Modular iodinated carboxybetaine copolymers as charge-sensitive contrast agents for the detection of cartilage degradation

Abstract

Accurately assessing cartilage tissue degradation is a big challenge in osteoarthritis (OA) research, as histology only provides information about a 2D tissue section, and currently available contrast agents for tomographic evaluation suffer from low specificity. In this study, we present a modular platform based on zwitterionic carboxybetaine (CBAA) to create multivalent polymeric contrast agents for x-ray computed tomography (CT) with high specificity towards the anionic glycosaminoglycans in the cartilage tissue. By copolymerizing CBAA with different ratios of anionic and cationic iodinated comonomers, we created a library of polymers with net charges ranging from strongly anionic to strongly cationic. The polymers were applied onto osteochondral plugs with different degradation states and the resulting CT images compared to histological stainings. In healthy tissues, the bulk contrast enhancement was strongly correlated with polymer charge, with cationic polymers reaching a 2-fold stronger contrast compared to established small molecule contrast agents. While a further increase in cationic charge slowed the penetration, it increased the polymer's specificity, thereby enabling the most cationic polymer C40 (40 mol% cationic iodinated comonomer) to discriminate accurately between tissues treated with IL-1β for 0, 1, 2 and 3 weeks. Moreover, this polymer also showed a strong local specificity, visualizing local differences in GAG distribution with significantly increased accuracy compared to the controls. Our polymer contrast agents show the importance of multivalency and charge control for the accurate, volumetric detection of GAGs in the cartilage tissue and paves the way towards new contrast agents in- and outside of the clinic.

Keywords: Cartilage; Contrast-enhanced computed tomography; Osteoarthritis; Zwitterionic polymers.

Graphical abstract

Fig. 1

Project overview: A) During cartilage degradation, anionic glycosaminoglycans (GAGs) are lost from the cartilage tissue, leading to changes in zonal charge distribution. By employing iodinated, charge-sensitive zwitterionic polymers that bind to the GAGs, we aim to visualize the local GAG distribution via x-ray micro-computed tomography to assess the degree of degradation within the tissue. B) Polymeric contrast agents were synthesized by RAFT copolymerization between zwitterionic carboxybetaine acrylamide (CBAA) and a combination of anionic (blue) and cationic (red) iodinated comonomers (ANIC/CATIC) to control the net charge of the polymers. Both ANIC and CATIC were synthesized from 3-amino-2,4,6-triiodoisophthtalic acid and contain three iodine atoms (green) per molecule to provide contrast. Figure created with BioRender. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Polymer contrast agents show charge-dependent uptake into healthy cartilage: A) Schematic illustration of the experimental setup. B) CECT cross sections of osteochondral plugs after incubation with contrast agents for 24 h in comparison to the unstained control. Scale bar: 500 μm. C) Averaged contrast enhancement over the whole cartilage volume indicates increased, charge-dependent contrast for most polymers compared to the reference samples CA2+ and iohexol. D) Penetration kinetics are also charge-dependent and reveal (E) increasing and decreasing penetration halftimes for increasing iodine loading for C and N polymers, respectively. F) As the polymers penetrate the cartilage tissue, their zonal distribution changes over time, finally reaching an equilibrium with higher concentration in the deeper zones compared to the surface, to match the local GAG concentrations. This is particularly striking for the most cationic polymer C40. N = 8. Figure created with Biorender.

Fig. 3

Polymer contrast agents detect different stages of cartilage degradation: A) Histological images of safranin O-stained cartilage tissue at different stages of degradation (left) in comparison to the respective CECT micrographs after 72 h of polymer incubation (right). Scale bar: 500 μm. B) Analysis of bulk averaged contrast enhancement after 24 h of polymer incubation reveals diagnostic potential of N20 and C40. C) After 72 h, the differences in N20 contrast are lost due to an unidentified mechanism, whereas the ones for C40 become more pronounced. D) Normalized polymer distribution profiles after 72 h show decreased surface staining for C40 and C20 polymers with increasing tissue degradation. The general trend for increasing contrast with increasing depth, however, remains visible for all contrast agents and tissue states. Penetration profiles are plotted in relative scale to accommodate differences in cartilage thickness between the different samples. N = 8.

Fig. 4

C40 polymer shows best correlation with safranin O staining: A) Side-by-side comparison of safranin O and polymer staining of the same cartilage sample. Scale bar: 250 μm. B) Overlay of local safranin O intensity profiles with CECT results for the plugs shown in panel A. Penetration profiles are plotted in relative scale to accommodate differences in cartilage thickness between the different samples. C) Averaged correlation results across the replicates within one tissue state and across all tissue states for the investigated contrast agents, indicating C40 as the most locally specific and sensitive contrast agent. N = 4.

figs1

Figure S1: Monomer synthesis: Overview of the synthesis strategies pursued for the synthesis of the CBAA (A), ANIC (B) and CATIC (C) monomers and the CA2+ reference contrast agent (D).

figs2

Figure S2: Partition coefficients after 24h of polymer incubation: The partition coefficients at the 24h timepoint reveal that most of the investigated polymers show an increased partition coefficient in comparison to CA2+ and iohexol. More concretely, the partition coefficient of CA2+ was measured at slightly above 1, indicating very poor cartilage targeting efficiency. For A20 and iohexol, the partition coefficient was even below 1, indicative of repulsive forces. N=8.