The plant leaves of E. odoratum were collected, washed with distilled water and subsequently dried at room temperature in dark conditions. After this, the dried leaves were ground to powder and 10 g of powder sample was mixed with 100 mL of ethanol for continuous stirring at 150 rpm for 24 h. In order to remove solid sediments, the mixture was then centrifuged at 10,000 rpm for 10 min. The supernatant was filtered using Whatman filter paper No. 1 and later, concentrated using rotary evaporator to obtain crude extract which was further lyophilized and stored at 4°C.

Zinc Oxide nanoparticles were prepared using phyto-assisted precipitation (Ranpariya et al., 2021) method using highly concentrated Zn precursor, ZnCl₂. Briefly, 0.2 M ZnCl₂ was prepared with total reaction mixture volume as 100 mL. Initially, only 80 mL distilled water was mixed with ZnCl₂ powder using magnetic stirring at 150 rpm at 70°C. After complete dissolution of ZnCl₂ crystals, the ethanolic extract of E. odoratum (10 mg/mL concentration) was added to the reaction mixture with a total volume of 5 mL and later, pH was adjusted to 6. After 1 h of continuous stirring, the mixture was transferred to centrifuge tubes for centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was discarded and the pellet was transferred to fresh tube and mixed with 50% ethanol (v/v in distilled water). The centrifugation cycle is repeated twice and the obtained pellet was kept in oven to dry for 12 h at 80°C.

The crystallographic phase of NPs was confirmed by X-ray diffraction analysis (PANalytical, EMPYREAN). The operating volage and current during XRD were 45 kV and 45 mA, respectively and, the diffraction pattern was recorded across the 2θ range of 20°–80° with Cu Kα source (1.5406 Å). The peaks obtained in XRD analysis was corroborated with JCPDS database to determine the phase formation of ZnO nanoparticles. The surface morphological analysis of ZnO NPs was done by FE-SEM microscopy (FE-SEM, Sigma-300, Carl Zeiss) and elemental composition was investigated by EDAX spectroscopy. In order to find the band gap of synthesized ZnO NPs, diffuse reflectance spectroscopy was done (Lambda-365 UV-Vis Spectrophotometer Perkin Elmer) with the reflectance of light in 200–800 nm range. The photoluminescence emission spectrum was recorded at laser wavelength of 355 nm and 285 µW incident power using Witech Alpha 300 RA system. The hydrodynamic size and zeta potential of NPs was measured using Litesizer 500 (Anton Paar GmbH). ZnO NPs suspension in distilled water was passed through 0.22 µm nylon syringe filter and sonicated for 30 min at RT in ultrasonic cleaner (LMUC-3, Labman Scientific Instruments Pvt. Ltd., India) operating at 40 kHZ.

The coating suspension consisted of 0.2% (w/v) HPMC and 5% (w/v) ZnO NPs in 81.25% ethanol. The coating solution was infused inside the lumens of TIVAP (2005ISP, Vygon, Ecouen, France) with the help of syringe and immersed in the coating solution. The tubings of TIVAP were in contact with coating solution for 6 h at 60°C under continuous orbital shaking at 150 rpm. After this, the excess solution was removed by immersing the catheter in 1x PBS, flushing the lumen of TIVAP by 1x PBS and therefore, ensuring no occlusion inside the lumen of TIVAP. The coated TIVAP was dried in oven at 80°C for 16 h. In order to confirm the coating of ZnO NPs on surfaces of TIVAP, FE-SEM and EDAX analysis was done (Sigma-300, Carl Zeiss) to further compare uncoated and coated TIVAP surfaces in terms of surface morphology and elemental composition. The catheters were sterilized as described elsewhere (Chauhan et al., 2014) in absolute ethanol.

S. aureus MTCC 7443 was grown in Tryptic Soy Broth (TSB) and Gram- E. coli MTCC 119 and P. aeruginosa PAO1 (kind gift received from Dr. Mohan C. Joshi, Jamia Milia Islamia, N. Delhi, India) were grown in Luria-Bertani broth (LB) at 37°C. For enumerating bacterial cell viability, serially diluted culture was spotted on sterile TSB agar (S. aureus) or LB agar (E. coli or P. aeruginosa) plates and kept for incubation at 37°C for 12–16 h.

