Introduction

Metal bound organic compounds have gained interest as versatile materials in various biomedical and pharmaceutical applications [1]. Among different metal ions bearing antimicrobial activity [2,3,4], copper as an inexpensive metal has attained a great deal of interest over the years rather than other metals [5].

Copper ions can be easily complexed with polymers having suitable functionalities providing acceptable composites bearing appropriate physio-chemical properties. It has been reported that the antimicrobial effect of copper can be improved in the presence of different matrixes, such as polymers [5,6,7,8]. In this regard, copper-ions containing polymers have been prepared with different polymer matrixes, such as Chitosan [9], Cellulose [10], Cotton [11], Polypropylene [8], and Polyethylene [12], displaying valuable antibacterial properties. This approach further allowed development of novel polymer coatings with potential biocidal behavior [13].

Nanonization also endow copper-containing materials with high surface areas and improved antimicrobial properties [14]. Different mechanisms have been suggested for the antimicrobial activity of copper nanoparticles. They can affect cell functions through adhesion to the cell wall of microbes leading to the disruption of the bacterial membrane integrity and finally death of microbes [14]. Copper nanoparticles can also interact with proteins presented in the cell membrane as well as intracellular proteins through formation of copper-peptide complexes causing their denaturation [15]. It has been also reported that these nanoparticles can attached to the DNA via phosphorus- and sulphur-containing compounds leading to the death of the microbes [16]. Researches have demonstrated that alloys, salts, and oxides as forms of copper have strong antimicrobial effect against a broad range of bacteria [17,18,19]. Among different forms of copper NPs, CuO NPs have demonstrated an excellent ability to kill a wide range of bacterial pathogens, which causes infections more than that of the metallic copper NPs [20]. Study showed that they have great effect on the expression of genes involved in transcription, metabolism, translation, DNA replication and repair [21].

Curcumin (CUR), as a main yellow bioactive component of turmeric, has gained significant attention of researchers worldwide owing to a variety of biological activities [22, 23]. Because of the extended antimicrobial activity of CUR, it has been widely employed as a structural component to design new antimicrobial agents having improved antimicrobial activities [24]. CUR is a highly conjugated β-diketone having powerful chelating activity [24]. Hence, it is able to chelate various metal ions to form a metallo-complex exhibiting greater effects compare to free CUR alone [25]. Metals complexation with CUR synergistically improves the potency of the target antimicrobial agents due to the simultaneous intrinsic features of metal and CUR in the target structure. Several metal complexes of CUR have been so far introduced showing various biological activities [26]. However, there is no study on the preparation and antimicrobial activity of the copper containing pCUR.

The present research aimed to report a facile method for the preparation of CuO NPs in the presence of highly cross-linked pCUR nanospheres (CuO/hc-pCUR nanocomposites). CuO/hc-pCUR nanocomposites were fully characterized structurally, and their antimicrobial activity was evaluated and compared with free CUR and copper-free pCUR against two strains of bacteria, i.e., Gram-positive Enterococcus faecalis (E. faecalis) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa). The role of pCUR as reducing and stabilizing agent is also a novel concept of this research [27, 28].

Materials and methods

Materials

Curcumin (CUR) was bought from Acros Organics (USA) and used after recrystallization in 2-propanol. The 2,4,6-Trichloro-1,3,5-triazine (TCT), triethylamine (TEA), copper acetate dihydrate (Cu(OAc)2·2H2O), and acetonitrile (ACN) were obtained from Merck (Germany). ACN was dried over calcium hydride. E. faecalis (ATCC 29212) and P. aeruginosa (ATCC 27853) strains were provided by the Iranian Biological Resource Center (Tehran, Iran). Brain heart infusion (BHI) broth, trypticase soy agar (TSA) medium, and trypticase soy broth (TSB) were purchased from Merck (Germany). Applied methanol in the microbial study was procured from Pharmco-Aaper (USA).

