Background: Copper nanoparticles are of tremendous interest due to their strong antibacterial properties, thereby having potential to enhance not only the physical and biochemical characteristics of hydrogels, but also their antimicrobial activity.

Objective: the goal of this study was to develop a multifunctional hydrogel embedded with copper nanoparticles that were manufactured in an eco-friendly manner.

Material and Methods: Copper nanoparticles were biologically synthesized using Cinnamon extract which was mixed with the CuSO₄ (pentahydrate copper sulphate) solution (0.1 M) in a 1:5 ratio. The CuNPs suspension was primarily characterized by UV–Visible spectroscopy, the spectrums being recorded from 200 to 800 nm. Xanthan gum, a natural polysaccharides, was used as thickening agent in the formulation of the following hydrogels: control gel, with CuSO₄ alone), G1, with biogenic CuNPs and G2 with CuNPs and essential oils (oregano, cinnamon, clove, eucalyptus, thyme, and lavender). The Kirby-Bauer diffusimetric method was used to assess comparatively the antimicrobial activity against Gram-positive, Gram-negative bacteria and fungal species for the formulated gels.

Results: The presence of the copper nanoparticles was confirmed by the presence of a surface plasmon resonance (SPR) peak recorded at 234-255 nm. The lowest antimicrobial activity was observed in the case of control hydrogel. Hydrogel G1 showed significantly better antimicrobial activity, especially on Staphylococcus epidermidis and Candida albicans. Hydrogel G2, embedded with biogenic CuNPs and essential oils, presented a higher antimicrobial activity against both Gram-positive and negative bacteria. It also displayed antibiotic activity against MRSA ATCC 33591, a methicillin-resistant Staphylococcus aureus strain with public health significance.

Conclusions: When coupled with the specified essential oil blend, copper sulfate demonstrated strong synergistic antibacterial action. Altogether, this work assessed and validated the in vitro antibacterial activity of a polysaccharides-based composite hydrogel comprising biologically synthesized copper nanoparticles and essential oils with potential applications in both human and veterinary medicine.

Abstract:

Gold nanoclusters (AuNCs) are nanoscale structures consisting of a few to tens of gold atoms. AuNCs exhibit size and scaffold-dependent photoluminescence which allows their usage as analytical sensors or as fluorescent probes for biological imaging [1,2]. Typical synthesis consists in mixing a metal precursor (HAuCl₄) with bovine serum albumin (BSA), then adding sodium hydroxide (NaOH) to increase the pH of the reaction mixture (and starting the reduction of Au(III)), and finally exposing the solution to a microwave irradiation. In this contribution, different molar ratios of the reactants were used to optimize AuNCs fluorescent properties. The change in molar ratios affected the fluorescence intensity and position of fluorescence emission maximum of AuNCs; simultaneously, it altered the period which is necessary for reaching the fluorescence maximum. It was observed that with an increasing concentration of NaOH (in the pH range of 9-13), position of fluorescence maximum manifested itself by a bathochromic shift, maximum value of fluorescence intensity decreased and was reached in a shorter period than at lower pHs. Indeed, a lower pH value of the reaction mixture leads to an efficient increase of the fluorescence quantum yield of AuNCs. It might be caused by several simultaneous factors: (i) conformational changes of BSA are less pronounced for instance at pH 9 than at pH 13; (ii) faster reduction of Au(III) proceeds at higher pH values which may consequently lead to overgrowth of nanoclusters to non-fluorescent particles. Both factors may contribute to the generation of a higher number of Au nanoclusters of smaller sizes possessing excellent fluorescent properties while working at lower pH values. The choice of an appropriate fluorescent standard is tremendously important, but often underestimated by many researchers.

Acknowledgement:

This research was funded by Grant Agency of the Czech Republic, grant number 19-03207S and Internal Grant Agency of Palacký University, grant number IGA_PrF_2021_003.

References:

[1] Zhang L., Wang E. (2014) Metal nanoclusters: New fluorescent probes for sensors and bioimaging. Nano Today 9, 132-157.

[2] Shang L., Dong S., Nienhaus G.U. (2011) Ultra-small fluorescent metal nanoclusters: Synthesis and biological applications. Nano Today 6, 401-418.

Twenty EOs were examined for the first time against the Escherichia coli MS2 virus (surrogate of SARS-CoV-2). The most effective were the lemongrass (LGO), Niaouli (NO) and eucalyptus (ELO) with a virucidal concentration (VC) of 356.0, 365.2 and 586.0 mg/mL, respectively. PCL was processed via electrospinning, generating uniform, beadless fibrous mats. EOs loading was accomplished via (1) physisorption on pre-existing mats (PCLaEOs), and (2) blending with polymer solution prior to electrospinning (PCLbEOs). In both, 10% v/v VC was used. EOs presence and release was confirmed by UV-visible spectroscopy (≈5257-631 µg) and gas chromatography-mass spectrometry (≈14.3% EOs release over 4 h), respectively. PCLbEOs mats were mechanically and thermal resilient, with LGO promoting the strongest bonds with PCL (PCLbLGO). On the other hand, PCLaNO and PCLaELO were deemed the least cohesive combinations. EOs-loaded mats were identified as superhydrophobic. Air and water-vapor permeabilities were affected by the mats’ porosity (PCL<PCLaEOs<PCLbEOs). Antimicrobial testing revealed the mats’ ability to retain the virus and to inhibit its action. PCLbLGO was the most effective combination against MS2. These mats’ scent was also deemed the most pleasant. Overall, data demonstrated the potential of these EOs-loaded PCL fibrous mats to work as COVID-19 active barriers for individual protection masks. Other applications in individual protection equipment may also be envisaged, including disposable hospital gowns, head and shoe caps, etc.

Peroxidase was produced by a fungal isolate identified as Aspergillus flavus KIGC through ITS sequence and phylogenetic analysis on 11^th day of fermentation when grown on banana peel as the sole carbon source. It was purified through ammonium sulphate, ion exchange including gel filtration. The peroxidase was purified to 8.86 folds with percentage recovery of 49.72%. The purified enzyme was a monomer of 57KDa. The purified peroxidase was immobilized on iron oxide magnetic nanoparticle and characterized using DLS, DSC, XRD, FTIR and SEM analyses. The characterization results suggest adequate nanoparticle formulation and effective enzyme immobilization therein. Optimal temperatures for both free and immobilized peroxidase were 75 and 70 °C, respectively, while the optimum pH peaks at 5.0 and 9.0 were obtained for free enzyme and 6.5 and 9.0 for immobilized peroxidase, respectively. The Michealis constant (K_m) of 0.075mM and catalytic efficiency (turnover rate) of 90.66 Mol/S^-1 were achieved by the immobilized peroxidase on guaiacol as substrate. The immobilized enzyme showed relatively high residual activity in towards ethanol > acetone > DMSO > methanol > Chloroform, respectively. Furthermore, metallic nanoparticle immobilized peroxidase exhibited an enhanced synthetic dyes decolorizationability compared to the free enzyme. The immobilization of peroxidase produced using organic waste as carbon source on iron oxide magnetic nanoparticles conferred considerable stability on the enzyme with great potential for various industrial applications.