The efficacy of E. odoratum mediated or chemically synthesized (kind gift from Dr. Khanuja, Jamia Milia Islamia, N. Delhi, India) ZnO NPs in inhibiting growth of S. aureus, E. coli or P. aeruginosa was checked by recording OD 600 nm at different time intervals using Shimadzu UV-Vis Spectrophotometer. Each treatment was replicated thrice and each experiment was carried out 3 times. The exponentially (OD600 ≈ 0.3–0.5) grown bacterial culture of S. aureus, E. coli or P. aeruginosa was given treatment of different concentrations of ZnO NPs and allowed to incubate for 24 h at 37°C with continuous shaking at 150 rpm. The untreated wells were kept as controls. After the incubation, the colony forming units were estimated by plating the serial dilutions on LB agar or TSB agar media plates for Gram-negative and Gram-positive bacteria respectively, and incubated at 37°C for 12–16 h (Lebeaux et al., 2014a).

The antibiofilm efficacy of surface modified TIVAP was evaluated using continuous flow system described previously (Chauhan et al., 2012) with few modifications. Briefly, bacterial biofilms of S. aureus, E. coli or P. aeruginosa were allowed to form on the ZnO NPs coated, HPMC coated and uncoated TIVAP. Under sterile conditions in laminar airflow, the catheters were supplied with fresh media from reservoir bottles. The intraluminal sections of TIVAP were filled with S. aureus, E. coli or P. aeruginosa at a cell density 100 cells/50 µL and allowed to adhere on catheter’s internal surface at 37°C for 3 h. Later, non-adherent bacteria were removed by flushing out 1x PBS for a duration of 10 min followed by continuous supply of media at a speed of 300 μL/min for 48 h. The non-adherent cells and spent media were collected in the discard bottle from the catheters. After 48 h, the biofilm bacterial cells from intraluminal section of TIVAP were harvested by a vigorous process as described previously (Chauhan et al., 2012). The TIVAP catheter surface was wiped using 70% ethanol to remove any contaminants present on the outer lumen of the TIVAP catheter. Under sterile conditions, the lumen of the catheter was cut cross sectionally in small pieces followed by transversal cuts to expose the inner lumen of the catheter. The cut sections were immersed in 1 mL 1xPBS containing tube and vortexed for 1 min. This was followed by sonication for 5 min using water bath ultrasonic cleaner (LMUC-3, Labman Scientific Instruments Pvt. Ltd., India) operating at 40 kHZ followed by vortex mixing for 1 min. Later, the bacterial cells in the 1xPBS suspension were diluted serially and plated on TSB agar (S. aureus) or LB agar (E. coli or P. aeruginosa) for viable cell count estimation.

The phase of the ZnO NPs synthesized using ethanolic extracts of E. odoratum was identified with the help of X ray diffraction pattern as shown in Figure 1. A series of diffraction peaks due to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), (202) planes were observed from the synthesized ZnO NPs. A JCPDS file 36-1451 was used to identify the hexagonal phase with wurtzite structure in the synthesized ZnO NPs. Spurious low intensity peaks were also observed specifically between 20° and 35°. These peaks could be due to the intermediate product or impurities. Confirmation of multiphase or impurity was done by EDAX measurements. Only “Zn” ad “O” signals were observed in sample indicating that unidentified peaks in observed XRD pattern corresponds to the intermediate phase only. Similar observation has been reported by Luković Golić et al. (2011), and others (Tokumoto et al., 2002).

Average crystallite size of the ZnO NPs have been determined from the Williamson-Hall plot (W-H plot) (βCosθ versus 4Sinθ) (Jeffery, 1957) after determining the FWHM of the XRD peaks and considering instrumental broadening. β T o t a l = β S i z e + β S t r a i n = 0.9 λ t cos ⁡ θ + 4 Δ d Sin θ d C o s θ β T o t a l cos ⁡ θ = 0.9 λ t + 4 Δ d Sin θ d (1)

Where, θ denotes the Bragg angle, t represents the crystal or particle size of ZnO NPs synthesized using ethanolic extracts of E. odoratum, d represents interplanar lattice spacing, β _Size and β _Strain represent the FWHM contributions pertaining to the size and strain, respectively. ∆d/d is the measure of strain. FWHM was obtained by fitting individual XRD peak to Lorentzian peak. Figure 2 shows the W-H plot yielding ZnO particle size = 50.4 nm.

