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.

Non-invasive detection and complementary diagnosis of liver metastases via chemokine receptor 4 imaging.

Noninvasive detection of early-stage liver metastases from different primary cancers is a pressing unmet medical need. The lack of both molecular biomarkers and the sensitive imaging methodology makes the detection challenging. In this study, we observed the elevated expression of chemokine receptor 4 (CXCR4) in uveal melanoma (UM) patient liver tissues, and high CXCR4 expression in liver metastases of UM murine models, regardless of the expression levels in the primary tumors. Based on these findings, we identified CXCR4 as an imaging biomarker and exploited a CXCR4-targeted MRI contrast agent ProCA32.CXCR4 for molecular MRI imaging. ProCA32.CXCR4 has strong CXCR4 binding affinity, high metal selectivity, and r1 and r2 relaxivities, which enables the sensitive detection of liver micrometastases. The MRI imaging capacity for detecting liver metastases was demonstrated in three UM models and one ovarian cancer model. The imaging results were validated by histological and immunohistochemical analysis. ProCA32.CXCR4 has strong potential clinical application for non-invasive diagnosis of liver metastases.

Adsorption of proteins on oral Zn2+ doped iron oxide nanoparticles in mouse stomach and in vitro: triggering nanoparticle aggregation.

Oral route is one of the most important portals of nanoparticle entry to the body. However, in vivo protein corona formed in the gastrointestinal tract has not been studied owing to the difficulty for the recovery of nanoparticles from the in vivo environment. In this study, by using the magnetic property of iron oxide nanoparticles (Fe3O4 NPs) and Zn2+ doped iron oxide nanoparticles (Zn0.4Fe2.6O4 NPs), the nanoparticles were separated from the gastric fluid after oral administration in mice. The effects of Zn2+ doping and static magnetic field (SMF) treatment on the protein adsorption on the nanoparticles were investigated in vitro and in vivo. Zn2+ doping decreases the adsorption of pepsin on the nanoparticles in vitro and affects the composition of the protein corona in vivo and enhances protein adsorption-induced aggregation of the nanoparticles in vitro and in vivo. SMF treatment affects the composition of the protein corona of Fe3O4 NPs and Zn0.4Fe2.6O4 NPs, and enhances the aggregation of Fe3O4 NPs and Zn0.4Fe2.6O4 NPs in vivo. Furthermore, the results demonstrate that electrostatic attraction is the crucial force to drive adsorption of proteins on Fe3O4 NPs and Zn0.4Fe2.6O4 NPs and protein adsorption-induced change in the surface charge of nanoparticles plays an important role in the pH-dependent aggregation of the nanoparticles. In addition, the work provides the evidence that the protein adsorption-induced aggregation of Fe3O4 NPs and Zn0.4Fe2.6O4 NPs has no effect on their magnetic susceptibility. The results highlight that Zn0.4Fe2.6O4 NPs may be used as a potential oral magnetic resonance imaging contrast agent in diagnosis of gastrointestinal disease.

Evidence of Translocation of Oral Zn2+ Doped Magnetite Nanoparticles Across the Small Intestinal Wall of Mice and Deposition in Spleen: Unique Advantage in Biomedical Applications.

Nanomaterials have been widely applied in oral drug delivery. A number of indirect evidences suggest that nanoparticles can pass across gastrointestinal walls to enter the blood circulation system. However, there is still no direct evidence to prove that the intact nanoparticles can pass across gastrointestinal walls and the nanoparticles can retain their original structure after translocation across gastrointestinal walls. In the present study, the potential toxicity of dimercaptosuccinic acid coated Zn2+ doped magnetite nanoparticles (DMSA-Zn0.4Fe2.6O4) in the spleen, stomach, and small intestine of mice has been investigated after 30 days of repeated intragastric administration. We provide first direct evidence that intact DMSA-Zn0.4Fe2.6O4 can pass across the small intestinal barriers to enter blood circulation system and arrive in the spleen. In addition, our findings provide direct evidence that although the biotransformation of DMSA-Zn0.4Fe2.6O4 occurs in vivo, some DMSA-Zn0.4Fe2.6O4 retain their original structure after translocation across the small intestinal wall and deposition in the spleen. The results indicate the safety of DMSA-Zn0.4Fe2.6O4 in the applications in mice at a 50 mg/kg dose and highlight the unique advantage of DMSA-Zn0.4Fe2.6O4 in biomedical applications.

