Green synthesis and antibacterial assessment of chitosan/silver nanocomposite conjugated with tobramycin against antibiotic resistant Pseudomonas aeruginosa

2.1 Materials

The Zingiber officinale rhizome (ginger) was purchased from a local shop in Jeonju, South Korea. Silver nitrate (AgNO₃), chitosan, tobramycin, 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA), 3-ethyldimethylaminopropyl carbodiimide (EDC), N-Hydroxy succinimide (NHS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), sodium hydroxide (NaOH), acetic acid, tris, Triton X-100, and sodium tripolyphosphate (STPP) were purchased from Sigma Aldrich. Nutrient agar and broth were obtained from Difco. The bacterial strain Pseudomonas aeruginosa was obtained from the Korea Collection for Type Cultures (KTCC).

2.2 Synthesis of AgNPs

Fresh ginger was obtained from a local market in Jeonju City, South Korea, and was cleaned with deionized water (D·H₂O). The ginger was sliced and dried in an oven. Subsequently, 5 g of the dried ginger were mixed with 50 mL of D·H₂O and boiled at 70 °C for 3 h. After cooling down, the resulting extract was filtered using Whatman filter paper, followed by sonication for 10 min at 40 kHz. To prepare the AgNO₃ solution, 8.5 mg (1 mM) of AgNO₃ was dissolved in 25 mL of D·H₂O, and this solution was then mixed with 25 mL of the ginger extract. The mixture was heated at 70 – 80 °C. After 30 min, the reaction mixture changed color from transparent to sparkling yellow and subsequently to sparkling brown after 1 h, indicating the synthesis of AgNPs. The formation of AgNPs was confirmed by monitoring the UV–Vis absorption spectrophotometric (Hewlett Packard (HP 8453) throughout the reaction.

2.2.3

2.2.3 Synthesis of Cs/AgNCs/tbn

To conjugate Cs/AgNCs with tobramycin, EDC and Sulfo-NHS were used. Using Sulfo-NHS, carboxyl groups are turned into an amine-reactive ester, facilitating cross-linking or bioconjugation. It is a charged version of NHS (N-hydroxy succinimide) and can be used to modify carbodiimide crosslinking reactions. A carboxylate (–COOH) present in the polymer molecule is activated so that it can be conjugated with the primary amine (–NH₂) found in tobramycin (Armijo et al., 2020). Initially, 10 mg of tobramycin was dissolved in a mixture of 20 mL of DMSO and D·H₂O in a 1:1 ratio. The solution was then sonicated for 20 min at 50 kHz. Following this, 5 mg of EDC was added to the solution while continuously stirring. Subsequently, 5 mg of NHS was added, and the pH was adjusted to 6. The solution was kept in the dark for 3 – 4 h to allow the conjugation reaction to occur (forming “solution 1″). Next, 20 mg of Cs/AgNCs was suspended in 5 mL of a 1:1 mixture of DMSO and D·H₂O This suspension was added to ”solution 1,“ and the pH was adjusted to 8. The mixture underwent stirring for 12 h. Once the specified time elapsed, the solution was centrifuged at a speed of 7000 rpm for 12 min. The supernatant was removed, and the resulting pellet was washed three times using DMSO followed by ethanol. Later, the remaining pellet was dried at a temperature of 65 °C for 3 h to obtain the conjugate nanoparticles. The characterization of the conjugate nanoparticles was performed using FTIR. Finally, the nanoparticles were stored at 4 °C for further usage (Tyagi et al., 2020). Fig. 1. Describes the formation of Cs/AgNCs/tbn.

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Fig. 1

Describes the formation of Cs/AgNCs/tbn.

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2.3 Characterization of nanomaterials

