Biogenic Synthesis and Characterisation of Novel Potassium Nanoparticles by Capparis spinosa Flower Extract and Evaluation of Their Potential Antibacterial, Anti-biofilm and Antibiotic Development

Abstract

Scientific researches on the synthesis, characterisation, and biological activity of potassium nanoparticles (K NPs) are extremely rare. In our study, we successfully synthesised a novel form of K NPs using Capparis spinosa (C. spinosa) flower extract as a reducing and capping agent. The formation of K NPs in new form (K₂O NPs) was confirmed by UV–vis and XRD spectra. Furthermore, the FTIR results indicated the presence of specific active biomolecules in the C. spinosa extract which played a crucial role in reducing and stabilising K₂O NPs. SEM imaging demonstrated that the K₂O NPs exhibited irregular shapes with nanosizes ranging between 25 and 95 nm. Remarkably, the biosynthesised K₂O NPs displayed considerable antibacterial activity against a wide range of multidrug-resistant (MDR) pathogenic bacteria. K₂O NPs demonstrated considerable anti-biofilm activity against preformed biofilms produced by MDR bacteria. Combining K₂O NPs with conventional antibiotics greatly improved their efficacy in compacting the MDR bacterial strains. Industrially, bulk form of potassium oxides was commonly used in the preparation of various antimicrobial compounds such as detergents, bleach, and oxidising solutions. The synthesis of potassium oxide in nanoform has shown remarkable biological efficacy, making it a promising therapeutic approach for pharmaceutical and medical applications.

Introduction

In nanotechnology, innovative materials are designed and engineered at nanoscales between 1 and 100 nm, which possess unique properties and can be used for various fields in biology, medicine, physics, chemistry, agriculture, etc. [1–4]. Among the chemical and physical methods, green synthesis is considered one of the best approaches used for NPs reduction and stabilisation. Green synthesis involves the use of plant extracts for the synthesis of NPs, offering benefits such as ease of use, affordability, environmental non-toxicity, biocompatibility, low energy conditions and improved stability [2, 5]. Naturally, plant extracts are a hash of various bioactive molecules, such as proteins, saccharides, polymers, polyphenols and vitamins, which play a critical role in NPs biosynthesis [6].

The wrong and overuse of antibiotics have led to the emergence of MDR bacteria, posing a considerable global medical threat in the modern decades [2, 7, 8]. For this matter, there is a need to search for effective alternative bactericidal agents or develop new antibiotics to combat MDR pathogenic bacterial strains. Various NPs have displayed efficacy against various bacterial strains including MDR types [9, 10]. NPs can bind to the surfaces of bacteria and disrupt their cell walls [11]. Besides, disrupting metabolic pathways, internal organelles, or inducing the formation of free radicals when penetrate bacterial cell walls [12, 13]. By capping NPs with certain phytomolecules during green synthesis, numerous biological functions such as nucleic acid and enzyme synthesis can be halted [14]. Microorganisms cannot develop mechanisms to resist NPs, which makes them effective against bacteria in the long-term [15].

Biofilms are communities of microorganisms that are embedded in a matrix of extracellular polymeric substances (EPS). The structure of EPS is consist of extracellular DNA (eDNA), proteins, and polysaccharides, which act as a protective barrier for biofilm-associated bacteria against antibacterial drugs, the immune system and adverse conditions [16, 17]. Alongside of their antibacterial activity, NPs are consider a promising therapeutic approach due to their ability to effectively and safely deliver drugs to target sites without causing significant side effects [18, 19]. Based on their extremely small size, NPs enable to penetrate biological barriers such as biofilms and cell walls, while their high surface area allows for efficiency in drug hold and deliver [16, 20].

Within this, we sought to biosynthesis of K NPs using C. spinosa flower extract, followed by comprehensive physical characterisations to confirm their formation. Estimate the effectiveness of K NPs in inhibiting bacterial growth and disrupting biofilm structure. Also exploring their potential in the synergistic effects of combining biogenic K NPs with conventional antibiotics.

