Polyol-synthesized bismuth oxide nanorods: Interaction with blood protein and antibacterial activity

2.1. Materials

Bismuth nitrate (Bi(NO₃)₃), ethylene glycol (EG), poly(vinyl pyrrolidone) (PVP) with a molecular weight of 58000, and cetyltrimethylammonium bromide (CTAB), were purchased from Merck Company (Shanghai, China). Human hemoglobin (lyophilized powder, H7379) was purchased from Sigma (USA). All reagents were used without further purification and were of analytical grade.

2.2. Synthesis of Bi₂O₃ NRs

It is important to note that the synthesis temperature and precursor concentration play key roles in determining the morphology and dimensions of the Bi₂O₃ NRs. Higher temperatures may accelerate nucleation and growth, favoring anisotropic rod-like structures, while precursor concentration influences the degree of supersaturation, which can affect the uniformity and aspect ratio of the NRs. These morphological features directly impact surface area, electrostatic interactions, and van der Waals forces, which in turn influence both protein binding and antibacterial activity. Understanding these effects is crucial for optimizing the functional performance of Bi₂O₃ NRs in biomedical applications.

Bi(NO₃)₃ was reduced by the polyol method as reported previously [17,18]. Briefly, the synthesis of Bi₂O₃ NRs was achieved by preparing a mixed solution of PVP (0.8 mmol) and EG (10 mL), heating at 120 °C for 2 h, injection of CTAB solution (2 mL, 0.09 M), incubation for 15 min, and addition of aqueous Bi(NO₃)₃ (0.110 M) under vigorous stirring.

CTAB as a surfactant controls the shape and size of the Bi₂O₃ NRs, while PVP serves as a stabilizing agent that prevents the aggregation of NRs. EG also acts as a solvent that facilitates the growth of the Bi₂O₃ NRs. Then, the sample was kept at 120 °C for 45 min and cooled to ambient temperature. After precipitation by acetone, excess materials were washed away with ethanol three times. The final sample was redispersed in ethanol and used for further studies.

2.3. Analytical characterization of Bi₂O₃ NRs

Physical techniques such as transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to determine the morphology and size of NRs. Chemical techniques including X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) were used to explore the composition, bonding and surface chemistry.

The morphology and diameter of the as-synthesized Bi₂O NRs, both in the absence and presence of Hb at a 1:1 ratio, were analyzed using TEM (FEI Tecnai, G2 Spirit Twin, USA, operated at 120 kV). The TEM grids used for analysis were prepared by applying a drop of the diluted NR solution on a carbon-coated copper grid and later drying it at room temperature for 45 min. The hydrodynamic size and charge distribution of as-synthesized Bi₂O₃ NRs in the absence and presence of Hb (1:1) were determined through DLS using Zetasizer Nano S (Malvern Instruments, UK). The laser wavelength was set to 632.8 nm with a 90° scattering angle. The XRD analysis was performed by XPERT-PRO with Cu kα radiation (λ = 1.5406 Å) operated at 40 kV and 30 mA at a 2θ angle in the region of 10°-80°. The XRD findings were compared with the Joint Committee on Powder Diffraction Standards (JCPDS) library to determine the crystalline structure. To identify the functional groups and chemical bonds present in the as-synthesized Bi₂O₃ NRs, the FTIR spectrum was recorded using a KBr pellet on a Shimadzu FTIR spectrometer (8400S, Japan) in the range of 400-4000 cm⁻¹. Briefly, a small amount of Bi₂O₃ NRs was mixed with dry KBr powder, ground to a fine mixture, and pressed into a transparent pellet. The resulting spectrum provides insight into the surface functional groups of Bi₂O₃ NRs, which influence van der Waals and other non-covalent interactions with biomolecules such as Hb.

2.4. Determination of the concentration of Bi₂O₃ NRs

The molar concentration of Bi₂O₃ NRs was calculated with the length (r) and diameter (h) determined by TEM, density of Bi₂O₃ ≈ 8.9 × 10⁶ g∙m^-3, molar mass of Bi₂O₃ = 465.96 g∙mol^-1, and Avogadro’s number = 6.022 × 10²³ particles∙mol^-1.

