In vivo multicolor molecular MR imaging using diamagnetic chemical exchange saturation transfer liposomes

Abstract

A variety of (super)paramagnetic contrast agents are available for enhanced MR visualization of specific tissues, cells, or molecules. To develop alternative contrast agents without the presence of metal ions, liposomes were developed containing simple bioorganic and biodegradable compounds that produce diamagnetic chemical exchange saturation transfer MR contrast. This diamagnetic chemical exchange saturation transfer contrast is frequency-dependent, allowing the unique generation of "multicolor" images. The contrast can be turned on and off at will, and standard images do not show the presence of these agents. As an example, glycogen, L-arginine, and poly-L-lysine were encapsulated inside liposomes and injected intradermally into mice to image the lymphatic uptake of these liposomes. Using a frequency-dependent acquisition scheme, it is demonstrated that multicolor MRI can differentiate between different contrast particles in vivo following their homing to draining lymph nodes. Being nonmetallic and bioorganic, these diamagnetic chemical exchange saturation transfer liposomes form an attractive novel platform for multicolor imaging in vivo.

Figure 1. Multi-color spectrum of DIACEST liposomes (DLs)

a) Artificial colors are assigned according to the exchangeable proton chemical shifts for a variety of diamagnetic agents, which range from 0 to 7 ppm. b) Cartoon displaying the components of DLs; c) MTR_asym plot showing the water signal intensity reduction as a function of frequency for three DLs (~30 nM) in vitro (pH=7.3 and 37°C), with red assigned to OH in Glyc DL, yellow assigned to NH₂ in L-Arg DL, and green assigned to NH in PLL DL.

Figure 2. Time course of DL uptake in PLN from ¹¹¹In SPECT imaging

Liposomes (140 or 100 nm) were injected into the footpads of a vaccinated mouse. a) CT image showing the injection sites indicate by yellow arrowhead. b) Average number of radioactive counts per voxel within the PLN as a function of time after injection of ¹¹¹In encapsulated liposomes. c-f) Planar SPECT images 5, 7, 23, and 46 hours after injection of liposomes (arrow=PLN). g) 3D tomographic CT/SPECT overlay image 46 hours after injection. Arrows indicate PLN locations.

Figure 3. Representative color MR images using different DL combinations

a) Unilateral Glyc liposomes, b) Unilateral L-Arg liposomes c) Unilateral PLL liposomes, and d) Bilateral LArg and PLL liposomes. Arrowheads in a-d) indicate the location of PLNs. For a-c), shown are from left to right: T2-weighted anatomical image, MTR_asym image at the frequency of interest with CEST contrast displayed using the jet color map in Matlab, MTR_asym/T2w image overlay with CEST contrast highlighted using a 64-bit scaled single color (i.e. red, yellow or green) map, and mean MTR_asym plot. In the mean MTR_asym plot, the values shown are of DL(+) PLN (solid line), DL(−) PLN (dashed line), and their difference (colored line). d) Representative Two-color CEST image demonstrating simultaneous detection and visualization of PLL (green, left PLN) and L-Arg (yellow, right PLN), with the mean MTR_asym values of the left (L) and right (R) PLN plotted in green and yellow, respectively. e) Comparison of CEST contrast in DL(+) nodes (grey columns) and in DL(−) nodes (white columns) for all three animal groups studied (n=3 mice for Glyc and n=4 mice for L-Arg, PLL) with the black columns showing the difference between them. The p values were calculated using a student t test (type II, two tails with SD marked by error bars); f) H&E stain and g) fluorescent image of a PLN with rhodamine-labeled liposomes in red and cell nuclei in blue (DAPI stain); h) merged immunofluorescent image of anti-CD45.1-allophycocyanin (pan-leukocyte marker, green) and rhodamine-labeled liposomes (red) demonstrates extracellular location of DLs without uptake by dendritic cells or macrophages.