Methods

Preparation of C. Spinosa Flower Extract

The flower buds of C. spinosa were collected from different regions of the western desert of Al-Anbar, Iraq. Subsequently being cleaned to remove any other plant parts, C. spinosa flowers were air-dried at room temperature for 7 days. Then, dried flowers were transformed into a powder form using a high-speed blender (Kinematica-England blender). For the extraction process, 5 grams of the resulting powder were stirred with 100 ml of distilled water (DW) for 30 min at 60 °C. Finally, the aqueous extract was filtered using filter paper to remove any solid residues and the resulting filtrate was collected and stored in a sealed, dark container at 4 °C for the next step [10].

Green Synthesis of K₂O NPs

For the biosynthesis K NPs using a green approach, the following procedure was performed. Firstly, 100 ml of a 1 mM potassium chloride (KCl, Sigma-Aldrich-USA) was stirred with 50 ml of C. spinosa flower extract at 50–55 °C for 1 day. Throughout this period, the color of the solution gradually transformed from dark brown to light green shade, which indicated the effective biosynthesis of K NPs. Following the biosynthesis step, the  mixture underwent a centrifuging-washing cycle three times at 12,000 rpm for 15 minutes. Finally, the pure pellet was stored in the dark at 4 °C (Fig. 1) [10, 21].

Fig. 1.

Green synthesis and characterisation of K₂O NPs

Characterisation of K₂O NPs

The optical characteristics of K₂O NPs were examined using a UV–vis spectrophotometer (Shimadzu UV-160A). X-ray diffraction (XRD) (Philips PW1730) was used to investigate the structural features of K₂O NPs. Fourier-transform infrared spectroscopy (FTIR) (Bruker ALPHA II) was employed to investigate the colloidal solution of K₂O NPs and C. spinosa flower extract powder. K₂O NPs particle size and surface morphology were observed by scanning electron microscope (SEM) and the NPs diameter estimated by image J software (Fig. 1) [22].

Antibacterial Activity of K₂O NPs

Seventy bacterial isolates were obtained from various clinical cases et al.-Ramadi Teaching Hospital in order to obtain different types of pathogenic bacteria that exhibit MDR ability and form biofilms. The identification of the isolates and the antibiotic sensitivity testing were performed using the VITEK-2 compact system. To screen the inhibitory activity of K₂O NPs, the agar well diffusion method was employed under incubation conditions of 37 °C for 18 h [23]. The main advantages of this method lies in its ease of performance, affordability, and ability to test a large number of bacteria and test materials. While the drawbacks of this technique is its inability to distinguish between bacteriostatic and bactericidal effects, as the inhibition of bacterial growth does not necessarily mean bacterial death. Also, the agar well diffusion method is not suitable for determining the MIC of tested materials such as NPs [24]. Gentamicin (CN) disc (10 µg) was used as a positive control and sterilised water was established as the negative control. The resazurin microtiter plate assay (REMA) was used to determine the minimum inhibitory concentration (MIC) of K₂O NPs against selected MDR pathogenic bacteria [25, 26].

Anti-biofilm Activity of K₂O NPs

The capability of bacterial isolates to form biofilms was evaluated using the Crystal Violet microtiter plate (CV) assay [27]. Also, the modified CV method was employed to determine the percentage of biofilm formation inhibition for 1MIC of K₂O NPs, with gentamicin serving as a positive control. Following the incubation duration, the bacterial cultures were removed from all wells and washed carefully 3 times with DW. Thereafter, the plates were dried using the oven for 50 min at 60 °C. After the drying, 100 μl of a 1% crystal violet stain (Thomas Baker) was added to all wells and incubated for 15 min at room temperature. After washing to remove excess stain, 125 μl of 95% ethanol was added to each well and allowed to react for 10 min. The absorbance was measured at 595 nm using a microplate reader (Humareader HS ELISA). The inhibition percentage was calculated using the following equation based on the optical density (OD) at 595 nm [28, 29]:

$$\text{Inhibition}\text{percentage}=100-\left[\left\{\text{OD595}\text{nm experimental}\text{well}\text{with}\text{test}\text{material}/\text{OD595}\text{nm}\text{control}\text{well}\text{without}\text{test}\text{material}\right\}\times 100\right].$$