First, the mass of a single Bi₂O₃ NR was calculated using (Eq. 1):

Then number of Bi₂O₃ NRs per liter was calculated (Eq. 2):

Finally, the molar concentration was calculated (Eq. 3):

2.5. Hemolysis assay

Bi₂O₃ NRs were assessed for their hemolytic activity on human erythrocytes. Fresh blood samples (from healthy volunteers, 20- to 30-year-old, male, non-smokers, and with no medication) were prepared by mixing 1 mL of human blood with saline buffer (9 mL, 0.9%; w/v), followed by washing three times with saline buffer, centrifugation (5000 ×g, 15 min, 4 °C), and resuspension in PBS. Then, 0.1 mL of blood was added to 0.9 mL of Bi₂O₃ NRs with different concentrations (0.1-3 mg/mL) and incubated for 60 min at 37 °C. Afterwards, the solutions were centrifuged (5000 ×g, 15 min) and the absorbance of the supernatant was read at 450 nm. Positive (100% hemolysis) and negative (0% hemolysis) controls were assessed through incubating erythrocytes with 1% Triton X-100 in PBS and saline solution, respectively.

Hemolysis percentage was estimated as reported previously as follows (Eq. 4):

where A, A₀, and A₁₀₀ denote the absorbance of the sample, negative, and positive controls, respectively.

2.6. Bi₂O₃ NRs –hemoglobin binding

The human whole blood was centrifuged (1400 ×g, 10 min), washed three times in saline buffer, and mixed (100 µL) with lysis buffer (500 µL, 2.5 M NaCl, 100 mM EDTA–Na salt, 1% Triton X-100, and 10% DMSO).

Then, the lysate was incubated with different concentrations of Bi₂O₃ NRs (0.1–3 mg/ml) for 60 min, followed by centrifugation (1400 ×g, 10 min). The degree of unbound Hb on Bi₂O₃ NRs was assessed by reading the absorbance of the supernatant at 540 nm. All the experiments were run in PBS (0.9% NaCl in 10 mM sodium phosphate buffer, pH 7.4) in a conventional quartz cell.

2.7. Bi₂O₃ NRs –Hb interaction study by fluorescence spectroscopy

Fluorescence emission spectra were recorded using a Cary Eclipse spectrofluorometer, with the excitation wavelength (λₑₓ) set at 295 nm and emission spectra collected between 300-450 nm across five temperatures ranging from 298 K to 314 K. Both excitation and emission slit widths were set at 5 nm. Quenching experiments were performed by titrating Bi₂O₃ NRs at varying concentrations (0.1, 0.5, 1, 5, and 10 μM) into Hb samples (5 μM) prepared in 50 mM phosphate buffer (pH 7.4). To eliminate the inner-filter effect, fluorescence intensities were corrected. Also, the fluorescence intensity of Bi₂O₃ NRs was subtracted from the protein signal.

2.8. Synchronous fluorescence spectroscopy

Synchronous fluorescence spectroscopy (SFS) was performed by simultaneously scanning the excitation and emission monochromators while maintaining constant wavelength intervals (Δλ) of 15 nm and 60 nm. All other experimental conditions were similar to those described in the fluorescence spectroscopy section.

2.9. Circular dichroism (CD) measurements

CD measurements were performed in the far-UV range (200–260 nm) using an Aviv 215 spectropolarimeter (USA) to assess the impact of Bi₂O₃ NRs on the secondary structure of Hb. The Hb concentration was maintained at 15 μM, and varying concentrations of Bi₂O₃ NRs (ranging from 0.1 to 10 μM) were incrementally added to the protein solution prepared in 50 mM phosphate buffer (pH 7.4). Spectra were recorded at room temperature, and an average of three scans was used to improve the signal-to-noise ratio. The obtained CD spectra were subsequently analyzed using CDNN software to estimate changes in secondary structure elements, including α-helix, β-sheet, and random coil content, providing insights into the structural alterations induced by nanoparticle binding.

