Davide Tamburro

Multifunctional Core–Shell Nanoparticles: Discovery of Previously Invisible Biomarkers

Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core–shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(N-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (KD < 10–11 M) for specific low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable.

Investigation of the Ovarian and Prostate Cancer Peptidome for Candidate Early Detection Markers Using a Novel Nanoparticle Biomarker Capture Technology

Current efforts to identify protein biomarkers of disease use mainly mass spectrometry (MS) to analyze tissue and blood specimens. The low-molecular-weight “peptidome” is an attractive information archive because of the facile nature by which the low-molecular-weight information freely crosses the endothelial cell barrier of the vasculature, which provides opportunity to measure disease microenvironment-associated protein analytes secreted or shed into the extracellular interstitium and from there into the circulation. However, identifying useful protein biomarkers (peptidomic or not) which could be useful to detect early detection/monitoring of disease, toxicity, doping, or drug abuse has been severely hampered because even the most sophisticated, high-resolution MS technologies have lower sensitivities than those of the immunoassays technologies now routinely used in clinical practice. Identification of novel low abundance biomarkers that are indicative of early-stage events that likely exist in the sub-nanogram per milliliter concentration range of known markers, such as prostate-specific antigen, cannot be readily detected by current MS technologies. We have developed a new nanoparticle technology that can, in one step, capture, concentrate, and separate the peptidome from high-abundance blood proteins. Herein, we describe an initial pilot study whereby the peptidome content of ovarian and prostate cancer patients is investigated with this method. Differentially abundant candidate peptidome biomarkers that appear to be specific for early-stage ovarian and prostate cancer have been identified and reveal the potential utility for this new methodology

The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease.

Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm)-acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via amidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine.

Analysis of urinary human growth hormone (hGH) using hydrogel nanoparticles and isoform differential immunoassays after short recombinant hGH treatment: preliminary results.

Successful application clinical-grade human growth hormone (hGH) immunoassays to the discovery of illegal doping cases has been rare. Indeed, the preferred biological matrix in doping control is urine, where the estimated baseline concentration of hGH falls well below the linear range and sensitivity threshold of all commercially available immunoassays, including hGH isoform differential immunoassays which can discriminate pituitary endogenous hGH from recombinant hGH. We employed hydrogel nanoparticles as a pre-processing step that concentrate urinary hGH into the linear range of isoform differential immunoassays. We explored the characteristics of immunoassays in urine spiked with both phGH or rhGH, after pre-treatment with the nanoparticles. Subsequently, pre-treatment was applied to urine obtained from 3 healthy volunteers administered during three days with daily subcutaneous injections of 0.026 mg/kg/day rhGH, Genotonorm(®). Linearity between both rhGH and phGH concentrations in urine measured by a chemoluminescent assay (Immulite) and in the particle eluate was evident for differential immunoassays (R square higher than 0.999). In case of treated individuals the recombinant/pituitary concentration ratios remained above the established World Anti-Doping Agency (WADA) criterion for hGH misuse up to 24h after the last administration dose, using both assays for volunteer 1 and 2 while in case of volunteer 3 results were inconclusive. The use of nanoparticles appears to open the possibility of assessing rhGH misuse in urine.

Mass Spectrometry-based characterization of the vitreous phosphoproteome

Purpose: The vitreous gel is a highly hydrated extracellular matrix containing many proteins. These proteins are likely accumulated in the vitreous by local secretion, filtration from the blood, or diffusion from the surrounding tissues and vasculature, and may be altered in disease state. In the last several years, several reports of large‐scale profiling of vitreous proteins have been published; however, there is little information on the characterization of the phosphoproteome of vitreous. Here, we sought to identify phosphopeptides and their phosphorylation sites from vitreous.

Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression.

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low‐molecular‐weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high‐grade STS sera using hydrogel core‐shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty‐two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non‐metastatic tumors. Cox‘s regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H‐related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.

Abstract 4787: Novel upfront sample enrichment nanotechnology identifies prostate, breast, colon, ovarian, lung, pancreatic, and melanoma cancer specific biomarker candidates