Antibiotic Efficiency with K₂O NPs

The Bauer-Kirby assay was employed to assess the efficacy of antibiotics (gentamicin 10 μg, ciprofloxacin 5 μg, trimethoprim 5 μg, and tetracycline 30 μg) both individually and in combination with K₂O NPs (at 1MIC) against selected MDR bacteria. After spreading the selected bacteria on the surface of the Mueller–Hinton agar medium (Himedia-India), the antibodies were placed at appropriate distances on the medium surface and incubated for 18 h at 37 °C. The inhibition area surrounding the antibiotic disc was measured and recorded in millimeters. This measurement provides an indication of the zone of inhibition, which represents the effectiveness of the antibiotic or combination treatment in inhibiting bacterial growth [10].

Results and Discussion

UV–vis Spectrum

The UV–vis spectrum of green synthesised K₂O NPs using C. spinosa flowers extract is shown in Fig. 2. The absorbance measurements of the K₂O NPs solution revealed large peak between 270 and 290 nm wavelengths. This peak indicates the existence of surface plasmon resonance on the surface of NPs. According to previous study, K NPs solution showed a peak of absorption at 270 nm [30].

Fig. 2.

UV–vis spectrum of K₂O NPs biosynthesised by C. spinosa extract

XRD Analysis

The XRD pattern of the biosynthesised K NPs using C. spinosa flower extract is illustrated in Fig. 3. The XRD peaks were indexed to monoclinic phase of K₂O which exhibit a face-centered cubic (FCC) structure according to the JPCDS data report no. 26–1327. The XRD pattern showed diffraction peaks at 2θ = 25.286°, 27.073°, 32.790°, 42.280°, 45.974°, and 49.470° are corresponded to the planes of (110), (102), (022), (113), (204), and (240). An in-depth XRD parameters where the crystallite size, in accordance with Dybe Scherrer module [10], was found ranged between 6.667 and 108.203 nm (Table 1).

Fig. 3.

XRD patterns of the biogenic K₂O NPs

Table 1.

In-depth XRD parameters of the biogenic K₂O NPs

2θ (deg.)	d-spacing (Å)	FWHM	Crystallite size (nm)	Micro strain (%)
25.286 27.073 32.790 42.280 45.974 49.470	3.519 3.291 2.441 2.136 1.972 1.841	0.101 0.224 0.283 0.372 0.968 0.157	108.203 39.450 25.401 8.703 6.667 12.014	0.163 0.417 0.481 1.227 1.479 0.766

FTIR analysis

FTIR analysis was conducted to identify the active biomolecules present in the C. spinosa extract that contribute to the biosynthesis of K₂O NPs. The FTIR results revealed various peaks corresponding to different functional groups, including OH (3700–3100 cm⁻¹), C=O (1700–1500 cm⁻¹), C=C (1600–1400 cm⁻¹), and C–OH (1200–1020 cm⁻¹) (Fig. 4). Comparing the FTIR spectra of the C. spinosa extract and the biosynthesised K₂O NPs, it can be observed that phytomolecules from the extract act as capping agents on the surface of the NPs. This indicates that the active biomolecules present in the C. spinosa extract play a significant role in both the reduction and stabilisation of the K₂O NPs during the synthesis process.

Fig. 4.

FTIR spectral results for A C. spinosa flower extract B biosynthesised K₂O

SEM Analysis

The shape and size of K₂O NPs biosynthesised using C. spinosa flower extract were examined using the SEM analysis. As indicated in Fig. 5, the K₂O NPs showed an irregular shape in terms of morphology and tended to accumulate, forming dense aggregations. Our observations are consistent with previous studies which have suggested that biosynthesised NPs tend to aggregate as a result of the presence of phytomolecules from the plant extract acting as capping agents on the NPs surfaces [31, 32]. The K₂O NPs particle sizes ranged between 25 and 95 nm, with an average size distribution of 64 nm. The SEM results provide insights into the size range and distribution of the biogenic K₂O NPs, indicating the effectiveness of the biosynthesis approach using C. spinosa flower extract in the NPs synthesis.