2.10. Fourier-transform infrared spectroscopy study

FTIR spectroscopy was employed to investigate the secondary structural changes of Hb upon interaction with Bi₂O₃ NRs. Measurements were performed using a PerkinElmer System 2000 FTIR spectrometer, focusing on the amide I region (1500–1700 cm⁻¹), which is particularly sensitive to alterations in protein secondary structure. Samples of free Hb and Hb–Bi₂O₃ NR bioconjugates (prepared at a final NR concentration of 10 μM) were analyzed. Spectral data were collected using an attenuated total reflectance accessory under dry film conditions to minimize water interference. The spectra were baseline-corrected, normalized, and the analysis was conducted to provide comparative insights into conformational shifts induced by nanoparticle binding.

2.11. UV-visible study

UV-visible spectroscopy was employed to investigate both the structural integrity of Hb and the microenvironmental alterations around its heme group. Measurements were performed across two spectral regions: 230–320 nm, to assess protein structural changes, and 380–430 nm, to monitor perturbations in the heme environment, using a UV–visible spectrophotometer. In these experiments, the Hb concentration was maintained at 15 μM in phosphate buffer (50 mM, pH 7.4), and titrations were carried out by incremental addition of Bi₂O₃ NRs at concentrations ranging from 0.1 to 10 μM. Changes in absorbance spectra were analyzed to elucidate possible conformational modifications and interactions between Hb and Bi₂O₃ NRs.

2.12. Molecular docking study

A Bi₂O₃ NR model with a diameter of 5 nm and a height of 25 nm was constructed by replicating the Bi₂O₃ crystal unit cell structure. Molecular docking simulations were performed using HEX 6.3 software to investigate the interaction between Bi₂O₃ NRs and Hb. The 3D X-ray crystallographic structure of human Hb (PDB ID: 2H35) was retrieved from the RCSB Protein Data Bank. Prior to docking, all water molecules and non-essential free atoms were removed from the Hb structure to prepare it for simulation. The docking was executed under standard conditions, and the binding poses with the lowest binding energies were selected for detailed analysis. Visualization and analysis of the docking results were carried out using UCSF Chimera and PyMOL molecular graphics tools.

2.13. Antibacterial assay

The “well diffusion method” was used to test the antibacterial activity of Bi₂O₃ NRs and Bi₂O₃ NR–Hb complexes on P. aeruginosa, E. coli, and S. aureus, as described previously [19]. To evaluate how Hb binding influences the antibacterial properties of Bi₂O₃ NRs, Bi₂O₃ NR–Hb complexes were prepared prior to testing. Bi₂O₃ NRs (1 mg/mL) were incubated with an equimolar concentration of Hb (10 µM) in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 2 h under gentle agitation. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Bi₂O₃ NRs were then determined based on the procedures reported previously [20].

2.14. Statistical analysis

All data are expressed as mean ± SD from three replicates. Data are analyzed by one way analysis of variance (ANOVA) using the SPSS program. P < 0.05 was used when differences were significant. 

3.1. Characterization of Bi₂O₃ NRs in the absence and presence of hemoglobin

Given the significance of protein-nanoparticle interactions in determining nanomaterial biocompatibility, this study focuses on Hb as a model macromolecule to evaluate the biosafety of Bi₂O₃ NRs. Bi₂O₃ NRs were synthesized and characterized to assess their morphology and their ability to interact structurally with Hb. As shown in Figure 1(a), TEM confirms that the synthesized Bi₂O₃ NRs exhibit a uniform rod-shaped morphology, with an average diameter of ∼8 nm and a length of ∼50 nm. This high aspect ratio may influence their surface reactivity and interaction dynamics with proteins. Other studies have shown that the diameter of the Bi₂O₃ NRs synthesized with O₂/Ar gas flow ratio of 0.2 had a wide range of 40-250 nm. That is why the authors claimed that the O₂/Ar gas flow ratio needs to be manipulated in order to fabricate homogeneous Bi₂O₃ NRs [21]. Also, Wang et al. through an electrospinning approach in the presence of polyacrylonitrile as reducing agent were able to synthesize Bi₂O₃ nanofibers (length: several µm, diameters: 200-300 nm) [22]. Furthermore, Sood et al. were able to produce well-crystalline α-Bi₂O₃ NRs through surfactant free sono-chemical process with diameters in the range of 100-120 nm and lengths in the range of 7-8 µm [23]. Therefore, different investigations have proposed that several parameters, including reaction temperature and time as well as reductant and surfactant concentration play key roles in determining the size and distribution of the as-synthesized Bi₂O₃ nanostructures.