The plant extract, CsNPs, AgNPs, and Cs/AgNCs were dissolved in D·H₂O and characterized by scanning the nanoparticle solutions using a UV–Visible spectrophotometer in the range of 250 – 600 nm (Hewlett Packard (HP 8453). Additionally, an extra 15 min of centrifugation at 14,000 rpm was performed on the synthesized mixtures. The resulting pellets were collected, cleaned, freeze-dried, and stored. For transmission electron microscopic (TEM) examination of the sizes and shapes of the nanoparticles, the nanoparticles were mixed with 1 mL of D·H₂O Then, 25 µL aliquots of CsNPs, AgNPs and Cs/AgNCs were placed on TEM grids, air-dried, and imaged using a Hitachi H-7650 electron microscope with an accelerating voltage of 80 to 100 kV. FE-SEM (Field Emission Scanning Electron Microscopy) and EDS (Energy-Dispersive X-ray Spectroscopy) analyses were carried out on freeze-dried powder. The samples were coated with carbon tape and then platinum-coated before analysis, using a Carl Zeiss SUPRA40VP microscope. Structural analysis of CsNPs, AgNPs, and Cs/AgNCs was performed using HR-XRD (High Resolution X-ray diffraction) through a Bruker D8 advanced instrument. FTIR (Fourier Transform Infrared) analysis was conducted using the powder of CsNPs, AgNPs, Cs/AgNCs, and Cs/AgNCs/tbn. The ATR method was employed, and the spectra were recorded in the range of 4000 – 500 cm⁻¹ using a (PerkinElmer) instrument.

2.4 Antibacterial potential of nanoparticles

The antibacterial activity of plant extract, CsNPs, AgNPs, Cs/AgNCs, and Cs/AgNCs/tbn against P. aeruginosa was evaluated using the Kirby-Bauer disk diffusion susceptibility test, following the method described by Bauer (1966). Single bacterial colonies were chosen and incubated overnight in Mueller-Hinton broth (MHB) at a temperature of 37 °C and a speed of 250 rpm. The optical density of the resulting bacterial suspension was determined at a wavelength of 600 nm using a spectrophotometer and then adjusted to a concentration of 1 × 10⁶ CFU/mL. Mueller-Hinton Agar (MHA) plates were prepared and subsequently inoculated with the adjusted bacterial suspension. Sterile 6 mm paper discs filled separately with sterile D·H₂O (control), tobramycin, CsNPs, AgNPs, Cs/AgNCs, Cs/AgNCs/tbn with a different concentration (5 to 20 µg/mL) were placed on the MHA plates. The plates were subsequently incubated at 37 °C for 24 h and visually inspected for the zone of inhibition (ZOI) around the discs, indicating the bioactivity of the substance in the discs.

To assess the dose-dependent antibacterial efficiency, a modified MTT assay was conducted following a protocol based on Krishnaraj et al., (2010). Briefly, 100 µL of sterile media was added to each well of a 96-well plate. Starting from the first well of each row, a 200 µg in 100 µL concentration of all the reactants was added, and serial dilutions were performed. Then, 100 µL of bacterial strains were added to all the wells, followed by incubation for 12 h at 37 °C. Control wells were maintained for all the test pathogens without the addition of nanomaterials. Subsequently, a known concentration of MTT solution (5 µL of 0.5 %) was added to all the wells, and the absorbance was measured using an ELISA reader at OD₅₉₅.

3.1 Synthesis of nanomaterials

This study employs the synthesis of CsNPs, AgNPs, and Cs/AgNCs from Zingiber officinale rhizome extract, which is reported for its antimicrobial properties (Menon et al., 2019). The biomolecules present in the extract, such as terpenoids, alkaloids, ascorbic acid, oxalic acid, and flavonoids, play a role in stabilizing and reducing the nanoparticles (Fig. 2). (Jahan et al., 2021). These green-synthesized nanoparticles offer a potential alternative to combat antibiotic-resistant bacteria, addressing issues related to low biocompatibility, short-term efficiency, and chemical instability commonly associated with conventional antibiotics.

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Fig. 2

Schematic Illustration of AgNPs reduction and capping using phytochemicals from Zingiber officinale rhizome.

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3.2 UV–Vis spectroscopic analysis

