Phyto-Engineered Gold Nanoparticles (AuNPs) with Potential Antibacterial, Antioxidant, and Wound Healing Activities Under in vitro and in vivo Conditions

Abstract

Background: A diabetic ulcer is one of the major causes of illness among diabetic patients that involves severe and intractable complications associated with diabetic wounds. Hence, a suitable wound-healing agent is urgently needed at this juncture. Greener nanotechnology is a very promising and emerging technology currently employed for the development of alternative medicines. Plant-mediated synthesis of metal nanoparticles has been intensively investigated and regarded as an alternative strategy for overcoming various diseases and their secondary complications like microbial infections. Hence, we are interested in developing phyto-engineered gold nanoparticles as useful therapeutic agents for the treatment of infectious diseases and wounds effectively.

Methods and results: We have synthesized phyto-engineered gold nanoparticles from the aqueous extract of Acalypha indica and characterized using advanced bio-analytical techniques. The surface plasmon resonance feature and crystalline behavior of gold nanoparticles were revealed by ultraviolet-visible spectroscopy and X-ray diffraction, respectively. High-performance liquid chromatography analysis of the extract demonstrated the presence of different constituents, while major functional groups were interpreted by the Fourier-transform infrared spectroscopy as the various stretching vibrations appeared for important O-H (3443 cm^-1), C=O (1644 cm^-1) and C-O (1395 cm^-1) groups. Scanning electron microscopy, high-resolution transmission electron microscopy results revealed a distribution of spherical and rod-like nanostructures with 20 nm of size. The gold nanoparticle-coated cotton fabric was evaluated for the antibacterial activity against Staphylococcus epidermidis and Escherichia coli bacterial strains which revealed remarkable inhibition at the zone of inhibition of 31 mm diameter against S. epidermidis. Further, antioxidant activity was tested for their free radical scavenging property, and the maximum antioxidant activity of the extract containing gold nanoparticles was found to be 80% at 100 µg/mL. The potent free radical scavenging property of the nanoparticles is observed at IC₅₀ value 16.25 µg/mL. Moreover, in vivo wound-healing activity was carried out using BALB/c mice model with infected diabetic wounds and observed the stained microscopic images at different time intervals (day 2, day 7 and day 15). It was noted that in 15 days, the wound area is completely re-epithelialized due to the presence of different morphologies such as spherical, needle and triangle nanoparticles. The re-epithelialization layer is fully covered by nanoparticles on the wound area and also collagen filled in the scar tissue when compared with the control group.

Conclusion: The pharmacological evaluation results of the study indicated an encouraging antibacterial and antioxidant activity of the greener synthesized gold nanoparticles tethered with aqueous extract of Acalypha indica. Moreover, we demonstrated enhanced in vivo wound-healing efficiency of the synthesized gold nanoparticles through the animal model. Thus, the outcome of this work revealed that the phyto-engineered gold nanoparticles could be useful for biomedical applications, especially in the development of promising antibacterial and wound-healing agents.

Keywords: antibacterial; antioxidant; gold nanoparticles; in vivo mice model; wound healing.

Figure 1

UV-vis absorption spectra of Neat Acalypha indica leaf extract (curve A in black colour) and AuNPs synthesized from Acalypha indica (curve B in pink colour). Abbreviations: AuNPs, gold nanoparticles; UV-vis, ultraviolet-visible.

Figure 2

XRD patterns of (A) Neat Acalypha indica leaf extract and (B) AuNPs synthesized from Acalypha indica. (* Bioinorganic constituents present in the extract). Abbreviations: XRD, X-ray diffraction; AuNPs, gold nanoparticles

Figure 3

HPLC chromatogram profile of aqueous extract of Acalypha indica leaf at 340 nm; UV spectrum of aqueous Acalypha indica leaf extract (Inset Figure 3A). Abbreviations: HPLC, high-performance liquid chromatography; UV, ultraviolet.

Figure 4

FT-IR spectra of (A) Neat extract and (B) extract containing AuNPs synthesized from Acalypha indica. Abbreviations: FT-IR, fourier transform-infrared; AuNPs, gold nanoparticles.

Figure 5

The proposed mechanism of reduction of gold ions to gold nanoparticles.

Figure 6

FE-SEM with EDAX images of AuNPs obtained from Acalypha indica (A and B) and HR-TEM with SAED images of AuNPs obtained AuNPs obtained from Acalypha indica (C and D). Abbreviations: FE-SEM, field emission scanning electron microscope; EDAX, energy dispersive analysis of X-rays; HR-TEM, high-resolution transmission electron microscopy; SAED, selected area electron diffraction; AuNPs, gold nanoparticles.

Figure 7

SEM with EDAX images of (A and B) uncoated cotton, (C and D) neat extract-coated cotton and (E and F) extract containing AuNPs-coated cotton. Abbreviations: SEM, scanning electron microscope; EDAX, energy dispersive analysis of X-rays; AuNPs, gold nanoparticles.

Figure 8

Antibacterial efficiency of AuNPs synthesized using Acalypha indica leaf extract prepared at 100 °C: (1) Uncoated cotton, (2) Extract-coated cotton, (3) Extract containing AuNPs-coated cotton. Abbreviation: AuNPs, gold nanoparticles.

Figure 9

In vitro antioxidant activity of extract containing AuNPs. Abbreviation: AuNPs, gold nanoparticles.

Figure 10

(A) Hematoxylin and Eosin stained microscopic images of regenerated structure of incised wounds on different days (3, 7 and 15) for treated with control, and extract containing AuNPs at 10x magnifications (scale bar 100 μm); (B) Histogram of re-epithelialization (in %); (C) Histogram of wound-cover area (in %). Abbreviation: AuNPs, gold nanoparticles.

Figure 11

(A) Masson’s Trichrome staining images of regenerated structure of incised wounds in different days (3, 7 and 15) for treated with control, and extract containing AuNPs at 10x magnifications (scale bar 100 μm); (B) Histogram of collagen deposition (in %). Abbreviation: AuNPs, gold nanoparticles.