A Nano-sized PARACEST-fluorescence Imaging Contrast Agent Facilitates and Validates in vivo CEST MRI Detection of Glioma

Meser M Ali; Mohammed PI Bhuiyan; Branislava Janic; Nadimpalli RS Varma; Tom Mikkelsen; James R Ewing; Robert A Knight; Mark D Pagel; Ali S Arbab

Disclosures

Nanomedicine. 2012;7(12):1827-1837. 

In This Article

Abstract and Introduction

Abstract

Aim: The authors have investigated the usefulness of in vivo chemical exchange saturation transfer MRI for detecting gliomas using a dual-modality imaging contrast agent.

Materials & methods: A paramagnetic chemical exchange saturation transfer MRI contrast agent, Eu-1,4,7,10-tetraazacclododecane-1,4,7,10-tetraacetic acid-Gly4 and a fluorescent agent, DyLight® 680, were conjugated to a generation 5 polyamidoamine dendrimer to create the dual-modality, nano-sized imaging contrast agent.

Results: The agent was detected with in vivo chemical exchange saturation transfer MRI in an U87 glioma model. These results were validated using in vivo and ex vivo fluorescence imaging.

Conclusion: This study demonstrated the merits of using a nano-sized imaging contrast agent for detecting gliomas and using a dual-modality agent for detecting gliomas at different spatial scales.

Introduction

Exogenous, relaxivity-based MRI contrast agents are often used to improve image contrast by altering the T1, T2 and/or T2* relaxation rates of tissues.[1] Similarly, fluorescent contrast agents can also generate high-contrast images.[2] These exogenous agents can improve diagnostic sensitivity for detecting pathological lesions that have abnormal pharmacokinetic rates of accumulating the agents, relative to normal tissues. However, all tissues have endogenous magnetic resonance (MR) relaxation contrast mechanisms and also produce auto-fluorescence. All vascularized tissues are capable of accumulating contrast agents. Thus, normal tissues with abnormal contrast may be mistaken for pathological lesions, so MRI and fluorescence imaging can suffer from poor diagnostic specificity.

Within the last decade, a new class of MRI contrast agents has been developed, which depend on the mechanism of chemical exchange saturation transfer (CEST).[3] These agents possess a chemical functional group with a labile proton, such as an amide or hydroxyl group, that has a MR frequency (chemical shift) that differs from the chemical shift of water by approximately 1–5 ppm. Selective radio frequency saturation of the chemical shift of the labile proton eliminates the MR signal from the proton in the sample. The subsequent exchange of this proton with a proton on a water molecule effectively transfers the saturation to water, which reduces the MR signal from water in the sample. The CEST effect was first demonstrated with small diamagnetic molecules, such as amino acids, sugars, nucleic acids and heterocyclic ring compounds.[4] To improve the difference in chemical shifts between the agent and water, metal ions have been incorporated into chemical structures to create paramagnetic CEST (PARACEST) agents.[5,6] Some PARACEST agents have been designed to noncovalently bind to a water molecule. For example, Eu-1,4,7,10-tetraazacclododecane-1,4,7,10-tetraacetic acid (DOTA)-Gly4 can generate CEST via the bound water.[5]

PARACEST agents have strong potential to improve the specificity of MRI diagnoses. For instance, the chemical exchange of amide protons is base-catalyzed and therefore PARACEST agents with amides can be used to measure pH and detect tumor acidosis, which may improve specificity for detecting tumors relative to normal tissues.[7,8] Furthermore, CEST agents that can detect the activities of tumor protease enzymes by monitoring the conversion of amides to amines during peptide cleavage have been developed, which may further improve diagnostic specificity.[9–11] Additionally, CEST agents can detect metabolites such as nitric oxide, which is an important paracrine signaling messenger for promoting tumor angiogenesis, and can further improve diagnostic specificity.[12] Importantly, the ability to selectively detect multiple PARACEST agents during a single MRI scan session may provide the ability to evaluate multiple biomarkers, which may multiply the specificity of tumor detection with MRI.[13]