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BACKGROUND: Routine laboratory blood tests for biomarkers could dramatically simplify and improve cancer diagnosis, prognosis and tailored therapy. The tumor and its microenvironment, produce cascades of information that enrich the blood proteome with potential cancer biomarkers that could be highly informative about the establishment, progression and metastatic dissemination of cancer. Nevertheless, the identification of low abundance and labile cancer specific biomarkers in blood remains extremely challenging. Low abundance markers are masked by high abundance proteins such as albumin, and by non-specific markers whose changes accompany many diseases. In this study, we analyzed a large cohort of human serum samples from a wide-range of cancer conditions and benign states, and utilized a rapid and powerful nanoparticle-based biomarker capture technology. METHODS- We utilized isopropylacrylamide (NIPAm) core shell hydrogel particles, functionalized with two chemical baits, Cibacron blue F3GA and vinylsulfonic acid, for biomarker capture from 450 serum/plasma samples. Twenty age and stage-matched cancer and 20 to 40 benign/inflammatory controls were processed for each of the 7 cancer types, and proteins eluted from the particles were analyzed by high resolution LC-MS/MS analysis using an LTQ-Orbitrap (Thermo Fisher). All specimens were analyzed in a randomized, batch fashion, followed by an accurate comparison of the results in order to 1) identify a panel of biomarkers for each cancer type and distinguish protein which are involved in commons cancer mechanism and proteins which could uniquely indicate a particular cancer type and 2) avoid the technological bias that could be associated with the comparison of separated studies. RESULTS: For each cancer type we obtained a panel of hundreds of candidates biomarkers, a large number of them not previous identified by MS and/or never measured in plasma. Within the proteins identified are kinases, cytokines, growth factors, structural and nuclear proteins. Each panel of candidate biomarkers includes a) proteins unique for a cancer type, such as the tyrosine kinase Tec, specific for prostate cancer; b) proteins common to many cancer types such as, PRKAG2, CD9, Lipocalin 2, Annexin I, and SDFR-1 and c) numerous proteins whose role in cancer is novel or not well-described. CONCLUSIONS: Cancer-specific low abundance marker candidates have been identified using NanoTrap ™ nanoparticles, a novel rapid upfront sample processing biomarker capture technique. We are developing a protocol for the validation of the biomarkers based on MRM-MS. Further investigation will be required to understand the biological meaning and clinical value of the proteins identified. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4787. doi:1538-7445.AM2012-4787

Abstract 4585: Verification of prostate cancer biomarker candidates by nanoparticle-capture MRM

Prostate adenocarcinoma (PCa) is highly diffuse between older men, and biomarkers that allow discrimination of cancerous pathologies from benign diseases, identification of clinical aggressive cancer with high specificity and monitor cancer progression are required. Recently we applied a new workflow for the discovery of candidate biomarkers based on hydrogel nanoparticle capture of LMW serum borne analytes. Comparing serum from pre and post prostatectomy patients we identified 112 candidates biomarkers. Our novel nanoparticle technology provides a rapid and near 100% efficient means to capture and greatly amplify the resting concentration of LMW protein analytes. Since most of these markers do not have well performing specific antibodies commercially available, we utilized MRM to verify their differential expression as first identified by Orbitrap-MS/MS. Hydrogel nanoparticle serum pre-processing was used for both Orbitrap-LTQ based discovery and MRM verification such that the same analyte fractionation workflow was used but different downstream mass spectrometry. MRM was performed on a Thermo Quantum triple quadrupole-MS using the nanoparticle trapped analyte archive as the input for analysis. The following biomarker candidates were selected for MRM verification: Apolipoprotein E, Diacylglycerol kinase alpha, complement 1 subcomponent q B chain precursor, complement component 1 q subcomponent gamma polypeptide, serpin peptidase inhibitor, vitronectin precursor, angiotensinogen preproprotein, H4 histone family member J, fibronectin 1 isoform 2, orosomucoid 1 precursor, alpha-2-HS-glycoprotein, annexin I. Several prostate candidate biomarkers were verified by Nanoparticle capture-MRM, thus providing data supporting the use and continued improvement of this novel biomarker discovery system for the identification of protein biomarker panels for early disease detection and therapeutic monitoring. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4585.

Abstract 4575: Covalent incorporation of a new class of dye-based affinity baits in sieving hydrogel nanoparticles overcome roadblocks to cancer biomarker discovery

Cancer biomarker discovery and clinical application has been seriously hampered by low abundance, instability and complex formation with high abundance proteins (e.g. albumin). N-isopropylacrylamide (NIPA) based hydrogel particles, consisting of a bait-containing core and a protective shell, overcome these major issues associated to biomarker discovery and measurement. Particles perform in one step, in solution, a) total sequestration and concentration, b) size sieving and c) complete protection form degradation of LMW protein biomarkers in blood and urine. In this study, novel dye molecules were selected as affinity ligands for broad classes of biomarkers; dyes were chosen for their ability to bind proteins, glycoproteins and nucleic acid for tissue histology, to bind textile proteins for fabric staining or for their known property to bind allergens. Dyes containing primary amine group (e.g. Acid Black 48, Basic Red 9) were coupled to NIPA/acrylic acid (AAc) particles by condensation of the amine group the carboxylic group. The ability of particles to capture and concentrate analytes was tested against a panel of low abundance, labile tumor relevant biomarker that include angiogenic growth factors (bFGF, VEGF, PDGF), IGF-1 and IGF binding proteins, interleukins (IL-6, IL-8), chemokines (CXCL12, CCL24), pro-apoptotic Bcl-2 proteins (Bak, Bax), and nucleic acids. Technologies used to screen dye functionalized particles include 1 D gel electrophoresis, western blotting, immunoassay, and mass spectrometry. Results showed that dye based baits have extremely high affinity for the target analytes so that particles capture all the analyte present in solution and that the affinity of biomarkers to the bait is unique and specific to the chemical structure of the bait molecule. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4575.