Methods

Preparation of hc-pCUR-NS

hc-pCUR-NS was prepared according to the reported procedure with some modifications [29]. Briefly, in a 250 mL round-bottomed flask, 1 g (2.7 mmol) of CUR and 0.22 g (1.2 mmol) of TCT were dissolved in 50 mL of freshly dried ACN. Then, to this mixture, 0.8 ml of TEA (5.7 mmol) was added and heated under microwave irradiation for 20 min (300 W, 82 °C). The precipitated pCUR was filtered and washed several times with deionized water and acetone; then, they were dried in a vacuum oven at 50 °C for 24 h to obtain a yellow powder of hc-pCUR-NS.

Preparation of CuO/hc-pCUR nanocomposites

Generally, in a 50 mL flat-bottomed flask, 0.01 g of as-prepared hc-pCUR-NS was dispersed into 100 mL of deionized water using sonication. Afterwards, 0.1 g of Cu(OAc)2 was added and kept in a reflux condition for 24 h (12 h at 100 °C and 12 h at 150 °C). The color of the mixture gradually changed from bluish green to black indicating the formation of CuO NPs. The product was then separated by centrifugation (8000 rpm) and washed via three centrifugation-redispersion cycles with deionized water. The precipitate was finally dried in a desiccator to yield CuO/hc-pCUR nanocomposites as dark brown powder.

Bacterial strains and culture conditions

Bacterial strains were cultured in a TSA medium and incubated at 37 °C for 24 h. The bacterial suspension was then prepared in TSB. In order to adjust the growth concentration to 1.5 × 108 colony forming units (CFU) mL−1, both spectrophotometry [optical density (OD) 600: 0.08–0.13] and colony counting procedures were applied [30].

Antimicrobial activity analysis

Antimicrobial effects of CuO/hc-pCUR nanocomposites in comparison with free CUR and hc-pCUR-NS against E. faecalis and P. aeruginosa were appraised using CFU and the disk diffusion method.

CFU method According to our previous study [31], 100 μL of the bacterial suspension (1.0 × 106 CFU mL−1) was added into wells of a 96-well sterile round-bottom microplate (TPP; Trasadingen, Switzerland). Next, 100 µL of free CUR, hc-pCUR-NS, and CuO/hc-pCUR nanocomposites (1 mg mL−1 of Cu, determined by ICP-MS) were added separately to the wells. Thereafter, the microplates were maintained in the dark at room temperature for 5 min in an aerobic atmosphere to allow the uptake of free CUR, hc-pCUR-NS and CuO/hc-pCUR nanocomposites by the bacteria. In order to assess bacterial viability, 10 µL of each well was cultured on TSA and incubated at 37 °C for 24 h, and the number of CFUs mL−1 of the test wells was determined using the Miles and Misra Method [32]. In each step, three wells of microplate containing bacterial suspension without free CUR, hc-pCUR-NS and CuO/hc-pCUR nanocomposites were employed as positive controls. Furthermore, three wells of a microplate containing TSA without the bacterial suspensions were used as negative controls.

Disk diffusion method According to the Kirby Bauer disk diffusion method [33], the bacterial suspensions with a concentration of 1.2 × 108 CFU mL−1 were cultured in Mueller–Hinton agar plates by the use of sterile swabs. The 7 mm size wells were punched into the agar plate using a sterile well cutter. Then 100 µL of free CUR, hc-pCUR-NS, and CuO/hc-pCUR nanocomposite colloidal solution (1 mg mL−1 of Cu, determined by ICP-MS) were then poured into each well on all plates. After incubation at 37 °C for 24 h, the bactericidal activity of nanocomposites was measured from the zone of inhibition and presented in mm as compared to the control (Ciprofloxacin).

Characterization

Microwave-assisted reactions were carried out via MicroSYNTH Microwave Labstation (Milestone, Italy). Size and morphology analysis of the prepared samples were done by field emission scanning electron microscope (FE-SEM, Tescan/Mira, Czech Republic). The Fourier transform infrared (FT-IR) spectra were recorded via Nicolet Magna 550 with the KBr pellet method at room temperature (25 °C). Zeta potential was determined using a ZEN3600 Zetasizer (Malvern Instruments, UK). Ultraviolet–visible spectra (UV–Vis) were recorded by a Jasco-530 spectrophotometer in the range of λ = 400–900 nm. The X-ray diffraction (XRD) patterns were collected by a STOE Theta–theta Powder Diffraction System (radiation: 1.54060 Cu, generator: 40 kV, 40 mA). Elemental analysis of the samples was performed by energy dispersive spectrometry, EDX (MIRAII Tescan), and inductively coupled plasma mass spectrometry, ICP-MS (7900, Agilent).