The lattice parameters of synthesized ZnO NPs were obtained using an interactive powder diffraction data interpretation and indexing program (Wu, 1989). The indexing program yields lattice parameters by least square fitting to the positions of x ray diffraction peaks in 20–80° range. Lattice parameters a = 3.25500 ± 0 Å and c = 5.21459 ± 0.00091 Å were fitted at figure of merit, F = 33.9 and R = 0.00011. The low value of the R-factor (∼10⁻³) and high value of F > 10 were indicative of the satisfactory estimate of the lattice parameters.

The morphology of ZnO NPs was investigated using FE-SEM microscopy. The spherical and hexagonal morphologies were observed (Figure 3). The SEM micrographs reveal particle aggregation and homogenous morphology distribution in agreement with earlier reports (Chaudhuri and Malodia, 2017; Naseer et al., 2020; Faisal et al., 2021; Iqbal et al., 2021). However, in the previous studies green synthesis procedure has also resulted in irregular crystal growth of ZnO NPs (Ogunyemi et al., 2019; Priyadharshini et al., 2022). The average grain size of E. odoratum leaves’ ethanolic extract mediated ZnO NPs was 34 ± 7.98 nm in our study which is in close agreement with XRD findings. As per previous findings, using SEM analysis different size ranges of bio-fabricated ZnO NPs have been reported such as 45–150 nm using cell free extract of Bacillus megaterium (Saravanan et al., 2018), 25–87 nm using fruit, seed and pulp extract of Citrullus colocynthis (Azizi et al., 2017), 10–20 nm (Doan Thi et al., 2020) using orange fruit peel extract, 30–43 nm using Withania somnifera root extract (Prasad et al., 2021), etc. In EDAX spectroscopy, the synthesized nanoparticles showed presence of Zn (65.35 wt%), O (28.13 wt%) and Au (6.53 wt%, due to gold sputter coating prior to SEM microscopy) indicating successful synthesis of ZnO nanoparticles.

DRS spectra for synthesized NPs showed strong reflection above 365 nm. Absorption spectra for the ZnO NPs synthesized using ethanolic extracts of E. odoratum is shown in inset of Figure 4. The absorption peak for synthesized ZnO NPs was observed at 380.5 nm. Researchers have presented reports of both, the red shift due to defect incorporations with extended localized states within the band gap region (Marotti et al., 2006; Kamarulzaman et al., 2015) and the blue shift of E_g (Tan et al., 2005; Debanath and Karmakar, 2013).

The bandgap energy of ZnO NPs is calculated using Kubelka-Munk (KM) relation as described in Wetchakun et al. (2012) and Yan et al. (2021). K S = 1 − R ∞ 2 2 R ∞ = F R (2)

Where, F(R) is the remission or Kubelka-Munk (KM) function.

In parabolic band structure, α h ν = C h ν − E g 1 / 2 (3) where, α denotes linear absorption coefficient of the material; hν denotes photon energy; C denoted proportionality constant.

For constant scattering coefficient (S) with wavelength, and using Eqs 1, 2, F R h ν 2 = B h ν − E g (3a)

E_g was measured by extrapolating the linear portion of modified KM function and hν, as shown in Figure 4. The optical bandgap for the synthesized NPs was found to be 3.25 eV. The smaller bandgap value as compared to bulk ZnO (∼3.3 eV) may be attributed to defects including, dislocations, stacking faults, zinc and oxygen vacancies, Zn and oxygen interstitial.

