Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease

Abstract

Biomarkers are increasingly important in the clinical management of complex diseases, yet our ability to discover new biomarkers remains limited by our dependence on endogenous molecules. Here we describe the development of exogenously administered `synthetic biomarkers' composed of mass-encoded peptides conjugated to nanoparticles that leverage intrinsic features of human disease and physiology for noninvasive urinary monitoring. These protease-sensitive agents perform three functions in vivo: target sites of disease, sample dysregulated protease activities and emit mass-encoded reporters into host urine for multiplexed detection by mass spectrometry. Using mouse models of liver fibrosis and cancer, we show that they can noninvasively monitor liver fibrosis and resolution without the need for invasive core biopsies and can substantially improve early detection of cancer compared with clinically used blood biomarkers. This approach of engineering synthetic biomarkers for multiplexed urinary monitoring should be broadly amenable to additional pathophysiological processes and to point-of-care diagnostics.

Point-of-care diagnostics for noncommunicable diseases using synthetic urinary biomarkers and paper microfluidics

“…We previously showed that synthetic biomarkers composed of long-circulating NWs accumulate in diseased tissue by diffusing across fenestrated vessels such as in liver fibrosis and cancer (8), or when the sites of disease are intravascular as in thrombosis, sense protease activity while in systemic circulation (9). These studies also showed that free peptides administered i.v.…”

“…To develop synthetic biomarkers for thrombosis and cancer, we first sought to design NPs for sensing the activity of the proteases thrombin and matrix metalloproteinase 9 (MMP9). We functionalized poly(ethylene glycol)-coated iron oxide nanoworms (NWs)-a long-circulating NP formulation previously characterized by collaborators and our laboratory (19,20)-with fluorescein-labeled derivatives of thrombin-and MMP9-cleavable substrates [PLGLRSW and PLGVRGK, respectively (8)] at a surface valency of 20-30 peptides per NW to induce intermolecular quenching (Fig. 1A).…”

“…To confirm that our synthetic biomarkers tailored for thrombin and MMP9 exhibit similar pharmacokinetics, we synthesized NWs with substrates labeled with carrier peptidelinked near-infrared fluorophores to monitor peptide traffic and cleavage by in vivo fluorescence imaging. To promote renal clearance and to enable in vivo fluorescent visualization of the peptidefluorophore reporter released by substrate proteolysis, we conjugated the near-infrared fluorophore VT750 (N-terminal) to the peptide glutamate-fibrinopeptide B (GluFib) (sequence eGvndneeGffsar), which we synthesized with D-amino acids (lowercase) to confer stability against protease activity (8,9,21).…”

“…Radiolabeled glucose is an example of a common exogenous agent used with PET to reveal the location of metabolically active tumors. Inspired by engineered approaches, our group recently outlined a framework whereby protease-sensitive nanoparticles (NPs) called "synthetic biomarkers" are administered to detect NCDs including liver fibrosis, cancer, and thrombosis noninvasively (8,9). These peptidecoated NPs probe diseased sites and, in response to cleavage by local dysregulated proteases, release mass-encoded reporters that then filter into the urine for analysis by mass spectrometry.…”

Stimuli-sensitive intrinsically disordered protein brushes

“…The extensive suite of proteases and protease recognition sites-any of which could be introduced into the sequence at one or more arbitrary positions-should provide a versatile toolbox for controlling IDP brush properties in a dynamic and specific manner. Analogous enzymatically addressable materials have been explored for a variety of applications such as payload delivery 42 , diagnostic monitoring 43 and ligand presentation [44][45][46] . Although much effort has been focused on understanding the biological function of endogenous IDPs, our study suggests that these proteins represent an untapped resource for biomaterial design.…”