Cell labeling via membrane-anchored lipophilic MR contrast agents

Abstract

Cell tracking in vivo with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are retained by cells. Most clinically approved Gd(III)-based contrast agents require high incubation concentrations and prolonged incubation times for cellular internalization. Strategies to increase contrast agent permeability have included conjugating Gd(III) complexes to cell penetrating peptides, nanoparticles, and small molecules which have greatly improved cell labeling but have not resulted in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd(III)-based MR contrast agents that label cell membranes in vitro. Two of the agents were synthesized with a multiplexing strategy to contain three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). These new agents exhibit significantly enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells (4 and Prohance).

Figure 1

Three lipophilic and two nonlipophilic contrast agents are described. 1 and 2 are multimeric MR contrast agents that contain three Gd(III) chelates while 3, 4, and Prohance are monomeric complexes. 4 and Prohance were selected as controls because they accumulate in the cytosol whereas the lipophilic agents label the cell membrane.

Scheme 1. Synthesis of Multimeric Lipophilic Contrast Agents That Contain a Single or Double Alkyl Chain

Agents were synthesized with “click” chemistry and contain three Gd(III) chelates. The resultant complexes have limited water solubility and were solubilized in 4 mM cholate.

Scheme 2. Synthesis of Monomeric Lipophilic Contrast Agent 3 Prepared with “Click”Chemistry

The agent is soluble in water and 4 mM cholate.

Figure 2

Concentration-dependent cell labeling for 1–4 and Prohance. HeLa cells incubated with normalized contrast agent (A) or normalized Gd(III) concentrations (B) for 24 h. MDA-MB-231-mcherry cells incubated with normalized contrast agent (C) or normalized Gd(III) concentrations (D) for 24 h. Error bars represent ± standard deviation of the mean of triplicate samples.

Figure 3

T₁-weighted cell pellet images of HeLa cells incubated with 1–4 and Prohance acquired at 7 T. A: Probe concentration was equalized at 20 μM. B: Gd(III) concentration was equalized at 90 μM. For both images TE = 11 ms, TR = 500 ms, MTX = 256 × 256, and slice thickness is 1.0 mm. Scale bars represent 1 mm. Error represents ±1 standard deviation of the mean of four 1-mm slices. These images show that 3 produces the most significant contrast enhancement compared to untreated cells.

Figure 4

Localization of contrast agents determined by cell fractionation. The internalized (cytosol + endosomes) and membrane fraction were analyzed by ICP-MS for Gd(III) content. All lipophilic complexes show higher membrane accumulation than Prohance with 2 having the greatest membrane localization. Error bars represent ± standard deviation of the mean of triplicate samples.

Figure 5

Cellular proliferation and retention of contrast agents 1–3 and Prohance after 72 h of leaching in HeLa, MDA-MB-231-mcherry, and NIH/3T3 cell lines. A: Cellular proliferation was determined by calculating the fold difference in cell count at t = 0 and 72 h. The data show that proliferation is not affected by labeling with lipophilic contrast agents. B: Cellular retention was determined by calculating the fold change in Gd(III) per cell at t = 72 and 0 h. The data show that, while retention is influenced by cell line selection, 1 and 2 have consistently enhanced retention compared to 3 and Prohance.