# Effect of Nanohydroxyapatite on Silk Fibroin-Chitosan Interactions--Molecular Dynamics Study
## 3 Results and Discussion  
This study examined two chitosan-silk fibroin complexes. One complex incorporated Ca\({}^{2+}\) ions and HPO\({}_{4}\)\({}^{2-}\) to mimic nanohydroxyapatite, while the other contained acetic acid. The selection of the acetic acid environment as a reference simulation condition was guided by two considerations: its well-documented role as a stabilizer for fibroin [67] and its ability to dissolve both fibroin and chitosan [67; 100]. The exemplary optimized structures of chitosan-silk fibroin complexes are presented in Figure 2. The detailed structural and geometric parameters, including hydrogen bond lengths, interaction distances, and binding energies, are provided in the Supplementary Materials. The analysis of the optimized structures revealed that basic amino groups in chitosan undergo protonation under all considered conditions. The role of charged ions in stabilizing these large biomolecular systems presents an intriguing aspect for investigation. Although the individual components of these complexes carry charges, the overall molecular assemblies themselves remain neutral due to the presence of counterions (CH\({}_{3}\)COO\({}^{-}\), HPO\({}_{4}\)\({}^{2-}\), Ca\({}^{2+}\)). Molecular dynamics simulations unveil that the formation of complexes with both CH\({}_{3}\)COOH and HPO\({}_{4}\)\({}^{2-}\)/Ca\({}^{2+}\) is energetically advantageous. However, the total binding energy, encompassing all stabilizing interactions, slightly favors the former system (7452 \(\pm\) 397 kJ/mol) over the latter (6483 \(\pm\) 561 kJ/mol). Despite these disparities, both values are remarkably high. Notably, the binding energies, calculated through similar procedures for chitosan-collagen complexes as reported in our previous studies [61; 63], did not exceed 850 kJ/mol. This suggests a significantly lower mutual affinity between chitosan and collagen compared to chitosan-fibroin systems. Notably, fibroin is known to form semi-interpenetrating polymer network (semi-IPN) arrangements with chitosan [101]. Moreover, thermal studies have shown high compatibility and thermal stability of the chitosan-fibroin mixture [102]. Similar to chitosan, fibroin undergoes protonation in acidic conditions [103; 104], as evidenced by computational results. While both biomolecules become positively charged through protonation, the formation of stable complexes is possible due to the presence of oppositely charged ions that neutralize the positive charge. Our previous studies [63] have demonstrated similar behavior in the case of collagen-chitosan systems. Importantly, both chitosan and proteins, such as collagen and fibroin, exhibit polyelectrolyte properties [105; 106; 107], leading to charge migration within the polymer chain and fluctuations in charge density within the biomolecule. These properties can be manifested in complex molecular systems. Nevertheless, similar calculations performed in previous studies [63] have shown that even oligomeric chitosan chains exhibit polyelectrolyte features. Since polyelectrolytes contain many ionic and polar groups, they are capable of forming various intermolecular contacts. Remarkably, the complexes investigated in this study reveal a dense network of interactions between chitosan and fibroin. The YASARA program (2021) employed in this study was capable of identifying various primary types of interactions in the considered fibroin-chitosan complexes, such as ionic contacts, hydrogen bonds, and hydrophobic interactions. Of particular significance are strong ionic contacts. These interactions are known to be crucial for stabilizing the fibroin-chitosan complexes [108]. As shown in Figure 3, in the case of both complexes, containing acetic acid and HPO4\({}^{2-}\)/Ca\({}^{2+}\) ions various ionic contacts between chitosan and fibroin appear. However, a significantly higher number of these interactions are observed under the influence of HPO4\({}^{2-}\)/Ca\({}^{2+}\) ions compared to the acetic acid solution.