Fluorescence-based Quantification of Gadolinium-bound Liposomes using Carbostyril 124-sensitized DTPA
Lindsay Kathleen Hill1,2, Stewart Russell3,4, Dung Minh Hoang1, and Youssef Zaim Wadghiri1

1Radiology, NYU School of Medicine, New York, NY, United States, 2Biomedical Engineering, SUNY Downstate Medical Center, Brooklyn, NY, United States, 3Thayer School of Engineering, Dartmouth College, Hanover, NH, United States, 4Department of Mechanical Engineering, The City College of New York, New York, NY, United States

Synopsis

Advancement in the field of Gadolinium-bound contrast agent discovery is reliant on the development and characterization of novel constructs made in-house. However, the assessment of physicochemical properties and in vivo pharmacokinetics, requiring highly sensitive measurements, is often impeded by the lack of analytical techniques that are simultaneously sensitive, affordable, and accessible. Here we demonstrate that Carbostyril 124-sensitized DTPA can be incorporated into a lipid-based microparticle, allowing for rapid quantification of Gadolinium concentration with nanomolar sensitivity using a readily available fluorescence plate reader. This sensitive and convenient technique could rapidly propel the characterization of novel MR contrast agents.

Purpose

Gadolinium (Gd)-based paramagnetic contrast agents, including Gd-bound micelles and liposomes, are increasingly used preclinically in MR for diagnostic studies. Advancement in this field relies on the in-house engineering of new constructs. Critical to this development is the characterization of physicochemical properties and in vivo pharmacokinetics, requiring highly sensitive Gd concentration measurements. Inductively coupled plasma mass spectrometry (ICP-MS) is the highly sensitive gold standard, but it is expensive and requires specialized equipment1. NMR relaxometry is more affordable, but requires a large sample volume due to limited sensitivity2. We previously demonstrated an alternative strategy using a Carbostyril 124 (cs124)-sensitized DTPA to quantify Gd agents at the nanomolar levels3 with a fluorescence plate reader widely available in research labs. Here we show that cs124-sensitized DTPA can be extended to multi-Gd based agents, as demonstrated in a lipid-based microparticle. As these agents are increasingly investigated for MR angiography and molecular imaging due to the amplification effect of multiple Gd ions on contrast enhancement, the need to quickly and accurately characterize their concentration and clearance kinetics is critical.

Background

Here the cs124-sensitized DTPA lipid construct, cs124-GdDTPA-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (cs124-GdDTPA-DPPE), shown in Fig. 1(A), is the energy donor and unsensitized Terbium-DTPA (TbDTPA) is the acceptor. Excitation of cs124-Gd does not result in fluorescence emission, but instead creates a long-lived excited electron energetic state that is transferred to Tb by collision. Because Tb is not fluorescent without a sensitizer, time-resolved detection of Tb’s characteristic fluorescence spectrum is proportional to the concentration of sensitized Gd.

Methods

The construct cs124-DTPA-DPPE was sonicated in deionized water and chelation of Gd was performed in deionized water for 1hour at 90°C (10:1 GdCl3:cs124-DTPA-DPPE). Unchelated Gd was separated from the construct through a 3kDa filter. Dynamic light scattering (DLS) was used to determine the size distribution of the filtered product. TbDTPA was generated by combining TbCl3 and DTPA 1:1 in deionized water for 1hour at 90°C.

Determination of Gd concentration

Absorbance measurements were made for cs124-GdDTPA-DPPE, and then for DTPA alone, which was subtracted from the raw signal to normalize the cs124 signal of the lipid-based particle. The concentration of cs124 was determined to be C=A ε-1 L-1, where A is absorbance, L=1cm, and ε was taken to be the same as that of cs124-DTPA alone, 1.07x104 M-1cm-1, as previously described3. Based on the construct design, the concentration of Gd is equal to that of cs124.

Fluorescence Assay

cs124-GdDTPA-DPPE particles were plated in triplicate in a black 96-well plate at concentrations ranging from 35μM to 10-10M in 100μl deionized water, followed by 10μl of 1nM TbDTPA per well. The plate was excited at 330nm and emission at 480nm was integrated from 600μs to 2000μs. The collision model of energy transfer3 was fit to fluorescence, showing that the energy transfer characteristics from these particles to Tb are the same as for molecular cs124-GdDTPA.