UV–Vis spectroscopy was performed to confirm the biosynthesis of nanoparticles. The plant extract, CsNPs, AgNPs, and Cs/AgNCs was scanned at a wavelength ranging from 250 − 600 nm as shown in (Supplementary Fig. S1). The plant extract absorption spectrum showed an absorption peak of 278. This is in agreement with the previous study of Mehata (2021), who reported an absorption peak of ginger extract at 277 nm. The absorption spectra of all nanoparticles ranged between 270 and 322 nm, except for AgNPs, which exhibited absorption between 400 and 500 nm. Each nanoparticle displayed distinct peak values. When 25 mL of ginger extract was mixed with 25 mL of 1 mM AgNO₃ solution, the previously transparent solution became a sparkling yellow, indicating the successful synthesis of AgNPs. Confirmation came from the maximum absorbance observed at 432 nm. This synthesis, indicating the presence of AgNPs due to Surface Plasmon Resonance, was further confirmed by the absorption spectrum. For the synthesis of CsNPs, a mixture containing 40 mg of chitosan in 100 mL of 1 % acetic acid was mixed with 25 mL of ginger extract and 8 mL of 0.1 % STPP, revealing a maximum absorbance at 276 nm within the range of 270 – 322 nm. Similar procedures were applied in the synthesis of Cs/AgNCs, differing only in the addition of 100 mL of AgNPs to a solution comprising 100 mL of 40 mg chitosan dissolved in 1 % acetic acid (pH > 6), along with 25 mL of ginger extract and 8 mL of 0.1 % STPP. Remarkably, the Cs/AgNCs exhibited a maximum absorbance at 275 nm, and an absorbance of 1.1893 at 436 nm, confirming the successful synthesis of Cs/AgNCs. These results agree with the previous study of Krishnaraj et al., (2022) who synthesized CsNPs, AgNPs, and Cs/AgNCs using the leaves extract of Cucurbita pepo.

3.2.1

3.2.1 Transmission electron microscopy (TEM) and high-resolution X-ray diffraction (HR-XRD) for size and crystallinity analyses

TEM and HR-XRD analyses were performed to characterize and confirm the size, shape, and crystallinity of AgNPs, CsNPs, and Cs/AgNCs. For crystallinity analysis, the diffracted intensities in the whole spectrum of 2θ were recorded between 20° to 90°. The TEM image of AgNPs showed that all the AgNPs are spherical (Fig. 3a) in shape which is very important because shape plays a vital role in the antibacterial activity of nanoparticles. Previous research studies show that AgNPs with spherical shapes are more effective than AgNPs with any other shape. Ciou et al., (2009) argued that spherical AgNPs are more effective because they can effectively interfere with and easily penetrate the bacterial cell membrane. The average diameter of AgNPs was 19 nm making AgNPs the smallest nanoparticles used in this study. The HR-XRD spectrum of AgNPs shows characteristic peaks at 2θ angles of ∼ 38.37°, ∼44.02°, ∼64.74°, and ∼ 77.54° which is equivalent to 111, 200, 220, and 311 crystal planes respectively. However, the wider peaks represent the poor crystallinity of AgNPs as shown in Fig. 4a. Fig. 3b showed that the diameter of CsNPs varied between 15–30 nm with an average diameter of 22 nm. The size of nanoparticles significantly affects their antibacterial activity. It has been concluded from previous literature that nanoparticles with a smaller sizes have more significant antibacterial potential than those with larger diameters because of their cell membrane penetration potential (Yin et al., 2020). The HR-XRD spectrum of CsNPs represents characteristic peaks at 2θ angles of ∼ 8.79°, ∼11.43°, ∼29.74°, and ∼ 45.69°. These sharp narrow peaks represent the crystalline nature of CsNPs as shown in Fig. 4b. Additional peaks related to Z. officinale compounds were also recorded. Fig. 3c. represented the TEM images of Cs/AgNCs showing that almost all the Cs/AgNCs were spherical with increased diameters ranging between 17.09 and 46.64 nm, with 28.61 nm as their average diameter. The average diameter of Cs/AgNCs was larger than that of AgNPs and CsNPs due to their conjugation. A similar study was reported by Butler et al., (2015), who concluded that the size of AgNPs increases upon conjugation. The HR-XRD pattern of Cs/AgNCs displayed the characteristic peaks of both AgNPs and CsNPs as shown in Fig. 4c. Very sharp narrow peaks of Cs/AgNCs at 2θ angle of ∼ 8.76°, ∼18.98° for CsNPs and at ∼ 37.50°, ∼44.52°, ∼66.01°, and ∼ 77.04° for AgNPs which equates to a 111, 200, 220 and 311 crystal peaks (respectively) represent the crystallinity of Cs/AgNCs. The additional peaks observed were due to the presence of secondary metabolites during the synthesis of the nanoparticles.

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Fig. 3

Transmission Electron Microscopy images of AgNPs (a), CsNPs (b), and Cs/AgNCs (c) for size and shape analysis.

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Fig. 4

X-ray diffraction graphs of AgNPs (a), CsNPs (b), and Cs/AgNCs (c) for crystallinity analysis.

