Protein MRI Contrast Agents as an Effective Approach for Precision Molecular Imaging.

ABSTRACT: Cancer and other acute and chronic diseases are results of perturbations of common molecular determinants in key biological and signaling processes. Imaging is critical for characterizing dynamic changes in tumors and metastases, the tumor microenvironment, tumor-stroma interactions, and drug targets, at multiscale levels. Magnetic resonance imaging (MRI) has emerged to be a primary imaging modality for both clinical and preclinical applications due to its advantages over other modalities, including sensitivity to soft tissues, nondepth limitations, and the use of nonionizing radiation. However, extending the application of MRI to achieve both qualitative and quantitative precise molecular imaging with the capability to quantify molecular biomarkers for early detection, staging, and monitoring therapeutic treatment requires the capacity to overcome several major challenges including the trade-off between metal-binding affinity and relaxivity, which is an issue frequently associated with small chelator contrast agents. In this review, we will introduce the criteria of ideal contrast agents for precision molecular imaging and discuss the relaxivity of current contrast agents with defined first shell coordination water molecules. We will then report our advances in creating a new class of protein-targeted MRI contrast agents (ProCAs) with contributions to relaxivity largely derived from the secondary sphere and correlation time. We will summarize our rationale, design strategy, and approaches to the development and optimization of our pioneering ProCAs with desired high relaxivity, metal stability, and molecular biomarker-targeting capability, for precision MRI. From first generation (ProCA1) to third generation (ProCA32), we have achieved dual high r1 and r2 values that are 6- to 10-fold higher than clinically approved contrast agents at magnetic fields of 1.5 T, and their relaxivity values at high field are also significantly higher, which enables high resolution during small animal imaging. Further engineering of multiple targeting moieties enables ProCA32 agents that have strong biomarker-binding affinity and specificity for an array of key molecular biomarkers associated with various chronic diseases, while maintaining relaxation and exceptional metal-binding and selectivity, serum stability, and resistance to transmetallation, which are critical in mitigating risks associated with metal toxicity. Our leading product ProCA32.collagen has enabled the first early detection of liver metastasis from multiple cancers at early stages by mapping the tumor environment and early stage of fibrosis from liver and lung in vivo, with strong translational potential to extend to precision MRI for preclinical and clinical applications for precision diagnosis and treatment.

Early Detection and Staging of Lung Fibrosis Enabled by Collagen-Targeted MRI Protein Contrast Agent.

Chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are major leading causes of death worldwide and are generally associated with poor prognoses. The heterogeneous distribution of collagen, mainly type I collagen associated with excessive collagen deposition, plays a pivotal role in the progressive remodeling of the lung parenchyma to chronic exertional dyspnea for both IPF and COPD. To address the pressing need for noninvasive early diagnosis and drug treatment monitoring of pulmonary fibrosis, we report the development of human collagen-targeted protein MRI contrast agent (hProCA32.collagen) to specifically bind to collagen I overexpressed in multiple lung diseases. When compared to clinically approved Gd3+ contrast agents, hProCA32.collagen exhibits significantly better r1 and r2 relaxivity values, strong metal binding affinity and selectivity, and transmetalation resistance. Here, we report the robust detection of early and late-stage lung fibrosis with stage-dependent MRI signal-to-noise ratio (SNR) increase, with good sensitivity and specificity, using a progressive bleomycin-induced IPF mouse model. Spatial heterogeneous mapping of usual interstitial pneumonia (UIP) patterns with key features closely mimicking human IPF, including cystic clustering, honeycombing, and traction bronchiectasis, were noninvasively detected by multiple MR imaging techniques and verified by histological correlation. We further report the detection of fibrosis in the lung airway of an electronic cigarette-induced COPD mouse model, using hProCA32.collagen-enabled precision MRI (pMRI), and validated by histological analysis. The developed hProCA32.collagen is expected to have strong translational potential for the noninvasive detection and staging of lung diseases, and facilitating effective treatment to halt further chronic lung disease progression.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. METHODS: Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. RESULTS: Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. CONCLUSIONS: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Positron Emission Tomography Imaging of Vessel Wall Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm.

BACKGROUND: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, 64Cu-RYM2. METHODS: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. 64Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. RESULTS: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. 64Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. CONCLUSIONS: 64Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for 64Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.

Differential BMP Signaling Mediates the Interplay Between Genetics and Leaflet Numbers in Aortic Valve Calcification.