INOS-mediated acute stomach injury and recovery in mice after oral exposure to halloysite nanotubes.

Natural halloysite nanotubes (HNTs) with a hollow lumen are already applied in numerous fields and enter the environment in increasing quantities, which may have effects on animal and human health. However their in vivo toxicity in mammals is still largely unclear. The aim of this study is to assess acute oral toxicity of HNTs in the stomach of mice and recovery. Oral HNTs at low dose (5 mg HNTs/kg BW) for 30 days increased in daily food and water intake and promoted mouse growth with no obvious adverse effect on the stomach. The promotive effect on mouse growth disappeared after cessation of oral administration of the nanotubes. Oral HNTs for 30 days at high dose (50 mg HNTs/kg BW) induced Si and Al accumulation in the stomach, which caused oxidative stress, inflammation and iNOS-mediated damage in the organ. The damage in the stomach led to slight atrophic gastritis and reduced mouse growth. Oral HNTs-induced changes at high dose were not observed after a 30-days recovery period. The findings provided the evidence that oral HNTs-induced acute toxicity in the stomach was reversible. More importantly, this research showed that Al and Si were cleared out of the mice by hepatic excretion and renal excretion, respectively, during the recovery period. The results suggest that HNTs at low concentration in environments have no adverse effect on mice, while there are health risks to mice under severe contamination by HNTs.

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.

Halloysite Nanotubes-Induced Al Accumulation and Fibrotic Response in Lung of Mice after 30-Day Repeated Oral Administration.

Natural halloysite (Al2Si2O5(OH)4· nH2O) nanotubes (HNT) are clay materials with hollow tubular structure and are widely applied in many fields. Many in vitro studies indicate that HNTs exhibit a high level of biocompatibility; however, the in vivo toxicity of HNTs remains unclear. In this study, the biodistribution and pulmonary toxicity of the purified HNTs in mice were investigated after intragastric administration for 30 days. HNTs have high stability in biological conditions. Oral administration of HNTs caused significant Al accumulation predominantly in the lung with relative slight effects on Si biodistribution. Oral administration of HNTs stimulated the growth of the mice at low dose (5 mg/kg BW) with no pulmonary toxicity but inhibited the mouse growth and resulted in oxidative stress and inflammation in lung at high dose (50 mg/kg BW). In addition, oral HNTs at high dose could be absorbed from the gastrointestinal tract and deposited in lung and could also induce pulmonary fibrosis.

Halloysite nanotubes-induced Al accumulation and oxidative damage in liver of mice after 30-day repeated oral administration.

Halloysite (Al2 Si2 O5 (OH)4 ·nH2 O) nanotubes (HNTs) are natural clay materials and widely applied in many fields due to their natural hollow tubular structures. Many in vitro studies indicate that HNTs exhibit a high level of biocompatibility, however the in vivo toxicity of HNTs remains unclear. The objective of this study was to assess the hepatic toxicity of the purified HNTs in mice via oral route. The purified HNTs were orally administered to mice at 5, 50, and 300 mg/kg body weight (BW) every day for 30 days. Oral administration of HNTs stimulated the growth of the mice at the low dose (5 mg/kg BW) with no liver toxicity, but inhibited the growth of the mice at the middle (50 mg/kg BW) and high (300 mg/kg BW) doses. In addition, oral administration of HNTs at the high dose caused Al accumulation in the liver but had no marked effect on the Si content in the organ. The Al accumulation caused significant oxidative stress in the liver, which induced hepatic dysfunction and histopathologic changes. These findings demonstrated that Al accumulation-induced oxidative stress played an important role in the oral HNTs-caused liver injury.