MRI

2D T1 and T2 maps were obtained on a 7-Tesla Bruker system. Phantoms were created using clinical Gd-DTPA (Magnevist) and cs124-GdDTPA-DPPE particles diluted in HEPES physiological buffer, pH 7, to concentrations of 5μM-0.5mM and 5μM-50μm, respectively. From the acquired relaxation times, T1 and T2, we determined the relaxivities, r1 and r2, of Magnevist and our particles at 7T. The ratio r2/r1 was calculated to demonstrate their effectiveness as T1 agents.

Results

DLS of Gd-chelated cs124-DTPA-DPPE revealed an average diameter of 1.2μm (Fig. 1(B)), suggesting the formation of liposomes of the giant unilamellar vesicle or multilamellar vesicle subtype4. The liposomes’ absorption spectrum revealed a maximum absorbance at 330nm, which was used to calculate the concentration of Gd in our sample to be 35μM (Fig. 2). Time-resolved fluorescence showed a detection limit of 10nM Gd (Fig. 3), which is comparable to ICP-MS1.

Both a T1-weighted image as well as T1 and T2 maps of the liposomes revealed profound T1-shortening as compared to Magnevist. The T1-weighted signal of 0.05mM Gd-liposomes, in red, was comparable to that of 10x the Magnevist concentration, 0.5mM (Fig. 4). Liposome relaxivity r1 was 26.5x higher than that of Magnevist and the liposomes’ r2/r1 of 1.04 shows promise for the sensitized lipid construct’s incorporation into novel T1 agents (Table 1).

Conclusion

We show that cs124-GdDTPA-DPPE lipid can be incorporated into mesoscale Gd-bound lipid-based particles, and can be used to quantify Gd concentration by fluorescence. Notably, all absorbance and fluorescence studies were conducted on a spectrophotometer equipped for time resolved studies, which delivers rapid results and is found in most laboratory settings. This expedient and sensitive system may significantly propel optimization studies of novel lipid-based MR contrast agents made in-house.

Acknowledgements

This work was supported in part by the NYU Applied Research Support Fund and resource support from NIH/NCI 5P30CA016087-32 and NIH P41 EB017183.

References

[1] Telgmann L, et al. Identification and quantification of potential metabolites exposed to different gadolinium-based MRI contrast agents: evaluation with inductively coupled plasma mass spectrometry (ICP-MS). J Chromatogr A. 2012; 1240: 147-155. [2] Bourasset F, et al. Comparison of plasma and peritoneal concentrations of various categories of MRI blood pool agents in a murine experimental pharmacokinetic model. MAGMA. 2001; 12(2-3): 82-87. [3] Russell S, et al. Quantification of the plasma clearance kinetics of a gadolinium-based contrast agent by photoinduced triplet harvesting. Anal Chem. 2012; 84(19): 8106-8109. [4] Torchilin VP, et al. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov. 2005; 4(2): 145-160.

Figures

Fig. 1 (A) Chemical structure of cs124-GdDTPA-DPPE, where cs124 is highlighted in orange. (B) Size distribution of the cs124-GdDTPA-DPPE construct in aqueous solution at 25°C determined by dynamic light scattering (N=4), showing monodispersed particles with an average radius of 575nm. This size is suggestive of giant unilamellar or multilamellar liposomes.

Fig. 2 Absorption spectra of DTPA (blue), cs124-GdDTPA-DPPE (red), and their difference (green and inset) showing the maximum absorption of cs124 at 330nm. The concentration of cs124 was calculated using ε(cs124330nm) = 1.07x104M-1cm-1.

Fig. 3 Calibration curve of the cs124-GdDTPA-DPPE construct determined by time-resolved fluorescence emission of TbDTPA. Data points (circles) each represent N=3 replicates, which were fit to a collision model (dashed line p < 5x10-5). Sensitivity ranged from the millimolar range to sub-micromolar.

Fig .4 Diagram and T1-weighted image of phantom tubes containing either HEPES or HEPES-diluted Magnevist (black text) or HEPES-diluted cs124-GdDTPA-DPPE particles (red text and circle). A water tube was used as reference (yellow arrow). The signal from 0.05mM cs124-GdDTPA-DPPE is comparable to that of a ten-fold higher Magnevist concentration (0.5mM).

Table 1 Relaxivity values, r1 and r2, for Magnevist and cs124-GdDTPA-DPPE as determined using T1 and T2 phantom maps at 7-Telsa. Each relaxation curve contained at least four concentrations of each agent. The ratio r2/r1 corresponds to the effectiveness as a T1-agent, with 1 being optimal.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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