Expression of a neuropilin-like protein, DCBLD2, is reduced in human calcific aortic valve disease (CAVD). DCBLD2-deficient mice develop bicuspid aortic valve (BAV) and CAVD, which is more severe in BAV mice compared with tricuspid littermates. In vivo and in vitro studies link this observation to up-regulated bone morphogenic protein (BMP)2 expression in the presence of DCBLD2 down-regulation, and enhanced BMP2 signaling in BAV, indicating that a combination of genetics and BAV promotes aortic valve calcification and stenosis. This pathway may be a therapeutic target to prevent CAVD progression in BAV.

Computed tomography imaging of macrophage phagocytic activity in abdominal aortic aneurysm.

Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe-/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe-/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe-/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.

Hydroxamate-Based Selective Macrophage Elastase (MMP-12) Inhibitors and Radiotracers for Molecular Imaging.

Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.

Molecular imaging of extracellular matrix proteins with targeted probes using magnetic resonance imaging.

The extracellular matrix (ECM) consists of proteins and carbohydrates that supports different biological structures and processes such as tissue development, elasticity, and preservation of organ structure. Diseases involving inflammation, fibrosis, tumor invasion, and injury are all attributed to the transition of the ECM from homeostasis to remodeling, which can significantly change the biochemical and biomechanical features of ECM components. While contrast agents have played an indispensable role in facilitating clinical diagnosis of diseases using magnetic resonance imaging (MRI), there is a strong need to develop novel biomarker-targeted imaging probes for in vivo visualization of biological processes and pathological alterations at a cellular and molecular level, for both early diagnosis and monitoring drug treatment. Herein, we will first review the pathological accumulation and characterization of ECM proteins recognized as important molecular features of diseases. Developments in MRI probes targeting ECM proteins such as collagen, fibronectin, and elastin via conjugation of existing contrast agents to targeting moieties and their applications to various diseases, are also reviewed. We have also reviewed our progress in the development of collagen-targeted protein MRI contrast agent with significant improvement in relaxivity and metal binding specificity, and their applications in early detection of fibrosis and metastatic cancer. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Biology-Inspired Nanomaterials > Peptide-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Chemokine receptor 4 targeted protein MRI contrast agent for early detection of liver metastases.

Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (r 1 = 30.9 mM-1 s-1, r 2 = 43.2 mM-1 s-1, 1.5 T; r 1 = 23.5 mM-1 s-1, r 2 = 98.6 mM-1 s-1, 7.0 T), strong CXCR4 binding (K d = 1.10 ± 0.18 µM), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm3. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.

Early detection and staging of chronic liver diseases with a protein MRI contrast agent.

Early diagnosis and noninvasive detection of liver fibrosis and its heterogeneity remain as major unmet medical needs for stopping further disease progression toward severe clinical consequences. Here we report a collagen type I targeting protein-based contrast agent (ProCA32.collagen1) with strong collagen I affinity. ProCA32.collagen1 possesses high relaxivities per particle (r1 and r2) at both 1.4 and 7.0 T, which enables the robust detection of early-stage (Ishak stage 3 of 6) liver fibrosis and nonalcoholic steatohepatitis (Ishak stage 1 of 6 or 1 A Mild) in animal models via dual contrast modes. ProCA32.collagen1 also demonstrates vasculature changes associated with intrahepatic angiogenesis and portal hypertension during late-stage fibrosis, and heterogeneity via serial molecular imaging. ProCA32.collagen1 mitigates metal toxicity due to lower dosage and strong resistance to transmetallation and unprecedented metal selectivity for Gd3+ over physiological metal ions with strong translational potential in facilitating effective treatment to halt further chronic liver disease progression.

Precision detection of liver metastasis by collagen-targeted protein MRI contrast agent.

The Liver is the most common organ for metastasis for various cancers, including uveal melanoma, the most common primary intraocular tumor. Uveal melanoma metastasizes to the liver in ~90% of patients, and results in death in almost all cases due to late detection and lack of effective treatment. There is a pressing unmet medical need to develop MRI contrast agents and imaging methodologies with desired sensitivity and specificity to overcome the high heterogeneous background and in vivo properties as well as reduced toxicity. Herein, we report the development of a collagen targeting protein contrast agent (ProCA32.collagen1), since collagen is a diagnostic biomarker and therapeutic target for many types of primary and metastatic cancers and the tumor microenvironment. In addition to a strong affinity to collagen I, ProCA32.collagen1 possesses high relaxivities (r1 and r2 are 68.0 ±â€¯0.25 and 100.0 ±â€¯0.32 mM-1 s-1 at 1.4 T, respectively, and 42.6 ±â€¯1.0 and 217 ±â€¯2.4 mM-1s-1 at 7.0 T per particle). ProCA32.collagen1 also has strong serum stability against degradation, resistance to transmetallation, and 102 and 1013-fold higher metal selectivity for Gd3+ over Ca2+ and Zn2+, respectively, compared to clinical contrast agents. ProCA32.collagen1 does not exhibit any cell toxicity for various cell lines. Sensitive detection of liver lesions in animal models can be achieved using multiple imaging methodologies, taking advantage of the dual relaxation property of ProCA32.collagen1. ProCA32.collagen1 enables sensitive and early stage detection of hepatic micrometastasis as small as 0.144 mm2 and two different tumor growth patterns. Further development of ProCA32.collagen1 has the potential to greatly facilitate non-invasive, early detection and staging of primary and metastatic liver cancers, and devising effective treatments.

