Enhanced Activity and Sustained Release of Protocatechuic Acid, a Natural Antibacterial Agent, from Hybrid Nanoformulations with Zinc Oxide Nanoparticles

Abstract

Hybrid nanostructures can be developed with inorganic nanoparticles (NPs) such as zinc oxide (ZnO) and natural antibacterials. ZnO NPs can also exert antibacterial effects, and we used them here to examine their dual action in combination with a natural antibacterial agent, protocatechuic acid (PCA). To produce hybrid nanoformulations, we functionalized ZnO NPs with four types of silane organic molecules and successfully linked them to PCA. Physicochemical assessment confirmed PCA content up to ~18% in hybrid nanoformulations, with a PCA entrapment efficiency of ~72%, indicating successful connection. We then investigated the in vitro release kinetics and antibacterial effects of the hybrid against Staphylococcus aureus. PCA release from hybrid nanoformulations varied with silane surface modification. Within 98 h, only 8% of the total encapsulated PCA was released, suggesting sustained long-term release. We used nanoformulation solutions collected at days 3, 5, and 7 by disc diffusion or log reduction to evaluate their antibacterial effect against S. aureus. The hybrid nanoformulation showed efficient antibacterial and bactericidal effects that also depended on the surface modification and at a lower minimum inhibition concentration compared with the separate components. A hybrid nanoformulation of the PCA prodrug and ZnO NPs offers effective sustained-release inhibition of S. aureus growth.

Keywords: Staphylococcus aureus; ZnO nanoparticles; antibacterial effect; delivery system; hybrid nanoformulation; natural agents; protocatechuic acid prodrug; sustained release.

Scheme 1

Schematic representation of all synthesis stages and biological evaluations against bacteria for a delivery system for the PCA prodrug depending on ZnO NPs.

Figure 1

ZnO nanoparticles as visualized using FE-SEM.

Figure 2

Thermal analysis results for materials at each stage by simultaneous thermal analysis, coupled with differential scanning calorimetry. (A) Weight loss measurements. (B) Derivative thermogravimetric (DTG) profiles. Materials were heated to 800 °C from RT at 10 °C/min in a mixture of artificial air and helium flowing through the furnace chamber.

Figure 3

Differential scanning calorimetry analysis of ZnO NPs and surface-functionalized NPs. An exothermic process associated with mass loss is indicated.

Figure 4

Results of X-ray diffraction analysis of dried powder materials for the PCA prodrug and before and after PCA loading in nanoformulations. The red-dotted circle indicates the presence of PCA in nanoformulations.

Figure 5

The spectra of ZnO NPs, modified ZnO NPs, and nanoformulations by Fourier-transform infrared spectroscopy. (A) ZnO NPs before and after surface modification. (B) Nanoformulations consisting of PCA and free PCA. In panel (A), the dotted green circle and lines show the changes following surface modifications with various silane groups compared to ZnO NPs. In panel (B), the dotted green circle and lines represent the detected new peaks in nanoformulations corresponding to free PCA.

Figure 6

Zeta potential measurements in deionized water for all prepared materials before surface modification and after, along with PCA prodrug-loaded nanoformulations. The measurements were performed for all materials suspended in deionized water at pH 7.4.

Figure 7

Release of the PCA prodrug from nanoformulations in the PBS medium (mean ± standard).

Figure 8

Results of the log reduction assay of ZnO NPs, surface-modified NPs, nanoformulations, PCA antimicrobial activity of the solutions (supernatants) obtained after three, five, and seven days against S. aureus. Columns labeled with different letters (a, b) had significantly different average values (p ≤ 0.05). Materials marked by the same letter (for instance, “a”) displayed no significant differences. Materials marked with different letters (for instance, “a” for one material and “b” for another material) displayed significant differences.

Figure 9

Antimicrobial activity of the solutions (supernatants) obtained after three and seven days of incubation of the tested materials at 37 °C in the PBS. Columns labeled with different letters (a, b) had significantly different average values (p ≤ 0.05). Materials marked by the same letter (for instance, “a”) displayed no significant differences. Materials marked with different letters (for instance, “a” for one material and “b” for another material) displayed significant differences.