Statistical analysis

Experiments were carried out three times, and the results were expressed as mean values ± standard deviation (SD). Statistically significant differences were evaluated using two-way analysis of variance (ANOVA) followed by Tukey’s test in SPSS statistical software version 23 (p < 0.05).

Results and discussion

Characterization of CuO/hc-pCUR nanocomposites

Considering the β-diketone moiety of the CUR as the chelating site for the metal ions, the general synthesis procedure and proposed chemical structure of CuO/hc-pCUR nanocomposites are shown in Scheme 1. Absence of any base source is the main characteristic of this synthetic method. CuO NPs are synthesized in situ after chelation and then deposition onto the hc-pCUR-NS matrix in a one-step reaction. In fact, copper ions reacted with yellow water suspension of hc-pCUR-NS to form a bluish green solution containing Cu(OH)2/hc-pCUR nano-complex in the absence of base. This complex is oxidized to form black crystalline CuO NPs. The color of hc-pCUR-NS changes to brown after fully washing the sample. These transformations could be easily seen by change in color of solution.

Scheme 1
scheme 1

General synthetic procedure and proposed structure of CuO/hc-pCUR nanocomposites

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UV–Vis spectroscopy

One of the most convenient techniques for the characterization of copper complexes is UV–Vis spectroscopy [34]. Figure 1 shows the absorption spectra of hc-pCUR-NS and CuO/hc-pCUR nanocomposites in water (1 mg mL−1). The spectrum of hc-pCUR-NS showed a maximum absorption (λmax) at 451 nm due to the π–π* transition of CUR units. For CuO/hc-pCUR nanocomposites, the λmax of hc-pCUR-NS exhibited a red shift (about 12 nm) that can be attributed to the pCUR-metal charge transfer indicating the involvement of the carbonyl group of CUR units in complexation with the metal NPs [35]. These results strongly confirm the successful interaction of CuO NPs with the β-diketone moiety of CUR units of hc-pCUR-NS in the complexation-growth procedure.

Fig. 1
figure 1

UV–Vis absorption spectra of hc-pCUR-NS (a), and CuO/hc-pCUR nanocomposites (b) in water (50 µg mL−1). Images of hc-pCUR-NS (c) and CuO/hc-pCUR nanocomposites (d) samples dispersed in water

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XRD analysis

The X-ray diffraction (XRD) patterns of hc-pCUR-NS and CuO/hc-pCUR nanocomposites are shown in the Fig. 2. Given the amorphous structure of pure hc-pCUR-NS, the crystal structure of CuO/hc-pCUR nanocomposites could be due to the presence of metallic species in their structure, as presented in the corresponding pattern (Fig. 2b). The diffraction peaks at 2θ = 32.5, 35.6, 38.8, 48.8, 53.5, 58.3, 61.6, 66.3, 68.0, 72.4, and 75.1 are related to the (110), (111), (111), (202), (020), (202), (113), (022), (220), (312), and (203) planes, respectively. This pattern is indexed to the monoclinic structure of CuO (tenorite) NPs (ICDD card no. 801916) [36]. Furthermore, no other peaks, resulting from impurities, such as copper hydroxide, were observed in the XRD pattern of CuO/hc-pCUR nanocomposites revealing successful formation of single phase pure CuO crystalline nanostructures onto the hc-pCUR-NS matrix.

Fig. 2
figure 2

XRD pattern of hc-pCUR-NS (a) and CuO/hc-pCUR nanocomposites (b)