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3.2.2

3.2.2 Field emission scanning electron microscopy (FE-SEM) to study surface morphology

The FE-SEM micrographs demonstrated spherical AgNPs with an average size of 40.3 nm (Fig. 5a). No agglomerates were found, revealing the stability of biosynthesized AgNPs, as they can be seen distinctly. The results presented that biosynthesized AgNPs possessed small grain-like particles, that were gathered to shape crystals with a smooth surface (Bharadwaj et al., 2021). The micrograph of CsNPs (Fig. 5b). showed a dense structure with agglomerated particles. The average size of CsNPs was measured to be 52.5 nm. Scattered particles on thin film were observed for Cs/AgNCs, with an average particle size of 49 nm (Fig. 5c). CsNPs were strongly attached to the surface of AgNPs which following the Chi-AgNPs synthesized by Senthilkumar et al., (2019). Being conjugate, Cs/AgNCs/tbn was found to be densely agglomerated (Fig. 5d). The conjugates were partly spherical with an average size of 66 nm, and similar morphologies were preserved throughout.

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Fig. 5

FE-SEM micrographs representing surface morphology of (a) AgNPs (b) CsNPs (c) Cs/AgNCs (d) Cs/AgNCs/tbn.

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3.2.4

3.2.4 FTIR study

The FTIR spectra of samples confirmed the formation of Cs/AgNCs and Cs/AgNCs/tbn by showing the presence of functional groups of compounds of AgNPs, chitosan, and tobramycin that are combined through conjugation (Fig. 6). The FTIR spectrum of Cs/AgNCs showed the absorption of infrared waves by functional groups that were similar to that of AgNPs and CsNPs within the same wavenumber range with slight shifts that indicated the formation of bonds between the compounds after conjugating them with tbn. The absorption bands of AgNPs could be seen at 1669, 1589, 1418, 1269, 1051, 659, and 575 cm⁻¹. These band at 1669 is attributed to C = C (alkene), 1589 to N–H bending (amine), 1418 to C-N stretching (aromatic amine), 1269 to O–H bending (alcohol), 1051 to C-O stretching (primary alcohol), 659 to C-Br stretching, and 575 cm-1 to C-I stretching referring to Halogen compounds. These functional groups were mostly compounds from the ginger extract that had capped silver ions to form silver nanoparticles (Yang et al., 2017). The absorption peaks for CsNPs were observed at 3001, 1573, 1409, 1150, 1043, 1013, 896, 647, 619, and 555 cm⁻¹. The absorption at the given wavenumber corresponded to the presence of C–H stretching (alkene), N–H bending (amine), O–H bending (alcohol), CO stretching (ether), CO-O-CO stretching (anhydride), C-F stretching (fluoro compound), C = C bending (alkene), C-Br stretching, and C-I stretching (halogen compounds), respectively. Tobramycin revealed the presence of N–H stretching (amine), C = H stretching (alkene), C–C stretching (alkene and aldehyde), C–H bending (alcohol), C-O stretching (alcohol), C = C bending (alkene), C–H bending (mono-substitute), O–H bending (alcohol), S-O stretching (sulfonate), C-O stretching, C-Cl stretching and C-Br stretching. These functional groups were identified based on their absorption at 3347, 2898, 1578, 1465, 1381, 1348, 1135, 1092, 840, 773, 659, and 603 cm⁻¹, respectively. The absorption by the same functional groups in the same range (spectra of AgNPs and CsNPs) with little shift was seen in the FTIR spectrum of Cs/AgNCs that showed the combination of AgNPs and CsNPs. The same results were also reported by (Govindan et al., 2012) who reported almost the same functional groups in the same range with little shifts when AgNPs and CsNPs were combined. For example, the bands obtained at 1573 (N–H bending (amine)), 1408 (O–H bending (alcohol)), 1128 (C-O stretching (alcohol)), 780 (C = C bending (alkene)), 619 (C-Br), and 514 cm^-1C-I stretching (halogen compounds) were due to the presence of AgNPs. While the bands at 3270 (C–H stretching (alkyne)), 1336 (S = O stretching (sulfonate)), 1232 (C-O stretching (ether)), 1043 (CO-O-CO stretching), 1013 (C-O stretching (anhydride)), 893 (C-F stretching (fluoro compound)), and 646 cm⁻¹ (C–H bending (trisubstitute) were due to the presence of CsNPs. The differences in the wavenumber range might be due to the difference in source for the synthesis of AgNPs. The same assumption was also reported by (Govindan et al., 2012, Ghasemzadeh et al., 2016, Dara et al., 2020) who observed similar functional groups in Cs/AgNCs with somehow little variation in the wavenumber range. Similarly, Cs/AgNCs/tbn is the combination of Cs/AgNCs and tobramycin, so the functional groups detected by the FTIR spectra of tobramycin and Cs/AgNCs could also be seen on the spectrum of Cs/AgNCs/tbn. Almost all the bands observed were the same as that of Cs/AgNCs FTIR spectra except N–H stretching (amine) at 3384, C–C stretching (alkene) at 1670 and 1659, S = O stretching (sulfonate) at 1322, C-O stretching (primary and secondary alcohol) at 1091 and 1059, C = C bending (alkene) at 799, 725 and 705, and C-Br stretching (halogen compounds) at 657. The disappearance of bands similar to tobramycin and composite and the emergence of new bands in the spectrum of conjugation could be due to the occurrence of conjugation. Helal et al., (2014) also observed in their study that some of the original bands of tobramycin disappeared after conjugation and new bands emerged confirming conjugation. Similarly, our FTIR results also align with the previous study of (Armijo et al., 2020), who reported the formation of an amide bond as an indicator of the formation of a conjugate with nanoparticles which here is observed at 1670 cm⁻¹.

