Alginate & Lead-Based Nanoparticle Contrast Agent for Post-Mortem Vascular MicroCT Imaging

Introduction: : Effective vasculature visualization is critical for advancing the understanding of biological processes, disease mechanisms, and treatment efficacy. While histology has traditionally been employed for pre-clinical or post-mortem vessel analysis, it is limited by sectioning bias and a lack of three-dimensional context. Microcomputed tomography (micro-CT) offers a non-destructive, high-resolution alternative; however, its application is restricted by the inability to distinguish soft tissues without the use of contrast agents. An ideal post-mortem vascular contrast agent must possess sufficient radiopacity, low viscosity for capillary perfusion under physiological pressures, minimal diffusion into surrounding tissues, thermal compatibility, and structural stability throughout tissue processing. Current solutions, such as iodinated benzine solutions, lead or barium doped silicon (i.e. Microfil and Vascupaint), and barium sulfate-gelatin formulations, exhibit limitations in at least one of these parameters, leading to inadequate small vessel visualization and limited application in mineralized tissues. To address these challenges, we have developed an abellaite (AB)-based nanoparticles (NP, < 60nm) suspended in an alginate (ALG) matrix, with post-perfusion gelation initiated by glucono-δ-lactone (GDL) which forms a highly stable vascular cast. Concentrations greater than 0.3g/mL were adequately radiopaque to be segmented from bone tissue via thresholding in a single microCT scan. We hypothesize that substitution of ANPs with lead-tungstate nanoparticles (LT), which lack crosslinking capacity but exhibit superior X-ray attenuation, is expected to enhance imaging contrast while preserving acceptable delivery viscosity and compressive properties. This platform represents a promising advancement in post-mortem vascular imaging for micro-CT applications.

Materials and

Methods: : 1% ALG (Thermo Fisher Scientific) without NPs or with an NP total of 0.4g/mL or 0.2g/mL with LT:AB ratios of 0:1 to 5:1 were used for all experiments. Rheological testing was performed to assess the viscosity behavior and detect any shear-induced gelation without GDL addition (1% strain; 10 rad/s at 25°C; 30 minutes, n=3/formulation). For mechanical testing, GDL (0.06 g/mL) was added to initiate gelation, and samples were cast into cylindrical molds (12.7 mm Ø × 12.7 mm). After one hour compression testing (10 mm/min) was performed on half of the samples. The remaining half were soaked in 2 mM CaCl₂ for one hour to mimic in vivo calcium exposure before identical mechanical testing (n=3-4/formulation/calcium exposure). To evaluate calcium-induced stability effects, matched GDL-only and GDL+CaCl₂-exposed gels were stored in humidity chambers at room temperature. Images were taken over the following weeks to observe structural changes. To better assess radiopacity, samples and a reference bone were scanned via microCT (70kV, 114uA, 1000ms integration, 15um resolution, n=3/formulation/batch, 3 batches each). Long-term performance was evaluated by x-ray imaging mouse organs perfused with 0.4 g/mL AB-ALG immediately post-fixation and again after 8 months of storage in 70% ethanol at 4°C. Because of current imbalances in some data sets and importance of comparison to controls, all data was analyzed via one-way ANOVA (factor(s): Group, [NP], ratio, calcium exposure) or repeated measures ANOVA (factor: rheology time points).

Results, Conclusions, and Discussions:: RESULTS & DISCUSSION
Rheological testing to date found ALG’s viscosity significantly increased ~10% over 30 minutes (p=0.002) even without NPs. Time (p < 2.2e-16), concentration of LT (p = 2.77e-13), and the interaction between AB and LT concentrations (p = 0.03219) were significant factors affecting viscosity change, while AB concentration alone (p = 0.07711) was not. Notably, time only became a significant factor after 245 seconds. While all are much more viscous than Microfil (~0.025 Pa-s), Microfil’s viscosity starts increasing prior to injection becoming effectively gelled after 20 minutes. All tested NP-ALG did not appreciably gel within a longer timeframe. Calcium treatment markedly improved mechanical properties at strains < 25% to exceed those of both controls; although break strain was much less. MicroCT intensity measurements revealed that gels exhibited greater radiopacity than both bone and the 1,000 mgHA/cm^3 K_2 HPO_4 phantom. For 0.4 g/mL gels, intensity may have exceeded the 16-bit detection threshold. As the LT:AB ratio increased, intensity decreased across all 0.4 g/mL samples and 0:1 to 1:1 of 0.2 g/mL, likely due to tungsten replacing lead and the scan occurring near tungsten’s K-edge. However, the 0.2 g/mL 2:1 sample unexpectedly showed an intensity increase, for reasons not yet understood. Organs perfused with AB-ALG showed minimal gel degradation after eight months, supporting long-term stability. Gels exposed to calcium retained their shape over time, while untreated gels liquefied within 24 hours. Organs perfused with AB-ALG showed minimal gel structural degradation after eight months in storage, also supporting long-term stability.

CONCLUSIONS
This study supports further investigation of dual-NP and ALG contrast agents for post-mortem applications. While viscosity did spontaneously increase over time, significant gelation did not occur until after pH modification allowing for delivery flexibility impossible with silicon or gelatin-based products. Further NP-ALG gels demonstrated superior radiopacity and suitable mechanical properties, with calcium exposure from biological environments further enhancing gel rigidity and long-term stability. Work has begun to evaluate at-scale performance (i.e. microfluidic vascular mimetics and in vivo) more fully as well as optimize in situ gelation.

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