Gain of a 500-fold sensitivity on an intravital MR contrast agent based on an endohedral gadolinium-cluster-fullerene-conjugate: a new chance in cancer diagnostics

Abstract

Among the applications of fullerene technology in health sciences the expanding field of magnetic resonance imaging (MRI) of molecular processes is most challenging. Here we present the synthesis and application of a Gd(x)Sc(3-x)N@C(80)-BioShuttle-conjugate referred to as Gd-cluster@-BioShuttle, which features high proton relaxation and, in comparison to the commonly used contrast agents, high signal enhancement at very low Gd concentrations. This modularly designed contrast agent represents a new tool for improved monitoring and evaluation of interventions at the gene transcription level. Also, a widespread monitoring to track individual cells is possible, as well as sensing of microenvironments. Furthermore, BioShuttle can also deliver constructs for transfection or active pharmaceutical ingredients, and scaffolding for incorporation with the host's body. Using the Gd-cluster@-BioShuttle as MRI contrast agent allows an improved evaluation of radio- or chemotherapy treated tissues.

Keywords: BioShuttle; Magnetic Resonance Imaging (MRI); Molecular Imaging; Rare Earth compounds; fullerenes; gadolinium; intracellular imaging; intravital Imaging; inverse Diels Alder Reaction; metallofullerenes; nitridecluster fullerenes.

Figure 1

(Scheme 1) Demonstrates the synthesis steps of the N-(2-Aminopropyl)-4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)benzamide 4 used as diene reaction partner.

Figure 2

(Scheme 2) Illustrates the classical chemical reaction of the cyclooctotetraene (COT) 5 with maleic anhydride 6 to the resulting reaction product tetracyclo-3,5-dioxo-4-aza-9,12-tridecadiene called “Reppe anhydride” 7 used as dienophile reaction partner.

Figure 3

(Scheme 3) Shows the reaction of Reppe anhydride 7 after 1,3-dipolar cycloaddition reaction of the Boc protected N-1.3.-diamino propane linker substituted glycine 9 with the Gd_xSc_3-xN@C₈₀ 8. The product 11 acts as the dienophile reaction partner with diene tetrazine-NLS-S^∩S-CPP conjugate 13 in the final DAR_inv. as pointed out in Figure 5/scheme 5).

Figure 4

(Scheme 4) The resulting molecule 13 consists of the diene compound ligated by N-(2-aminopropyl)-4-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazine-3-yl)benzamide linker to nuclear localization sequence (NLS) which in turn is covalently connected by disulfide bridge formation with the cysteines of the C-terminus of the cell penetrating peptide (CPP) and the NLS. This diene tetrazine-NLS-S^∩S-CPP conjugate 13 was ligated with the functionalized MR imaging component GdSc₂@C_80n cargo 11 (Figure 3/scheme 3).

Figure 5

(Scheme 5) Depicts the Diels Alder inverse as the terminal ligation step to the Gd-cluster@-BioShuttle as final product 14 after purification ready for use in MR imaging studies.

Figure A

illustrates a demonstration of the T1 weighted relaxation times. The ordinate of the graph in the upper part shows the relaxation times [msec], the axis of the abscissa represents the concentrations of the dilution series [nmol/100µL] of the Gd-cluster@-BioShuttle 14. As a reference Gd-DTPA was used in a final concentration of 0.5 nmol/ 100 µL] the lower limiting concentration at which a barely tomographic signal is produced. The lower part of the figure reveals the corresponding tomographic signals in collected cells in reaction tubes. The Gd-DTPA control is in the lower panel on the right side.