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Fig. 6

FTIR spectra of CsNPs, AgNPs, tobramycin, Cs/AgNCs, and Cs/AgNCs/tbn.

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3.3 Bacterial cell susceptibility results were determined through disk diffusion and MTT assay

The disk diffusion assay revealed a noticeable trend referring to the increase in the size of the inhibition zone with an increase in the applied volume of each sample (Supplementary Fig. S3). Such a trend has been presented in previous studies showing the dose-dependent activity of antibacterial agents (Zienkiewicz-Strzalka et al., 2019). The largest inhibition zones were observed for Cs/AgNCs/tbn with 20.3 ± 0.33 mm at 20 µL followed by tobramycin (12.6 ± 0.33 mm), Cs/AgNCs (9.66 ± 0.33 mm), and AgNPs (8.33 ± 0.19 mm) (Table 2). Somehow, similar results were obtained by Volova et al., (2018) where silica composite conjugated with antibiotic were found to be more active than the composite conjugated with AgNPs alone. Thus, it's known that antibiotics penetrate the cell membrane and bind irreversibly to their receptors, alter proteins’ function, and inhibit the synthesis of bacterial membranes.

The MTT assay used to determine bacterial cell viability with the application of AgNPs, Cs/AgNCs, and Cs/AgNCs/tbn showed significant results that agree with the results of the disk diffusion assay. Importantly, the results might vary with the type of strain being tested. The lowest MIC was recorded for the conjugate of composite with tobramycin i.e., 0.4 µg/mL. Such a low concentration of conjugate was effective because the currently tested strain of bacteria is tobramycin susceptible (inhibited by 1.56 µg/mL of tobramycin). It has been reported in previous literature that MIC of tobramycin could indicate the susceptibility of the bacteria, such as inhibition of the bacterial cell growth at a concentration of ≤ 4 µg/mL shows the specific strain to be susceptible (Armijo et al., 2020). The MIC of AgNPs (12 µg/mL) was lower than that of Cs/AgNCs (25 µg/mL). The antibacterial activity of Cs/AgNCs was most probably due to the presence of AgNPs because CsNPs alone did not show any antibacterial effect (Dara et al., 2020). The nano-silver can disrupt the integrity of the cell membrane, produce oxidative stress, deactivate bacterial proteins by binding to the thiol group, and deactivate metabolic processes such as osmoregulation, electron transport chain, and respiration (Zienkiewicz-Strzalka et al., 2019). The Cs/AgNCs conjugated with tobramycin showed a remarkable antibacterial effect that proves the synergic effect of tobramycin, CsNPs, and AgNPs together. The MIC of Cs/AgNCs/tbn was four times lesser than that of tobramycin alone indicating the reduction of doses by 75 %. A similar synergism was reported in a study where chitosan-AgNPs in combination with different antibiotics were tested against Gram-positive and Gram-negative bacteria (Brasil et al., 2018).