Designing Calcium-Binding Proteins for Molecular MR Imaging.

Early diagnosis, noninvasive detection, and staging of various diseases, remain one of the major clinical barriers to effective medical treatment and prevention of disease progression toward major clinical consequences. Molecular imaging technologies play an indispensable role in the clinical field in overcoming these major barriers. The increasing application of imaging techniques and agents in early detection of different diseases such as cancer has resulted in improved treatment response and clinical patient management. In this chapter we will first introduce criteria for the design and engineering of calcium-binding protein (CaBP) parvalbumin as a protein Gd-MRI contrast agent (ProCA) with unprecedented metal selectivity for Gd3+ over physiological metal ions. We will then discuss the further development of targeted MRI contrast agent for molecular imaging of PSMA biomarker for early detection of prostate cancer.

Direct visualization of interaction between calmodulin and connexin45.

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI.

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd(3+) contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd(3+) binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 µM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10(-22) M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM(-1) s(-1) and r2 of 37.9 mM(-1) s(-1) per Gd (55.2 and 75.8 mM(-1) s(-1) per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM(-1) s(-1) per Gd (188.0 mM(-1) s(-1) per molecule) and r1 of 18.6 mM(-1) s(-1) per Gd (37.2 mM(-1) s(-1) per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.

GRPR-targeted Protein Contrast Agents for Molecular Imaging of Receptor Expression in Cancers by MRI.

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd(3+) binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM(-1)s(-1) at 1.5 T and 25 °C) and strong Gd(3+) selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.

Protein MRI contrast agent with unprecedented metal selectivity and sensitivity for liver cancer imaging.

With available MRI techniques, primary and metastatic liver cancers that are associated with high mortality rates and poor treatment responses are only diagnosed at late stages, due to the lack of highly sensitive contrast agents without Gd(3+) toxicity. We have developed a protein contrast agent (ProCA32) that exhibits high stability for Gd(3+) and a 10(11)-fold greater selectivity for Gd(3+) over Zn(2+) compared with existing contrast agents. ProCA32, modified from parvalbumin, possesses high relaxivities (r1/r2: 66.8 mmol(-1)⋅s(-1)/89.2 mmol(-1)⋅s(-1) per particle). Using T1- and T2-weighted, as well as T2/T1 ratio imaging, we have achieved, for the first time (to our knowledge), robust MRI detection of early liver metastases as small as ∼0.24 mm in diameter, much smaller than the current detection limit of 10-20 mm. Furthermore, ProCA32 exhibits appropriate in vivo preference for liver sinusoidal spaces and pharmacokinetics for high-quality imaging. ProCA32 will be invaluable for noninvasive early detection of primary and metastatic liver cancers as well as for monitoring treatment and guiding therapeutic interventions, including drug delivery.

Application of mercapto ordered carbohydrate-derived porous carbons for trace detection of cadmium and copper ions in agricultural products.

In this paper, we have introduced nanoporous carbon modified with mercapto groups as a new solid-phase method for extraction of cadmium(II) and copper(II) ions. The modified nanoporous carbon sorbent was characterised by thermogravimetric analysis, differential thermal analysis, transmission electron microscopy, Fourier transform infrared spectrometry, X-ray diffraction, and nitrogen adsorption surface area (BET) measurements. Effects of pH value, flow rates, type, concentration and volume of the eluent, breakthrough volume, and effect of other ions were studied. The experimental results show that simultaneous trace cadmium(II) and copper(II) ions can be quantitatively preconcentrated at pH 6.0 with recoveries >97%. Under optimised conditions, limits of detection are 0.04 and 0.09 ng mL(-1) for the ions of cadmium and copper respectively, and the precision of the method for analysis of cadmium and copper ions (5.0 µg of each target ions, N=8) are 2.4% and 2.1%, respectively. The obtained capacities of mercapto-nanoporous carbon were found to be 145 and 95 mg g(-1) for cadmium and copper ions, respectively. The accuracy of the proposed procedure was verified by analysing standard reference material. Finally, the introduced sorbent was successfully applied for trace determination of cadmium and copper ions in food samples.