Periodic Reporting for period 1 - INPATT (INorganic Photochemical PATTerning)
Reporting period: 2022-04-01 to 2023-09-30
INPATT is a Proof-of-Concept project aimed at developing the photo-induced precipitation of calcium minerals. The main idea behind the project is that light-controlled precipitation can be achieved using molecules that release functional moieties (such as carbonate or phosphate groups), which subsequently precipitate in the presence of calcium ions in a medium as low-soluble calcium mineral. The absorbance of these cleavable molecules can be tuned to the preferred wavelength and therefore, these processes can be adapted to non-invasive conditions, using aqueous solutions, bio-compatible matrices, and a non-harmful irradiation wavelength. The work was performed cooperatively by three different teams, at the University of Konstanz (UKON), at the University of Bologna (UNIBO), and at the IACT-CSIC in Granada. The work in Konstanz focuses on the light-induced precipitation of calcium phosphates with application in medicine, covering four major objectives: (1) Establishment of molecules suiting this process, (2) Characterization of the resulting precipitate, (3) Embedment of the formed calcium phosphate in a matrix, (4) Characterization of the calcium phosphate inside the matrix. The investigation at the UNIBO was devoted to (1) the design of novel approaches and formulations, suitable for the photo-precipitation of calcium-carbonate-based materials, (2) testing the formulas by the 3D-printing of simple objects, and (3) the chemical characterization of the materials. The team of Granada managed the project and focused on the textural and crystallographic characterization of mineral phases, optimizing photo-dissolution protocols, and the management of patents related to INPATT.
The results from this ERC-PoC could be the basis for further exploitation and eventual licensing by stakeholders in the field. It was also already achieved on the part of the University of Konstanz to publish a paper on one part of the topic, which was accepted in Q3 of 2023 containing the calcium phosphate formation by the use of 4NPP inside a matrix [Besirske et al., 2023] Furthermore, it is planned to file a draft for the organic solvent-based matrix and calcium phosphate precipitation for patentability, as well as further publications on this promising topic. On the same wave, the work done at the University of Bologna implemented new approaches to precipitate calcium carbonate through aggregation and conversion of calcium-based nanoparticles upon luminous stimulus. Such results can be a promising base for future publications. Finally, CSIC-Granada has developed an optimized protocol for the local-small-scale photo dissolution of calcium carbonate and prepared the work for further patents.
The results from this ERC-PoC could be the basis for further exploitation and eventual licensing by stakeholders in the field. It was also already achieved on the part of the University of Konstanz to publish a paper on one part of the topic, which was accepted in Q3 of 2023 containing the calcium phosphate formation by the use of 4NPP inside a matrix [Besirske et al., 2023] Furthermore, it is planned to file a draft for the organic solvent-based matrix and calcium phosphate precipitation for patentability, as well as further publications on this promising topic. On the same wave, the work done at the University of Bologna implemented new approaches to precipitate calcium carbonate through aggregation and conversion of calcium-based nanoparticles upon luminous stimulus. Such results can be a promising base for future publications. Finally, CSIC-Granada has developed an optimized protocol for the local-small-scale photo dissolution of calcium carbonate and prepared the work for further patents.
At the UKON, 4-Nitrophenylphosphate (4NPP) was used as a photolabile molecule for phosphate release. By adjusting the pH and the concentrations of the calcium source and the photolabile molecule, as well as the accessibility of CO2 from the air, we were able to find the perfect conditions to precipitate calcium phosphate. Also, we were switching to the synthesis of larger ring structures as photoactive agents, with more red-shifted absorbance than 4NPP. We performed full characterization of the molecules and the splitting mechanism (Absorption, NMR and titration experiments). We calculated that 4NPP has a photochemical quantum yield of 2 %, whereas the larger ring structures according to the NMR studies split off almost completely. Subsequently, a calcium source was inserted and in the case of 4NPP, a solid precipitate was obtained. Characterization (IR, pXRD, SEM/ EDX) doubtlessly shows that the precipitate consists of amorphous carbonate-apatite Type B. For the hard matrix in the case of 4NPP, an agarose-sucrose mixture was used, transparent to visible light; we also established other matrices: polyacrylamide and organic solvent-based matrices for the larger organic ring structures. We were able to cover wavelength ranges 390-460 nm, establishing a huge toolbox of photoinitiators, matrix precursors, and photolabile molecules to adapt the system for user needs. Nonetheless, the characterization of calcium phosphate inside the matrix, hard to achieve, is still under ongoing research. To sum up, we were able to precipitate a calcium phosphate locally inside a matrix and characterize it, which can be of great interest in the future for research on implant materials. At the University of Bologna, two novel routes were explored to reach the precipitation of calcium carbonate through the irradiation of a precursor in the liquid-state. (1) As a first approach, the source of calcium ions was modified from the original formula [Menichetti A. et al, 2021]: an aqueous suspension based on calcium ions and ketoprofen in the form of a fluid paste was investigated. The main work concerned the optimization of formulations. Upon LED irradiation the paste was quickly converted into amorphous calcium carbonate, as proven by IR-spectroscopy. Nonetheless, significant amount of the photo-reaction sub-products and water still remain in the final materials, affecting its mechanical properties and not making it actually suitable for 3D-printing. Indeed, further studies will be necessary to tackle these drawbacks. (2) As a second approach, the light-induced aggregation was performed by mixing calcium carbonate powder and a photocurable-polymeric resin. New formulas were studied by mixing calcium carbonate powder and a photo-polymer resin with different methods. The stability of the blend over time was checked, and it was confirmed that mixtures were stable in a time range suitable for 3D-printing processes. Simple 3D-models were printed by loading the 3D-printer with the novel formulas. The models appeared compact and regular in shape, and the chemical-homogeneity all over the objects was confirmed by IR spectroscopy. These sounded like great background for the next investigations.
The idea of the photo-induced precipitation of calcium phosphates done by the UKON for the PoC INPATT is based on previous studies on the photo-induced precipitation of calcium carbonate [Menichetti et al, 2021]. The UKON aimed to explore other minerals to be deposited in the same way. Within INPATT, UKON continued by establishing the method of localizing the precipitate inside the matrix and the verification of the very. All these results on 4NPP and the respective matrix culminated in the publication of the results in Chemistry – A European Journal. Also, larger ring systems and organic solvent-based matrices were established during the timespan of INPATT, using different light sources for their activation. In the meantime, at the UNIBO the main aim was to go one step further than the previous study and try to photo-precipitate calcium carbonate in a massive way. To reach this aim, light-induced aggregation of calcium-based mineral powders was studied by using photocurable-polymeric resins and photo-decarboxylation strategies. In both cases, promising results were reached. The last experiments before the end of the INPATT included the combination of matrix precursor, photoinitiator, calcium source, and photolabile molecule, which did not lead to publishable results yet. Nonetheless, we will continue this promising research in the future as this can be of great interest for medical applications in the field of implants.