Comprehensive Characterization and Biological and Safety Evaluation of Zinc Oxide-Curcumin Nanoconjugates: Unraveling Synergistic Effects for Enhanced Therapeutic Applications

Abstract

Nanoparticles have been widely synthesized and used in various biological applications. Among the minerals, zinc (Zn) alters numerous biological pathways, and curcumin (Cur) functions mainly as the antimicrobial, anti-inflammatory, and anticancerous agent. In this study, nano-zinc oxide (nZnO), nano-curcumin (nCur), and its conjugate (nZnOcur) were synthesized through the chemical reduction method. The particles were characterized using UV-visible (UV-visible) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy-energy-dispersive X-ray spectroscopy (SEM-EDS), high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and ζ-potential measurements. The UV-visible spectrum revealed that nZnO had an absorbance peak at 370 nm, while nCur had peaks at 400-600 nm, and the nZnOcur conjugate had peaks at 550-650 nm and a smaller peak in the 300-400 nm range. The key functional groups identified by FTIR confirm the successful conjugation of nZnOcur nanoparticles. XRD patterns indicated the crystalline wurtzite phase of nZnO with variations in peak intensity, suggesting structural changes upon curcumin incorporation. SEM-EDS and DLS analyses provided average particle sizes of 88.41 and 122 nm; 53.81 and 98 nm; 43.41 and 102 nm for nZnO, nCur, and nZnOcur conjugate, respectively. HR-TEM images showed that nZnO (∼70 nm, spherical) transformed into rod-like nZnOcur (∼105 nm) upon conjugation with curcumin. nZnOcur had higher antibacterial activity against bacterial strains, demonstrating synergistic effects. The antioxidant capacity was greater in both nCur and nZnOcur conjugates, while anti-inflammatory assays indicated significant protein denaturation inhibition with all three nanoparticles. Cytotoxicity tests revealed that nZnO had higher toxicity, and the acute oral toxicity study indicated nZnOcur conjugate as least toxic, with LD₅₀ value of 425.4 mg/kg. Overall, the findings underscore the nZnOcur conjugate as a promising and safer nanoparticle for antimicrobial, anti-inflammatory, and antioxidant applications with least side effects.

1

UV–visible spectroscopy analysis of synthesized nano-zinc oxide (nZnO), nano-curcumin (nCur) and nano-zinc oxide–curcumin (nZnOcur) conjugate. The UV–visible absorption spectra confirm the formation of the synthesized nanoparticles. nZnO exhibits a peak at 370 nm. nCur shows a broad absorption between 400 and 600 nm. The nZnOcur conjugate displays a broad absorption peak at 300–400 nm (zinc) and a small peak at 500–600 nm (curcumin), indicating the presence of both components in the conjugated nanoparticles.

2

FTIR analysis of synthesized nano-zinc oxide (nZnO), nano-curcumin (nCur), and nano-zinc oxide–curcumin (nZnOcur) conjugate. The presence of different fuctional groups was identified with their characteristic peaks. The nCur had eight peaks with stretching vibration of {−C–O–}. The presence of −C–O and −C–O–C groups indicates the presence of the nZnOcur conjugate.

3

Powder XRD analysis of synthesized nano-zinc oxide (nZnO), nano-curcumin (nCur), and nano-zinc oxide–curcumin (nZnOcur) conjugate. The XRD patterns of nZnO, nCur, and nZnOcur conjugate confirm the crystalline nature of ZnO, matching the JCPDS card no. 01-079-2205 with characteristic 2θ peaks.

4

SEM–EDS analysis of synthesized (a, b) nano-zinc oxide (nZnO), (c, d) nano-curcumin (nCur), and (e, f) nano-zinc oxide–curcumin (nZnOcur) conjugate. SEM–EDS analysis confirmed the particle size and composition of synthesized nanoparticles. nZnO (77.82–97.59 nm), nCur (44.64–63.00 nm), and nZnOcur (37.94–50.21 nm) showed distinct distributions. EDS revealed the elemental composition of the synthesized nanoparticles.

5

HR-TEM analysis and SAED pattern of synthesized nano-zinc oxide (nZnO), nano-curcumin (nCur), and nano-zinc oxide–curcumin (nZnOcur). (a–e) Various magnifications (1 μm, 100 nm, 50 nm, and 5 nm) and SAED pattern of nZnO. (f–j) Various magnifications (0.5 μm, 200 nm, 100 nm, and 20 nm) and SAED pattern of nCur. (k–o) Various magnifications (1 μm, 200 nm, 50 nm, and 20 nm) and SAED pattern of nZnOcur.

6

Mean particle size of the synthesized nanoparticles of nano-zinc oxide (nZnO), nano-curcumin (nCur), and nano-zinc oxide–curcumin (nZnOcur) conjugate analyzed using the dynamic light scattering assay. The DLS values measure the core particle along with its surrounding molecules. The DLS values of nCur and nZnOCur were lower than that of nZnO.

7

Stability of the synthesized nanoparticles (nano-zinc oxide, nZnO; nano-curcumin, nCur; and nano-zinc oxide–curcumin conjugate, nZnOcur), as determined by the ζ-potential, and the values were expressed in mV. The values could be expressed in positive or negative changes. All of the synthesized nanoparticles had negative charges, and the conjugate had a higher negative value than the individual zinc oxide and curcumin nanoparticles, indicating their higher stability.

8

Antimicrobial activity of nZnO against E. coli, S. aureus, and Salmonella spp. The antimicrobial effects of synthesized nanoparticles (nano-zinc oxidenZnO, nano-curcuminnCur, and nano-zinc oxide–curcumin conjugatenZnOcur) were in a dose- dependent manner against E. coli, S. aureus, and Salmonella spp., as evaluated by the disc diffusion method.

9

Antioxidant activity of synthesized nanoparticles (nano-zinc oxidenZnO, nano-curcuminnCur, and nano-zinc oxide–curcumin conjugatenZnOcur) assessed as scavenging activity against DPPH in vitro. The nCur exhibited higher antioxidant activity followed by the conjugate; nZnO had the least activity.

10

Anti-inflammatory activity of synthesized nanoparticles (nano-zinc oxide, nZnO; nano-curcumin, nCur; nano-zinc oxide–curcumin conjugate, nZnOcur). The synthesized nanoparticles (nZnO, nCur, and nZnOcur conjugate) exhibited concentration-dependent anti-inflammatory activity via protein denaturation inhibition. nZnO showed increased response at 500 μg/mL, while nCur had 93.86% inhibition, and the nZnOcur conjugate demonstrated the highest inhibition (94.05%). These results highlight their potential for anti-inflammatory applications.

11

Cytotoxicity analysis of synthesized nanoparticles (nano-zinc oxide, nZnO; nano-curcumin, nCur; nano-zinc oxide–curcumin conjugate, nZnOcur). The cytotoxicity study on the BHK-21 cell line showed higher viability for nCur (79.01%) and nZnOcur (73.43%) at 50 μg/mL concentration, with decreased viability at higher concentrations. nZnO exhibited strong cytotoxicity (IC₅₀ = 34.34 μg/mL), while nCur (IC₅₀ = 98.42 μg/mL) and the nZnOcur conjugate (IC₅₀ = 73.07 μg/mL) demonstrated lower toxicity, indicating their biocompatibility.

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Synthesis of nanoparticles (nano-zinc oxide, nZnO; nano-curcumin, nCur; and nano-zinc oxide–curcumin conjugate, nZnOcur) through the chemical reduction method. (a) nZnO synthesis from zinc nitrate; (b) nCur from extracted curcumin; (c) nZnOcur conjugate from curcumin and zinc nitrate.