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Figure 1.

Characterization of Bi₂O₃ NRs synthesized through a one-pot solution approach method. TEM analysis in the (a) absence and (b) presence of Hb, DLS study for determination of (c) hydrodynamic size, (d) PDI, and (e) zeta potential of Bi₂O₃ NRs in the absence and presence of Hb, (f) XRD analysis, (g) FTIR analysis.

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Upon exposure to human Hb, as depicted in Figure 1(b), Bi₂O₃ NRs display marked agglomeration, indicating a strong interaction between the nanoparticles and the protein. This aggregation behavior suggests the formation of Bi₂O₃ NR-Hb complexes, likely mediated by non-covalent interactions such as electrostatic forces and hydrophobic contacts between the nanoparticle surface and Hb’s functional groups. Such interactions may induce structural perturbations in Hb, potentially affecting its conformational integrity and biological function.

These findings underscore the importance of probing nanoparticle-induced changes in protein structure as a key factor in evaluating the biomedical applicability and safety of nanomaterials. By centering the investigation on Hb as the primary macromolecular target, this study contributes to a deeper understanding of how inorganic nanostructures can influence protein behavior at the molecular level.

To further investigate the interaction between Hb and Bi₂O₃ NRs, DLS analysis was performed to monitor changes in particle size distribution, polydispersity index (PDI), and surface charge (zeta potential) upon Hb binding. In the absence of Hb, Bi₂O₃ NRs displayed a hydrodynamic diameter of approximately 151.57 nm (Figure 1c), with a low PDI (Figure 1d), indicating good colloidal stability and a uniform particle population. However, upon introduction of Hb, both the hydrodynamic diameter and PDI increased significantly, suggesting that Hb binding alters the physical state of the nanoparticle suspension.

This increase in size and polydispersity is indicative of surface adsorption and/or partial aggregation, pointing to strong nanoparticle-protein interactions. The formation of Bi₂O₃ NR-Hb complexes likely involves non-covalent forces, which induce secondary structural rearrangements in Hb or promote clustering of nanoparticles through protein bridging. These findings are consistent with the agglomeration patterns observed in TEM imaging (Figure 1b), reinforcing the hypothesis that Hb directly interacts with Bi₂O₃ NRs in a manner that affects both protein conformation and nanoparticle dispersity.

Collectively, these DLS results highlight Hb’s role not merely as a passive binder but as a dynamic participant in nanoparticle interaction, with potential implications for its biological function and the safety profile of Bi₂O₃ NRs in biomedical contexts

Nanostructure-based products usually display strong van der Waals attraction forces, resulting in unwanted agglomeration. Therefore, optimizing the steric hindrance as well as electrostatic repulsion among nanostructures could be considered as a potential strategy to intensify their colloidal stability. Enhancing the zeta potential could be used as a well-known approach to manipulate the electrostatic repulsion among nanostructure materials. NRs with a high surface charge distribution demonstrate a high electrostatic repulsion force and significant colloidal stability. Figure 1(e) presents the zeta potential measurements of the synthesized Bi₂O₃ NRs. The initial zeta potential was found to be approximately -25.56 mV, indicating a moderately high negative surface charge, which suggests a favorable initial dispersion in aqueous environments. This negative charge suggests that the surface of the Bi₂O₃ NRs may be coated with negatively charged functional groups or ions, contributing to electrostatic repulsion between particles. Such repulsion could prevent the Bi₂O₃ NRs from aggregating, thereby promoting steric hindrance and resulting in a stable colloidal suspension. Good colloidal stability under environmentally relevant conditions is a critical factor affecting the mobility, transformation, and ultimate fate of nanomaterials once released into natural ecosystems.