To study the possible defects in the synthesized ZnO NPs, PL spectra was investigated at room temperature. PL spectra of the synthesized sample was recorded using excitation wavelength at 355 nm wavelength with 285 µW power. Emission signal was recorded with CCD (charge Coupled Detector). Optical properties of synthesized ZnO NPs were investigated for its use as coatings in central venous catheter. The nanoparticle size (Choopun et al., 2005; Wang et al., 2005) and surface morphology (Ye et al., 2005; Zhao et al., 2016) as well as defects and synthesis process (Kumar Jangir et al., 2017) affect the PL properties of ZnO. Generally, room temperature PL of ZnO exhibits sharp transition in UV range and a broad transition in visible range. The sharp transition in UV range corresponds to optical transition between electrons in conduction band (CB) and holes in valence band (VB), including excitonic effect (band to band transition). PL originates due to recombination of surface states (Chestnoy et al., 1986). The broad emission is related to dopant/impurities or point defects such as zinc interstitial and oxygen vacancies (Kumar Jangir et al., 2017), etc. Figure 5A shows PL spectra of ZnO nanomaterial. The UV emission at 381.8 nm is attributed to near bandgap excitonic emission (band to band transition). Free exciton in ZnO occur when electron hole pair forms between CB and VB. Second observed peak corresponding to crystalline defects in PL spectra was fitted to Gaussian peak function to obtain corresponding energy level and to access types of defects present in the synthesized sample and their influence on the optical properties.

The number of fitted peaks (Figure 5A) indicate the presence of defect states. The parameters obtained from fitted peaks including peak position, FWHM, area under the curve, are shown in the Figure 5A. Figure 5B shows energy band diagram for the synthesized ZnO NPs. The PL spectra of NPs show intense red emission at 669.85 nm, along with orange and green emission at 599.3 nm and 541.3 nm, respectively. Green emission is attributed transition from surface traps (ST) to oxygen vacancy level (V_O). Orange emission is due to transition from Zn_i level to O_i level and red emission is attributed to transition from ex Zn_i level to V_O level.

The particle size distribution of green synthesized zinc oxide nanoparticles is moderately multimodal with polydispersity index as 0.26 and the hydrodynamic diameter is 142.82 nm. Moreover, d₉₀, i.e., 69.95 nm (Figure 6A) represents the size of 90% of particles in the suspension to be below the d₉₀ value which is larger than the average particle size, i.e., 34 nm observed in SEM. This is possibly due to the bias of the characterization technique to measure bigger size particles or aggregates (Modena et al., 2019).

The correlogram of ZnO nanoparticles samples decays rapidly (Figure 6B) indicating the composition of NPs suspension by small sized particles, therefore, changing their relative positions rapidly and also, bringing about rapid intensity fluctuations (Figure 6C). The colloid suspensions with ζ-potential in range ∈ (∞, −15] + [15, ∞) are known to be stable (Modena et al., 2019). The colloids are strongly stable if ζ-potential modulus is greater than 25 governed by adequate mutual repulsive forces (Zhou, 2012; Morais et al., 2013). The ζ-potential of ZnO NPs is −15.61 mV (Figure 6D), possessing anionic surface charge and can form a moderately stable colloid. There is a close relationship between the plant extract metabolites and ζ-potential (Lynch et al., 2009). Moreover, the presence of negative charge on NPs due to adsorption of plant extract metabolites reduces aggregation among particles making it a stable dispersion (Vimala et al., 2014).

The surface of TIVAP showed functionalization of ZnO nanoparticles using SEM-EDAX analysis (Figures 7A, B). The zinc oxide nanoparticles along with HPMC form polymer encapsulated nanoparticles coatings on catheter surfaces as observed in SEM micrograph (Figure 7A). Recently, HPMC based films were developed incorporated with ZnO Nanoparticles for antibacterial wound dressing application (Pitpisutkul and Prachayawarakorn, 2022). Further, more detailed investigations are required to find exact mechanism of chemical interaction between HPMC, ZnO and PDMS surfaces.