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FT-IR analysis

Further characterizations of the prepared samples were done by FT-IR spectroscopy (Fig. 3). For hc-pCUR-NS (Fig. 3a), the broad peak at about 3440 cm−1 could be related to the stretching of the O–H bond of CUR moieties, and the absorption peak at around 2923 cm−1 could be attributed to the C–H stretching vibrations of the methyl groups. Also, the peak at around 1629 cm−1 could be ascribed to the C=O stretching vibration, and the characteristic peaks at around 1504 cm−1 and 1605 cm−1 could be related to the stretching vibration of C=C phenyl rings [37]. Moreover, the strong band at around 1365 cm−1 and the stretching band at about 1158 cm−1 are related to the stretching of C–O–Ph [38] and the C–O–C bonds of the CUR units [37], respectively. For the CuO/hc-pCUR nanocomposites (Fig. 3b), the characteristic peaks of hc-pCUR-NS, involved in the coordination between the β-diketone moiety of CUR units and CuO NPs, underwent a slight shift in wavenumber and thus a relative change in absorbance [34]. Figure 3 depicts a comparison between the spectral features of bare hc-pCUR-NS and CuO/hc-pCUR nanocomposites. It can be seen that the stretching vibration band of the C=O bond of CUR at 1629 cm−1 shifts to a lower wavenumber (1594 cm−1), and the relative absorbance of the 3420 cm−1 band, associated to the stretching vibration of O–H bond, decreases. The new appeared peaks at 489 cm−1 confirm formation of the CuO NPs [39,40,41]. This band could be related to the stretching vibrations of Cu–O bond and corroborate the XRD analysis, where the corresponding plane (202) represents the Cu–O stretching mode of CuO NPs. The results are in accordance with previously reported analysis regarding the CUR-Cu(II) complex [42].

Fig. 3
figure 3

FT-IR spectra of hc-pCUR-NS (a) and CuO/hc-pCUR nanocomposites (b)

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FE-SEM analysis

The morphology of hc-pCUR-NS before and after CuO deposition was studied by FE-SEM analysis (Fig. 4). As seen, hc-pCUR-NS is in a spherical structure with the mean diameter of ~ 246 nm (Fig. 4a). Also, Fig. 4b shows many small spherical CuO NPs agglomerated on the surface of the hc-pCUR-NS matrix. The mean diameter of CuO NPs was evaluated ~ 34 nm. This result clearly showed CuO NPs anchoring on the surface of the hc-pCUR-NS matrix.

Fig. 4
figure 4

FE-SEM images of pure hc-pCUR-NS (a) and CuO/hc-pCUR nanocomposites (b) size distribution histograms of hc-pCUR-NS (c) and CuO NPs (d) determined by Image J 1.44p software

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EDX analysis

As depicted in the typical EDX spectrum (Fig. 5a), CuO/hc-pCUR nanocomposite contains carbon (0.27 keV), oxygen (0.39 keV), nitrogen (0.52 keV), and copper (0.92, 8.04, and 8.9 keV). Meanwhile, the sharp peaks related to copper confirm significant loading of CuO on the surface of hc-pCUR-NS.

Fig. 5
figure 5

EDX spectrum of CuO/hc-pCUR nanocomposites

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Zeta potential analysis

The surface charge of hc-pCUR-NS and CuO/hc-pCUR nanocomposites in deionized water was investigated using zeta potential analysis (Fig. 6). CuO/hc-pCUR nanocomposites exhibited higher negative surface charge (− 11 mV) as compared to hc-pCUR-NS (− 2.07 mV), which was probably due to the anionic form of the enolic hydroxyl group of CUR units involved in the chelation with metal [42]. Since aggregate stability of colloidal solutions is related to their surface charge [43], CuO/hc-pCUR nanocomposites' dispersion showed better stability relative to hc-pCUR-NS. In this regard, it could be predicted that CuO/hc-pCUR nanocomposites would have better antimicrobial activity than that of hc-pCUR-NS.

Fig. 6
figure 6

Results of zeta potential analysis for hc-pCUR-NS and CuO/hc-pCUR nanocomposites

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Comparative analysis of antimicrobial activity

The antimicrobial activity of free CUR, hc-pCUR-NS, and CuO/hc-pCUR nanocomposites was evaluated against Gram-positive E. faecalis and Gram-negative P. aeruginosa. Figure 7 shows the capacity of hc-pCUR-NS and CuO/hc-pCUR nanocomposites to inhibit the growth or to reduce the number of viable bacteria in planktonic form. For both experimental bacteria, CuO/hc-pCUR nanocomposites showed the highest capacity to reduce their viability in culture medium.