Unfortunately, PARACEST agents have relatively poor detection sensitivity. As a rough estimate, a detectable CEST effect that saturates 1% of approximately 110 M water protons requires approximately 10 mM of a PARACEST agent, with one labile proton and a chemical exchange rate of approximately 100 Hz. Small molecule PARACEST agents with multiple labile protons at the same chemical shift and/or with faster chemical exchange rates can generate a detectable CEST effect at concentrations of approximately 1 mM in chemical solutions (note: a faster exchange rate cannot exceed the chemical shift difference between the agent and water, otherwise the CEST effect will be decreased).[3] However, this minimum concentration (1 mM) of exogenous PARACEST agents is not suitable for in vivo studies, because endogenous proteins have labile protons that generate a magnetization transfer effect that is similar to CEST, which competes with the CEST agents for exchange with water.[14] To date, few reports have demonstrated in vivo detection of exogenous small molecule PARACEST agents.[7,8,13,15–21]

To improve in vivo detection sensitivity, PARACEST agents have been conjugated to nanocarriers such as dendrimers, linear polymers and other high molecular weight macromolecules, such as adenovirus particles.[22–24] For example, the authors have previously conjugated an Eu-DOTA-Gly4 PARACEST agent to a generation 5 polyamidoamine (G5PAMAM) dendrimer via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/Nhydroxysuccinimide (NHS) coupling, and demonstrated that injection of the agent into a mouse model of mammary carcinoma generated CEST in tumor tissue.[17] Although this preliminary result showed that the CEST image contrast was statistically significant, the result was not validated using other methods, which raised concern that the change in image contrast may have originated from conditions other than the CEST agent, such that the agent had not necessarily accumulated in the tumor tissue. Other conditions that may dynamically change image contrast, include T2 relaxation effects, endogenous magnetization transfer or magnetic susceptibility that affects the direct saturation of water. Similarly, all other in vivo MRI studies with PARACEST agents that have been published to date have not been validated using other imaging methods.[7,8,13,15–21]

Dual-modality contrast agents are ideally suited for the validation of imaging results, because the conditions that may confound the interpretation of one imaging modality rarely affect the other. Furthermore, a dual-modality contrast agent can be designed to exploit the strengths of two complimentary imaging modalities, which can multiply the value of the agent for biomedical imaging.[25] MRI is often used for presurgical planning to identify the macroscopic features of pathological tissues, and fluorescence imaging is emerging as a useful intrasurgical tool to identify microscopic margins of pathological tissues.[26,27] Therefore, conjugating fluorescent agents and PARACEST agents to a dendrimer is a synergistic approach.

In order to validate the in vivo detection of a PARACEST agent, the authors investigated the development of a fluorescent PARACEST MRI contrast agent using a G5PAMAM dendrimer as the nanocarrier (Figure 1). The author and colleagues used a rat model of U87 human glioma for the in vivo study, in order to apply their imaging methods to a major diagnostic problem.[28] The accumulation of a nano-sized imaging agent in a hypervascular glioma can improve diagnostic specificity by distinguishing the glioma from peritumoral edema.[29] The glioma can compromise the blood–brain barrier and allow accumulation of polar, nano-sized contrast agents that are typically impermeable to this barrier, which further improves diagnostic specificity for detecting the tumor.[30] Therefore, the development of a nano-sized, dual-modality MRI contrast agent may have excellent utility for the diagnoses of gliomas, especially the tumor margin, and may improve treatment planning.

Figure 1.

Synthesis of the dual-modality contrast agent. (1) Eu-DOTA-Gly4, NHS (1.8 equivalents), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (4.5 equivalents), 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 0°C, 1 h; (2) G5PAMAM (0.072 equivalent), phosphatebuffered saline, pH 7.4, room temperature, 24 h; 37% yield (42 Eu-DOTA-Gly4 units among 128 NH3 + polyamidoamine chain termini); and (3) Eu-G5PAMAM (70 µmol), DyLight® 680 NHS ester (1 equivalent), phosphate-buffered saline, room temperature, 24 h, 93% yield. The average stoichiometry is listed for each ligand. The chemical exchange saturation transfer effect arises from the bound water of EuDOTAGly4.
Eu-DOTA-Gly4: Eu-1,4,7,10-tetraazacclododecane-1,4,7,10-tetraacetic acid-Gly4; G5: Generation 5; G5PAMAM: Generation 5 polyamidoamine; NHS: N-hydroxysuccinimide.

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