PDGF eluted from core-shell particles (85.27+/−2.24 pg/mL). (B) PDGF concentration in the core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.

Figure 6. Core shell particles increase the concentration of extremely dilute PDGF approximately 10-folds (1000 percent) as measured by ELISA assay.

(A) ELISA readings of the starting solution of PDGF in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 63.69+/−1.448 pg/mL and PDGF eluted from core-shell particles (491.14+/−4.818 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.

Figure 7. Core shell particles increase the concentration of native PDGF in serum as measured by ELISA assay.

(A) ELISA readings of the starting serum solution in Calibrator diluent RD6-3 (R&D Systems, animal serum with preservatives) at a concentration of 170.91+/−4.66 pg/mL and PDGF eluted from core-shell particles (1743.43+/−11.06 pg/mL). (B) PDGF concentration in core-shell particle eluate plotted against the quantity of particles utilized for the incubation, duplicate experiments. (C) ELISA standard curve of PDGF concentration versus absorbance. The standard curve was generated with two repeats for each PDGF calibrator concentration.

Figure 8. SDS PAGE analysis showing chemokines uptake by particles.

Core-shell particles were incubated with the following chemokines, mucosae-associated epithelial chemokine (MEC/CCL28), stromal cell-derived factor-1 beta, (SDF-1β/CXCL12b), and eotaxin-2 (CCL24), in presence of bovine serum albumin (BSA). Solutions of the chemokines and BSA are shown in lanes 1, 4, and 7. After incubation with the particles, no chemokine was left in the supernatant (S, lane 2, 5, and 8) and all the chemokine was captured by particles (P, lanes 3, 6, and 9). BSA was totally excluded by particles.

Figure 9. Immunoblot analysis showing that core-shell particles protect captured PDGF from tryptic degradation.

(A) Sypro ruby total protein staining and (B) Immunoblot analysis with anti-PDGF antibody of the same PVDF membrane are presented. Lane 1) control PDGF+BSA solution; 2) content of particles incubated with PDGF+BSA (IN); 3) supernatant of particles incubated with PDGF+BSA (OUT); 4) content of particles incubated with BSA+PDGF+trypsin (IN+TRYPSIN); 5) supernatant of particles incubated with BSA+PDGF+trypsin (OUT+TRYPSIN); 6) BSA+PDGF+trypsin without particles incubated for 40 minutes (+TRYPSIN 40′); 7)) BSA+PDGF+trypsin without particles incubated for 20 minutes (+TRYPSIN 20′); 8)) BSA+PDGF+trypsin without particles incubated for 10 minutes (+TRYPSIN 10′); 9)) BSA+PDGF+trypsin without particles incubated for 0 minutes (+TRYPSIN 0′).

Figure 10. SDS PAGE analysis showing that core-shell particles protect chemokines from enzymatic degradation.

Core-shell particles were incubated with the following chemokines, mucosae-associated epithelial chemokine (MEC/CCL28), stromal cell-derived factor-1 beta, (SDF-1β/CXCL12b), and eotaxin-2 (CCL24), in presence of trypsin. Solution of the chemokines (control) are shown in Lanes 1, 4, and 7. Chemokines incubated with particles (Lane 3, 6, and 9) are protected from tryptic degradation whereas chemokines not incubated with particles (Lane 2, 5, and 8) are susceptible to proteolytic digestion.

Figure 11. Immunoblot analysis demonstrating recovery of PDGF spiked in human serum.

Lane 1) Human serum plus PDGF (5 ng/µL): when serum is not incubated with particles, PDGF cannot be detected; 2) particle supernatant (OUT); 3) particle content (IN); 4) Human serum plus PDGF (2 ng/µL): when serum is not incubated with particles, PDGF cannot be detected; 2) particle supernatant (OUT); 3) particle content (IN).