ZnO exhibits improved antibacterial activity at the nanoscale (Padmavathy and Vijayaraghavan, 2008). The antibacterial properties of ZnO nanoparticles have been reported as a function of its characteristic features. ZnO Nanoparticles when doped with Fe results in significant antibacterial activity against Gram-negative bacteria like P. aeruginosa, E. coli (Kayani et al., 2018). ZnO nanoparticles consisting flower like morphology (hierarchical structures) make Gram-positive bacteria more susceptible than Gram-negative bacteria (Babayevska et al., 2022). The differential antimicrobial activities of ZnO NPs are due to influence of physical and chemical properties of ZnO NPs obtained by varying synthesis methods, modification of NP surface using doping with metals or capping agent (Gudkov et al., 2021; da Silva et al., 2019). To assess the antibacterial efficacy of ZnO NPs synthesized using ethanolic extracts of E. odoratum or chemically synthesized ZnO NPs, exponentially growing bacteria were treated with different concentrations (50 μg/mL to 750 μg/mL) of ZnO NPs for 24 h at 37°C. The viable cell count was estimated by plating the S. aureus on TSB agar plates, and E. coli and P. aeruginosa on LB agar plates. Untreated cultures were used as controls (Figures 8A–F; Supplementary Figure S1). Although a concentration dependent killing was observed upon exposure of all the bacterial strains to either E. odoratum mediated or chemically synthesized ZnO NPs, there was no significant difference in the antibacterial activity between the ZnO NPs synthesized by two methods. Although the growth of both Gram-positive as well as Gram-negative strains was inhibited significantly with increasing concentration, growth of S. aureus was inhibited up to 99.9% at a concentration of 250 μg/mL E. odoratum mediated ZnO NPs. Maximum killing of 99.98% was seen in case of S. aureus at 500 μg/mL ZnO NPs whereas 99.9% killing could be achieved only at higher concentration of 750 μg/mL in case of Gram-negative bacteria. This could be due to presence of secondary metabolites (rich in phenols, saponins and tannins) (Inya-agha et al., 1987) in E. odoratum extracts, as previously reported, maybe specifically active against Gram-positive bacteria. The literature suggests broad spectrum antibacterial and antifungal activity of E. odoratum plant extract. Venkat Raman et al. (Venkata raman et al., 2012) have shown broad spectrum antibacterial activity against Gram-negative and Gram-positive bacteria including MTCC736 Bacillus subtilis, MTCC2807 Corynebacterium glutamicum, MTCC1572 E. coli, MTCC7028 Klebsiella pneumonia, MTCC733 Salmonella typhi, MTCC87 S. aureus, MTCC1938 Streptococcus thermophilus, MTCC1771 P. vulgaris, MTCC451 Vibrio parahaemolyticus. E. odoratum extract shows significant (p < 0.001) antibacterial activity against all bacterial species excluding Proteus vulgaris and S. typhi. Besides, E. odoratum also show antifungal activity (Ramesh and Subramani, 2018). Chromolaena odorata (synonym of E. odoratum) extract in combination with antibiotics inhibit growth of P. aeruginosa isolated from wound infections. Moreover, E. odoratum mediated ZnO NPs are able to kill efficiently both the both the Gram-positive and Gram-negative bacteria, indicating the broad-spectrum activity of these NPs and further warrants the evaluation for probable application in clinical settings. Furthermore, as suggested by other research groups, focused studies are needed to compare the antibacterial efficacies of metal and/or metal oxide NPs using optimized techniques more relevant to nanoparticles (Kourmouli et al., 2018; Masri et al., 2022).

Zinc oxide nanoparticles are well recognized antibacterial (Liu et al., 2009; El-Masry et al., 2022) and antibiofilm agents, and used to develop antifouling surfaces of medical devices, for example, modifying dental resins (Wang et al., 2019) and denture bases in dental implants (Cierech et al., 2019) and deposition (Wang et al., 2021) and patterning of ZnO NPs on titanium (Ye et al., 2022) for orthopedic implants. We have developed coating of ZnO NPs, synthesized using ethanolic plant extract of E. odoratum, on silicone elastomer-based surface of the commercial TIVAP with help of hydroxypropyl methylcellulose (HPMC) as binding agent. ZnO-HPMC based coatings result in formation of hydrophilic surfaces (Rao et al., 2014). The ZnO NPs coated CVCs showed more than 97% inhibition of S. aureus biofilm formation and up to 90% inhibition of E. coli and P. aeruginosa biofilm formation (Figure 9). Hydrophilic surfaces are known to reduce bacterial adhesion and biofilm formation (Lorenzetti et al., 2015; Verhorstert et al., 2020). ZnO NPs based coatings can be tuned to attain hydrophilic surfaces reducing bacterial colonization and inhibiting biofilm formation upon the surfaces (Yong et al., 2015; Kusworo et al., 2021). Our results demonstrated the application of ZnO NPs for the first-time and successful reduction in bacterial formation which can successfully reduce risk of central venous catheter associated infections. Further, in vivo studies would validate the antifouling ability in the presence of trilateral interaction between host, device and bacteria.