3.4 Bio-transmission electron microscopy results determine the mode of action of CsNPs, AgNPs, Cs/AgNCs, and Cs/AgNCs/tbn

To support the antibacterial activity, the interaction of CsNPs, AgNPs, Cs/AgNCs, and Cs/AgNCs/tbn with bacteria was studied through Bio-TEM. Generally, all the tested samples were found to have strong interaction with bacteria which most probably would be the reason for their toxic effect on bacteria. AgNPs adhered to the cell wall of bacteria and then penetrated bacterial cells disrupting the integrity of the cell that ultimately led to bacterial cell death (as shown in Fig. 7a) (Krishnaraj et al., 2015). CsNPs exhibit electrostatic interactions between the amino groups of positively charged glucosamine and the negatively charged bacterial cell membranes. This interaction triggers changes in the permeability of the membrane that results in the efflux of cell components leading to cell death (Chandrasekaran et al., 2020). In Fig. 7b. The interaction of chitosan with the cell wall of P. aeruginosa satisfies its proposed antibacterial mechanism. Cs/AgNCs due to their small size and large surface area to volume ratio were shown to interact and penetrate bacterial cell walls (Fig. 7c). Similar results were found in a study where chitosan derivative combined with AgNPs was applied against E. coli. When they examined bacterial cells after treatment with Cs/AgNPs, severe detachment of the cell wall from the outer membrane was observed to allow the cytoplasmic content to leave the cell (Huang et al., 2017). In agreement with the results of disk diffusion and MTT assay, Cs/AgNCs/tbn demonstrated a strong bacterial interaction leading to cell disruption (Fig. 7d). The synergic antibacterial effect of AgNPs, CsNPs, and tobramycin enables the conjugate theoretically to target multiple sites of action causing rapid bacterial cell death (Baptista et al., 2018).

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Fig. 7

Bio-TEM micrographs representing biological interaction of a) AgNPs b) CsNPs c) Cs/AgNCs d) Cs/AgNCs/tbn.

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3.5 Reactive oxygen species (ROS) quantification

To evaluate the level of oxidative damage caused to bacteria by nanoparticles, ROS was quantified using Confocal Laser Scanning Microscopy (CLSM) after treating the P. aeruginosa with plant extract AgNPs, CsNPs, Cs/AgNCs, and Cs/AgNCs/tbn. For this purpose, the level of CHS1, (chilling-sensitive mutant) was determined after treatment and compared to control (untreated bacterial cells), as the level of CHS1 indicates the generation of ROS (Fig. 8). While fluorescence intensity in Fig. 9. showed that all the types of nanoparticles were very effective and resulted in a significant generation of ROS. However, the level of significance varied in the nanoparticles. Cs/AgNCs/tbn was most effective with the highest CHS1 count of 49809, followed by AgNPs (33321). In addition, the results obtained from DNA degradation, MTT, and disc diffusion assays were also confirmed that Cs/AgNCs/tbs were most effective in antibacterial activities, indicating the direct dependence of all types of nanoparticles on their ROS-generating potential. Plant extract and CsNPs generated the lowest levels of ROS with the highest CHS1 counts of 20,217 and 18,650 respectively. Again, this supports the previous assumption because CsNPs caused the least oxidative damage to the macromolecules of bacteria as shown in DNA degradation, disc diffusion, and MTT assays. The ROS generation potential of Cs/AgNCs (29749) was almost the same as that of AgNPs. A nanoparticle generation of ROS and its antibacterial activity is one of the most researched mechanisms. Also, previous literature suggests that nanoparticles affect bacteria by targeting bacterial lipid and protein membranes by inducing ROS generation leading to bacterial cell death (Gold et al., 2018). Nemčeková et al., (2022) reported that AgNPs-induced oxygen species kill bacteria by altering their DNA by interacting with the charged phosphate backbone of DNA disrupting the electrostatic forces that aid the stability of double helix resulting in the weakened stability of hydrogen bonds between complementary bases. However, Radzig et al., (2013) suggested that ROS targets the bases rather than phosphate groups. When they treated P. aeruginosa with AgNPs, they noted that AgNPs generated ROS by inhibiting the enzymes, DNA lesions were formed from the oxidized precursors of DNA. Zhang et al., (2018) argue that the generation of ROS by AgNPs causes oxidative damage to bacteria due to the strong affinity between the positive charge of AgNPs and the negative charge on the peptidoglycan layer of bacterial cell membranes.

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Fig. 8

ROS quantification of P. aeruginosa treated with AgNPs, CsNPs, Cs/AgNCs, Cs/AgNCs/tbn.

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Fig. 9

Fluorescence microscopy images of P. aeruginosa treated with plant extract (b), CsNPs (c), Cs/AgNCs (d), AgNPs (e), and CS/AgNCs/tbn (f) compared to Control (a).