Fig. 5.

The SEM image of biogenic K₂O NPs synthsised using the C. spinosa flower extract at 20.00 kv and 120,000 × magnification

Bacterial Strains

Among the seventy bacterial isolates, the identification and antibiotic sensitivity test reports generated by the VITEK-2 compact system revealed the presence of ten MDR bacterial isolates. These isolates included Salmonella typhi, Enterococcus faecalis, Proteus mirabilis, Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Streptococcus agalactiae. These specific bacterial isolates were chosen for further analysis to evaluate the antibacterial and anti-biofilm activities of K₂O NPs. Additionally, the efficiency of antibiotics, both alone and in combined with K₂O NPs, were assessed. The selection of these isolates allowed for a comprehensive evaluation of the antimicrobial properties of K₂O NPs and the potential synergistic effects when combined with antibiotics.

Antibacterial Activity of K₂O NPs

The well diffusion assay was conducted to evaluate the antibacterial activity of the green synthesised K₂O NPs against the selected pathogenic MDR bacteria. The zone of inhibition, calculated in millimeter, around the K₂O NPs exhibited varying diameters depending on the certain types of MDR bacterial species. A comparison was made with gentamicin (CN) as a positive control and sterilised water as a negative control (Table 2). Potassium oxides is commonly used in the industrial synthesis of soap and detergent products, oxidising solutions, bleach, and oxygen-generating agent [33–35]. Research has demonstrated that metallic NPs exhibit greater antibacterial activity compared to their bulk counterparts [36].

Table 2.

The inhibition zones (in millimetre) of biogenic K₂O NPs, gentamicin (positive control) and sterilised water (negative control) against MDR bacteria of the well diffusion assay

MDR bacteria	K2O NPs	Gentamicin (CN)	Sterilised water
	Inhibition zone in millimeter (mm)
S. typhi E. faecalis P. mirabilis A. baumannii S. aureus P. aeruginosa S. epidermidis K. pneumoniae E. coli S. agalactiae	16 14 16 14 14 12 12 14 14 10	0 0 12 10 0 0 12 14 10 0	0 0 0 0 0 0 0 0 0 0

Biogenic metallic NPs have shown remarkable efficacy in killing various pathogenic bacterial species that exhibit resistance to many antibiotic types [2]. The antibacterial activity of metal NPs against bacteria can be attributed to their nanoscale size, which enables them to attach and disrupt bacterial cell walls or penetrate into the interior, leading to disruption of biological molecules involving ribosomes, nucleic acids, and enzymes [11, 37, 38]. Furthermore, NPs can induce the formation of reactive oxygen species (ROS), which can damage vital cell macromolecules through oxidative stress [12, 13].

The REMA assay results showed that the MIC of the K₂O NPs were 6.25, 25, 6.25, 25, 25, 50, 25, 12.5, 25 and 12.5 U/mL for pathogenic MDR S. typhi, E. faecalis, P. mirabilis, A. baumannii, S. aureus, P. aeruginosa, S. epidermidis, K. pneumonia, E. coli, S. agalactiae, respectively (Fig. 6). Some studies have revealed that the variability in the MIC of the NPs are due to several factors, such as the structure of bacterial cell walls, the NPs average size, and the specific phytomolecules used in the NPs biosynthesis. Also, some NPs have more antibacterial activity against Gram-negative bacteria than Gram-positive bacteria, which can be attributed to variations in cell wall charge and composition [2, 39].

Fig. 6.

