Multifunctional polydopamine - Zn2+-curcumin coated additively manufactured ceramic bone grafts with enhanced biological properties

Abstract

The lack of site-specific chemotherapeutic agents after osteosarcoma surgeries often induces severe side effects. We propose the utilization of curcumin as an alternative natural chemo-preventive drug for tumor-specific delivery systems with 3D printed tricalcium phosphate (TCP) based artificial bone grafts. The poor bioavailability and hydrophobic nature of curcumin restrict its clinical use. We have used polydopamine (PDA) coating with Zn²⁺ functionalization to enhance the curcumin release in the biological medium. The obtained PDA-Zn²⁺ complex is characterized by X-ray photoelectron spectroscopy (XPS). The presence of PDA-Zn²⁺ coating leads to ~2 times enhancement in curcumin release. We have computationally predicted and validated the optimized surface composition by a novel multi-objective optimization method. The experimental validation of the predicted compositions indicates that the PDA-Zn²⁺ coated curcumin immobilized delivery system leads to a ~12 folds decrease in osteosarcoma viability on day 11 as compared to only TCP. The osteoblast viability shows ~1.4 folds enhancement. The designed surface shows the highest ~90 % antibacterial efficacy against gram-positive and gram-negative bacteria. This unique strategy of curcumin delivery with PDA-Zn²⁺ coating is expected to find application in low-load bearing critical-sized tumor-resection sites.

Keywords: Additive manufacturing; Antibacterial surface; Calcium phosphate; Curcumin; Drug delivery; Polydopamine coating.

Fig. 1:

Fabrication of 3D printed substrates with different designs, optical microscopic images of the substrates after PDA coating, the chemical structure of dopamine, and resultant properties at a glance after PDA-zinc-curcumin coating; (a) Schematic of the binder jetting-based 3D printing process and CAD designs of complex structures, printed with TCP powder in this machine (b) optical microscopic images of different 3D printed substrates after PDA coating (c) the chemical structure of dopamine, the monomer of PDA (d) resultant properties of the coated surface at a glance

Fig. 2:

Multi-objective optimization strategy with different PDA coating time, PDA: Zn²⁺ molar ratio, and the amount of curcumin as an input and osteoblast viability (5 d), antibacterial efficacy (24 h), and osteosarcoma viability (5 d) as an output; (a) PDA coating time of 10 h-15 h shows the optimum output (b) the optimization results predict a ratio of PDA: Zn²⁺ between 1.2–1.8 as the optimum, (c) the optimum output for biological properties are predicted for a curcumin amount in the range of 400–600 μg (d) validation of the predicted experimental design with day 7 osteoblast study of different compositions, such as TCP-PZ-Cur 1, TCP-PZ-Cur, and TCP-PZ-Cur 2; ** indicates a p-value of <0.0001.

Fig. 3:

The ¹H NMR and XPS results; (a) ¹H NMR of free curcumin; the prominent hydroxyl and methoxy peaks are marked with Δ symbol; (b) the prominent peaks in the ¹H NMR of PDA are marked with * symbol; (c) the chemical structure of the resultant PDA-Zn complex (d) chemical structure of curcumin (e) chemical structure of PDA after polymerization from dopamine (f) the full scan XPS spectra of PDA and PDA-zinc complex shows characteristics peaks such as C1s, N1s, and O1s. The presence of Zn2P peaks is noticed in the PDA-zinc complex (g) the high-resolution spectra of Zn2P indicate peaks at ~ 1046.06 eV and ~ 1022.8 eV (h) the deconvoluted O1s spectra of the complex shows indication of zinc-oxygen interaction at ~ 531.2 eV.

Fig. 4:

The cumulative release profiles at (a) pH 7.4 and (b) pH 5.0 of free curcumin, immobilized curcumin on the PDA-Zn²⁺ coating, and curcumin incorporated within the PDA-Zn²⁺ matrix; the obtained results show that at pH 7.4, curcumin immobilization on PDA-Zn²⁺ coated surface leads to the highest release of 40 ± 2.3 % curcumin for 30 days. In contrast, the free curcumin shows a release of only 23±2 %, and the incorporation of curcumin leads to a higher release of 33±1.5 %; curcumin immobilization leads to the highest release of 51 ± 1 % curcumin at pH 5.0. In contrast, the free curcumin shows a release of only 30±2 %, and the incorporation of curcumin leads to a higher release of 42±3 % (c) power law fit at pH 7.4 for the first 24 h data (d) FESEM images after drug release at pH 7.4 show no significant degradation of the grafts (e) similar observation is noticed at pH 5.0. However, the generation of porosity is noticed and marked with arrows.

Fig. 5:

The osteoblast-3D printed graft substitutes interactions (a) MTT assay results indicate cytocompatibility of all the tested compositions; on day 3, all compositions show similar cell viability; on day 7 and day 11, the cell viability of the treatment samples enhances significantly compared to that of control. * denotes a p-value of <0.05 and the ** indicates a p-value of <0.0001 (b) The ALP assay result shows that TCP-PZ-Cur results in ~ 2 times higher cell differentiation than the control; (c) The FESEM images show good cellular attachment on each sample surface. Extend filopodia is noticed in the TCP-PZ-Cur sample. The dotted circles show cellular attachments and filopodia is marked with the arrows in the images.

Fig. 6:

The antibacterial assay results for 24 h, 48 h, and 72 h against S.aureus (a) The agar plate images show that both TCP-PZ and TCP-PZ-Cur lead to a significantly lesser number of bacterial colonies compared to that of control (b) The TCP-PZ composition shows up to ~ 52 % antibacterial efficacy and the TCP-PZ-Cur shows up to ~ 90 % antibacterial efficacy. The ** indicates a p-value of <0.0001 (c) The FESEM results show a lesser number of bacterial colonies in the treatment samples. The bacterial colonies are marked with a dotted circle and the debris are marked with arrows (d) The punctured cell membranes and bacterial debris are marked with arrows in the image. (e) the proposed antibacterial mechanism.

Fig. 7:

Interaction of P. aeruginosa with the fabricated scaffolds. (a) The agar plate results at 48 h and, (b) 72 h indicate that TCP-PZ-Cur shows a significant reduction in bacterial colonies than the control, (c) the confocal microscopic images at 72 h indicate a significant number of dead bacteria colonies in the treatment as opposed to living colonies in the control, (d) the FESEM images at 48h, and (e) 72 h respectively show lesser bacterial densities in the TCP-PZ-Cur surface. Ruptured bacterial cell membranes are noticed for this composition and marked with white arrows in the image (f) quantification results by agar plate colony count indicate that the TCP-PZ-Cur sample shows the highest ~ 85% antibacterial efficacy; ** indicates a p-value of <0.0001 (g) schematic of the bacterial cell membrane rupture. It is expected to reduce the chances of post-surgical infections.

Fig. 8

(a) MTT assay results after osteosarcoma cell culture indicate significantly reduced cell viability at each time point, in the presence of curcumin. The TCP-PZ-Cur shows ~ 4 times reduction in cellular viability on day 7, which further increases to ~ 12 folds reduction on day 11, compared to the control. The ** indicates a p-value of <0.0001 (b) FESEM results show healthy osteosarcoma attachments on control TCP at each time point. In contrast, the curcumin-containing composition does not show a significant number of cells at each time point.