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3.6 DNA degradation assay

The purpose of the assay was to determine whether all the nanoparticles had caused damage to the DNA of P. aeruginosa. For this purpose, bacterial cells were treated with plant extract, AgNPs, CsNPs, Cs/AgNCs, and Cs/AgNCs/tbn. The treated cells were scanned at 200–300 nm using a UV–Vis spectrophotometer and their absorbance was compared to the control (untreated bacterial cells). The results showed that all the nanoparticles effectively damaged the bacterial DNA as shown in (Supplementary Fig. S4). However, the level of significance of all the nanoparticles and conjugates varied. The results also showed that Cs/AgNCs/tbn (abs = 0.108 at 280 nm) damaged the DNA of P. aeruginosa most significantly. Our results agree with the previous study of Ahmad et al., (2017), who argued that the composition of chitosan and silver nanoparticles significantly damaged bacterial DNA. Interestingly, Cs/AgNCs/tbn was also most effective in generating ROS as shown in (Supplementary Fig. S4). While CsNPs (abs = 0.29) and plant extract (0.325) were the least effective. This also aligns with the results of ROS quantification, as they generated the least ROS. This indicates that the DNA oxidation potential of nanoparticles is directly dependent upon their ROS-generating potential. AgNPs (abs = 0.121) were more effective than Cs/AgNCs (abs = 0.232). This might be due to an increase in the size of AgNPs when combined with CsNPs because the penetration ability of nanoparticles decreases as their size increases. This assumption agrees with the study of Butler et al., (2015), who reported that the DNA oxidation potential of nanoparticles decreased upon combination with antibiotics or other nanoparticles.

3.7 In vitro zebra fish embryo toxicity

In this in vitro study, the embryonic toxicity of various concentrations of AgNPs, CsNPs, CS/AgNPs, and Cs/AgNCs/tbn was assessed using zebrafish embryos. The activity of the embryos was monitored from 3 to 96 h, and different parameters were examined to determine if nanoparticles exhibited toxicity towards the fish embryos at a specific concentration (Table 3). The hatching rate of the embryos was evaluated using plant extracts in concentrations ranging between 200 μg/mL to 0.19 μg/mL. Results indicate that the hatching rate of the zebrafish embryos exposed in plant extracts (Fig. 10b), was comparable to the control group (Fig. 10a), from 24 to 96 h. 100 % of the zebrafish embryos hatched successfully, and there was no delay in hatching compared with the control group. The survival rate of the hatched embryos was also similar between the plant extract group and the control. Additionally, the heart rate of zebrafish embryos was assessed after 72 h and compared to the control group. Minor changes were observed in the heart rate of the embryos exposed to plant extract, with values ranging from 120 to 160 beats per minute (bpm), while the control group exhibited a heart rate of approximately 120 to 170 bpm. No structural changes were detected in the zebrafish embryos exposed to the plant extracts.

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Fig. 10

(a) Control (b) The effect of plant extract on the hatching rate of zebrafish embryos; (c) the effect of various concentrations of CsNPs on the hatching rate, survival rate of zebrafish embryos; (d) the effect of various concentrations of AgNPs on the hatching rate, survival rate of zebrafish embryos; (e) the effect of various concentrations of Cs/AgNPs on the hatching rate, survival rate, of zebrafish embryos; (f) the effect of various concentrations of Cs/AgNCs/tbn on the hatching rate, survival rate, of zebrafish embryos.

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The toxicity of CsNPs was examined as the same concentration of plant extracts; the CsNPs showed toxicity in higher concentrations ranging from 200 μg/mL to 100 μg/mL (Fig. 10c); no hatching was recorded at higher concentrations. Exposure to 50 μg/mL of CsNPs the hatch rate significantly declined at 48 hpf compared with the control results in a concentration-dependent reduction hatch rate of zebrafish embryos. They were then compared to the control group after being exposed to CsNPs concentration of 25 μg/mL to 0.19 μg/mL for 24 to 72 h of examination. After 72 h, the embryo hatching rate in lower concentrations was closer to the control group. The survival rate at 50 μg/mL was 80 % and from 25 μg/mL to 0.19 μg/mL was 100 % successful and the heart rate in CsNPs compared to the control group increased from 113 to 154 ± 6 bpm. Regarding the structural change in the zebrafish embryo in lower concentration, no significant changes were detected in 96 h of the study. These results are relatable to the results shown by Bhoopathy et al., (2021), where high concentrations of CsNPs (10 – 20 mg/mL) caused malformation of zebrafish embryos. At 20 mg/mL, the mortality rate of zebrafish was shown to be high. Thus, it can be concluded that the toxicity level of CsNPs is dose-dependent and increases with increasing concentration.

