Franziska Linss 1, Jacqueline Bodes 1, Dennis Mulac 1, Klaus Langer 1 Anja Feldmann 2, Yvonne Nitschke 2, Frank Rutsch 2
1 University of Muenster, Institute of Pharmaceutical Technology and Biopharmacy - 2 Department of General Pediatrics, Muenster University Children’s Hospital
Background: This study addresses pathological calcification processes that play a major role in the genesis of the rare hereditary metabolic diseases generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE), which are mainly characterized by calcification of the media of large- and medium sized arteries. Another focus is on calcific aortic valve disease with its end stage of calcific aortic valve stenosis representing one of the most common heart diseases in industrialized countries. Although drug formulations are in development to prevent ectopic calcifications, to this date no therapies exist to resolve these.
Objective: Development of a novel treatment for ectopic calcifications, which combines controlled drug delivery through nanoparticles and active targeting via antibody conjugation.
Methods: Human serum albumin (HSA)-based nanoparticles (NPs) were prepared using a desolvation technique. For visualization, the NPs were labelled with a fluorescent dye coupled to HSA. The active chelating ingredient diethylenetriaminepentaacetic acid (DTPA) was covalently bound to HSA using different strategies. Inorganic pyrophosphate (PPi), which strongly inhibits the formation of hydroxyapatite, was embedded into the matrix of NPs. Surface modification was performed by conjugating antibodies (anti-elastin or isotype IgG control) to the NPs. Porcine aortic valves and murine aortas served as ex vivo models used to simulate the pathologies mentioned above. For this purpose, tissues were harvested and directly incubated in culture medium. Osteogenic additives were added at defined time points to induce calcification. WST- and MTT-assays were performed to examine the tissue viability under experimental conditions. Pure DTPA/PPi as well as varying NP formulations were added to the medium to observe an effect on the inhibition or reversal of calcification. Thereafter, tissue sections were collected and analyzed. Part of the tissues were cryo-embedded and examined in sections via fluorescence microscopy. The extent of calcification was visualized by von Kossa staining. Calcium was quantified using o-cresolphthalein complexone (oCPC) method and atomic absorption / atomic emission spectroscopy.
Results: In this study, we successfully induced artificial tissue calcification ex vivo. A method to investigate the extent of calcification affecting porcine aortic valves and murine aortas was established. Induced calcification was effectively reversed at DTPA concentrations starting at 0.075 mg/ml. In addition, calcification of the aortic valves of pigs could also be inhibited from this concentration. The addition of PPi in concentrations as low as 0.1 mg/ml led to nearly absolute inhibition of tissue calcification. Success in reversing calcification was achieved with NPs, which were functionalized with DTPA. Increased reversion of calcifications by specific anti-elastin antibody-modified NPs was highly significant comparing to isotype IgG-NP control containing the same concentrations of DTPA.
Conclusion: The results represent a significant step towards the development of an effective, non-invasive therapeutic approach using nanoparticular formulations for the disintegration of soft tissue calcification.