Fig. 7
figure 7

Effects of free CUR, hc-pCUR-NS, and CuO/hc-pCUR on reduction of CFU mL−1 of E. faecalis and P. aeruginosa. (*) indicates significant difference from the compared groups, P < 0.05

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The disk susceptibility tests against experimental bacteria are depicted in Fig. 8. The diameter of the inhibition zone and the swelling amount from the edge of each disk in the agar plate are presented in Table 1. Compared to free CUR and hc-pCUR-NS, CuO/hc-pCUR nanocomposites showed the highest antibacterial activity, with mean diameters of around 15 mm and 20 mm against P. aeruginosa and E. faecalis, respectively. The nanocomposites also presented around 60% and 83% antibacterial activity against P. aeruginosa and E. faecalis, respectively, as compared with the antibiotic (Ciprofloxacin).

Fig. 8
figure 8

Antibacterial activity of free CUR, hc-pCUR-NS, and CuO/hc-pCUR against aP. aeruginosa and bE. faecalis are shown by the disk diffusion method

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Table 1 The diameter of inhibition zone from experimental samples (in mm)
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According to the literature, the cytotoxicity and oxidative stress of metal on bacteria may reduce the bacterial function and activity [44]. Large surface area along with the small particle size of CuO NPs might enhance the bactericidal nature of the metal giving rise to avoiding the presence and survival of such a bacterial community environment. According to a previously reported study, the bactericidal effect of CuO NPs immobilized into an interlayer of montmorillonite was better than that of CuO NPs alone. It was argued that because of the comparatively larger surface area of CuO–montmorillonite clay and smaller particle size of CuO in montmorillonite, the release of Cu2+ ions from CuO–montmorillonite was more than that from CuO NPs [45]. In the present study, it was demonstrated that the order of intensity of the materials’ antimicrobial activities was CuO/hc-pCUR > hc-pCUR-NS > free CUR. Therefore, hc-pCUR-NS not only did provide a suitable surface area, but also induced a synergistic antimicrobial effect on the nanocomposites. Another study stated that the synergistic effect of copper(II)-chitosan complexes also showed a better growth inhibition in Gram-positive and –negative bacteria [46]. In the current study, CuO/hc-pCUR inhibited both Gram positive (E. faecalis) and Gram negative (P. aeruginosa) bacteria in an observable manner probably due to the presence of CuO NPs.

The main reason behind the strong antimicrobial activity of CuO/hc-pCUR nanocomposites might be due to the release of Cu2+ ions when the materials interact with an aqueous phase [47]. In fact, Cu2+ ions could bind to functional groups (carboxyl, phosphate, and hydroxyl) of proteins and enzymes bringing about inactivation and inhibition in cellular processes [48, 49]. Also, regarding the influence of the microorganism on the antimicrobial property of copper, Gram-positive bacteria were more affected by Cu2+ ions (about + 100%) as compared to Gram-negative bacteria, as the latter encompasses a double membrane and a periplasmic space decreasing the transportation of the metal ions [50, 51]. This could explain the significant difference in antimicrobial effect of the nanocomposites against Gram-positive and Gram-negative bacteria observed in this study; whereas, the toxicity mechanisms reported for copper are the same for both types of the tested bacteria. Moreover, since anionic compounds are more active against Gram-positive species in comparison with Gram-negative ones [52], CuO/hc-pCUR, as an anionic nanocomposite, could also be a promising choice to inhibit the growth of E. faecalis cells (Fig. 7).

Conclusion

CuO/hc-pCUR nanocomposites were prepared as antimicrobial materials via in situ dispersion-precipitation applying the chelation ability of the of the β-diketone site of CUR. The size of the negatively charged CuO/hc-pCUR nanocomposites was about 246 nm. Most importantly, the results of the biological characterizations performed on Gram-positive (E. faecalis) and Gram-negative (P. aeruginosa) bacteria revealed the excellent antimicrobial effect of CuO/hc-pCUR nanocomposites, especially against E. faecalis, which probably resulted from their large surface area and the small particle size of CuO NPs. These promising findings encourage further investigation and characterization of hc-pCUR-NS complexes. The applied method was easy, cost-effective, and versatile; so the strategy could be potentially be employed for anti-infective surface coatings of biomaterials.