An analysis of the hatching rate of zebrafish embryos exposed to increasing doses of AgNPs ranging from 200 μg/mL to 50 μg/mL, (Fig. 10d), showed higher toxicity to embryos as observed 0 % hatching rate at 72 hpf. The hatch rate depends on concentration when the concentration decreases to 25–12.5 μg/mL, the embryo hatched delay compared with the control group. The delay in hatching may be due to Ag⁺ released from the dissolution of AgNPs or may be a result of other inhibition processes (Sharma 2020). After 72 h in lower concentrations ranging from 6.25 μg/mL to 0.19 μg/mL AgNPs showed a normal hatching rate. The survival rate of embryos also depends on the concentration of AgNPs. As the concentration of AgNPs increases the number of normally developed zebra fishes decreases, and the mortality rate increases (Krishnaraj et al., 2023). When they were treated with higher doses of AgNPs 200 μg/mL,100 μg/mL, and 50 μg/mL, they showed higher toxicity and they had no survival rate from 24 h to 96 h compared to low doses of AgNPs, the survival rate was increased. It has been reported in previous studies that 142.2 μg/L is critical concentration of AgNPs in adult zebra fishes (Krishnaraj et al., 2016). After this, the heart rate was also calculated and compared to the control, AgNPs significantly decreased the heart rate of embryo zebrafish which was the indicator of toxicity level (Xin et al., 2015). It was recorded after 72 h in 25 μg/mL to 0.019 μg/mL is 101 to 139 ± 4 bpm. AgNPs in 200 μg/mL to 50 μg/mL with higher toxicity demonstrate coagulation, which is not recorded in plant extracts, CsNPs, Cs/AgNPs, and Cs/AgNCs/tbn.

Cs/AgNPs were found to exhibit toxicity at higher concentrations ranging from 200 µg/mL to 100 µg/mL, while no adverse effects were observed at lower concentrations. Exposure to 200 µg/mL and 100 µg/mL of Cs/AgNPs resulted in no hatch rate of zebrafish embryos, particularly at 72 and 96 hpf (Fig. 10e). However, at lower concentrations (50 µg/mL to 25 µg/mL), the embryos exhibited delayed in hatching from 24 to 72 h but showed a hatching rate like the control group after 72 h. The survival rate was 75 % successful in concentrations that range from 50 µg/mL to 25 µg/mL and from 12.5 µg/mL to 0.19 µg/mL, the survival rate increased to 100 %, and the heart rate of the embryos exposed to Cs/AgNPs ranged from 108 to 152 ± 2 bpm, which was comparable to the control group. No significant structural changes were detected in the zebrafish embryos at lower concentrations after 96 h of examination. All the data for Cs/AgNPs shows less toxicity than AgNPs. The possibility for reduced toxicity is the presence of the Cs matrix around Cs/AgNPs (Pérez-Díaz et al., 2016).

The hatching rate of zebrafish embryos was examined using Cs/AgNCs/tbn at concentrations ranging from 200 μg/mL to 0.19 μg/mL (Fig. 10f). A results demonstrated the hatching rate of the embryos exposed to Cs/AgNCs/tbn was comparable to the control group throughout the 24 to 96 h period. The embryos exposed to Cs/AgNCs/tbn showed no delay in hatching referred to the control group, with 100 % of the embryos hatching successfully. The survival rate of the hatched embryos was similar between the Cs/AgNCs/tbn group and the control group. The heart rate of the zebrafish embryos was also assessed after 72 h and compared, to the control group. Minor changes in heart rate were observed in the embryos exposed to the Cs/AgNCs/tbn, ranging from 115 to 155 ± 4 bpm, while the control group exhibited a heart rate of approximately 120 to 170 bpm. No structural changes were detected in the zebrafish embryos exposed to the Cs/AgNCs/tbn. All the data shows that Cs/AgNCs/tbn is less likely to have toxicity toward zebrafish embryos compared to AgNPs, CsNPs, and Cs/AgNPs. Thus, Cs/AgNCs/tbn is the best antibacterial agent with no toxic effect and higher antibacterial activity even at very low concentrations.