The MIC assay of the K₂O NPs against (1) S. typhi, (2) E. faecalis, (3) P. mirabilis, (4) A. baumannii, (5) S. aureus, (6) P. aeruginosa, (7) S. epidermidis, (8) K. pneumonia, (9) E. coli, (10) S. agalactiae, (11) positive control (only medium) and (12) negative control (medium + bacterial growth)

The physical interaction between the bacterial surface and the NPs plays a crucial role in the antibacterial activity of NPs [40]. In the context of green synthesis, biogenic NPs are reduced by phytomolecules present in plant extracts, such as vitamins, enzymes, polyphenols, carbohydrates, and polymers [41]. These phytomolecules not only confer stability to the synthesised NPs but also introduce various functional groups onto the NPs surface. These functional groups facilitate the establishment of chemical bonds and provide active sites for physical interactions between the NPs and bacterial cells, which are crucial for their inhibitory effects [42].

Anti-biofilm Activity of K₂O NPs

The CV assay results for the MDR bacterial isolates indicated that the strongest biofilm producing isolates were P. aeruginosa (Gram-negative strain) and S. aureus (Gram-positive strain). Treatment of the biofilms with 1 MIC for each K₂O NPs and gentamicin (positive control) showed that the inhibition percentage of P. aeruginosa biofilms was 45% and 12% respectively, while S. aureus biofilms showed 57% and 18% inhibition percentage, respectively (Fig. 7). Biosynthesised NPs have significant activity against the issue of antibiotic resistance associated with biofilm formation [43]. The antimicrobial and anti-biofilm activities of NPs are influenced by their nano size-dependent characteristics, which provide advantages in eliminating biofilms [44, 45]. Biogenic NPs have proved anti-biofilm effects due to their effective production of ROS, and quorum sensing (QS) inhibition. ROS mediated cell membrane disruption and reduced extracellular polysaccharide formation, while QS regulates biofilm development. Metallic NPs have exhibited remarkable anti-biofilm activity against various pathogenic bacteria, including P. aeruginosa and S. aureus [43, 45–47].

Fig. 7.

The inhibition percentage (%) of K₂O NPs and gentamicin (CN) against 24 h preformed biofilms of P. aeruginosa and S. aureus as shown by the CV assay

Antibiotics Combination with K₂O NPs

The combination of antibiotic discs with K₂O NPs demonstrated remarkable antibacterial effectiveness against MDR bacterial strains, surpassing the activity of antibiotic discs alone (Fig. 8). In addition to their antibacterial activity, NPs possess unique properties, including a large surface area to volume ratio, stability, low melting temperature, and exceptional mechanical strength. These qualities make NPs highly suitable for serving as carriers for drugs [48]. Moreover, the capacity of NPs to accommodate both hydrophilic and hydrophobic substances further augments their potential as vehicles for drug delivery [49].

Fig. 8.

Antibacterial inhibition zones in millimeter (mm) of antibiotics A gentamicin, B ciprofloxacin, C trimethoprim, and D tetracycline alone and in combination with K₂O NPs against MDR bacterial isolates

Conclusion

Biogenic K₂O NPs were successfully synthesised using C. spinosa flower extract as a reducing and capping agent. The UV–vis spectral analysis of the K₂O NPs revealed the presence of large peak between 270 and 290 nm. X-ray analysis confirmed the formation of K NPs in the oxide form, namely K₂O NPs. FTIR results indicated the existence of phytomolecules on the surface of the biosynthesised K₂O NPs, which play a crucial role in stabilising the NPs. The morphology of the K₂O NPs was found to be irregular, with a nano size ranged between 25 and 95 nm as observed in the SEM images. The K₂O NPs exhibited significant antibacterial activity against various types of pathogenic MDR bacteria. Moreover, K₂O NPs demonstrated high anti-biofilm activity against preformed biofilms of MDR bacteria. The efficiency of antibiotics against MDR bacteria was enhanced when incorporated with K₂O NPs.

Funding

The authors did not receive support from any organization for the submitted work.

Data Availability

The datasets from this study are available on reasonable request.

Declarations

Conflict of interest

No potential conflict of interest was reported by the author(s).