This observation is consistent with the DLS results, which showed a low PDI and relatively uniform hydrodynamic size, supporting the idea of well-dispersed NRs in solution. While the exact chemical nature of the surface charge needs further confirmation, possibly through FTIR, the data clearly indicate good colloidal stability in simple aqueous media, suggesting that Bi₂O₃ NRs could remain suspended and thus bioavailable in biological media.

However, upon introduction of Hb, the zeta potential decreased to around −20.89 mV. This reduction in surface charge suggests partial neutralization of the Bi₂O₃ NRs’ negative surface by Hb, possibly due to protein adsorption or surface interaction. The diminished electrostatic repulsion leads to a lower colloidal stability, which is reflected in the DLS data as an increase in hydrodynamic radius and PDI, and is further corroborated by TEM images showing the onset of NR clustering or slight aggregation.

Altogether, these findings indicate that while Bi₂O₃ NRs are initially stable in suspension, the presence of biological molecules such as Hb alters their surface characteristics, promoting interparticle interactions and a tendency toward agglomeration. Such protein-nanomaterial interactions may also occur in biological environments where Bi₂O₃ NRs could encounter biomolecules, affecting their aggregation behavior, bioavailability, and toxicity profiles. The resulting agglomeration may lead to changes in the physicochemical properties of the NPs, which can affect their stability, cellular uptake, and biological behavior. Therefore, understanding these interactions is vital not only for biomedical applications but also for predicting the imposed risks associated with the unintentional utilization of Bi₂O₃ nanostructures.

XRD analysis (Figure 1f) of the Bi₂O₃ NRs revealed the composition of the final synthesized particle. It is evident from Figure 1(f) that the XRD data show well-resolved pattern peaks, confirming the crystalline and monophasic nature of the synthesized NRs. The prominent peaks of Bi₂O₃ NRs were detected in the range of 2θ (10° to 80°). The diffraction peaks attributing to planes (1 1 0), (2 1 0), (2 0 1), (0 0 2), (2 2 0), (2 2 2), (4 0 0), (2 0 3), (4 2 1), and (4 0 2) provide an important reason for the preparation of face centered cubic (FCC) Bi₂O₃ NRs [24]. It is evident that the Bi₂O₃ NRs are free from other phases, which is typically expected when nanostructures are pure. The XRD pattern also matches well with the JCPDS card no. 78-1793.

FTIR analysis was conducted for the study of the probable vibrational bonds in Bi₂O₃ NRs. The FTIR spectrum of as-synthesized Bi₂O₃ NRs has been presented in Figure 1(g). FTIR spectrum of Bi₂O₃ NRs produced with a one-post synthesis approach shows several peaks at 3420/1670, 1020, and 455 cm⁻¹, corresponding to O-H vibration, Bi-O, and Bi-O-Bi bond vibrations, respectively. This data suggested that the Bi₂O₃ NRs may carry a large content of OH⁻ moieties on their surfaces, which can be helpful for further surface modification with any organic/inorganic groups [25].

3.2. Hemocompatibility and hemoglobin binding assays

Based on the American Society for Testing and Materials (ASTM F 756–00, 2000), each nanomaterial can be categorized into three different classes: hemolytic (hemolysis percentage> 5%), slightly hemolytic (hemolysis percentage 2%-5%), and non-hemolytic (hemolysis percentage< 2%) [26]. In the present study, the hemolytic activity of Bi₂O₃ NRs was further assessed at a concentration range of 0.1-3 mg/mL, which was comparable to the concentrations of NPs reported previously [26]. Although an enhancement in the hemolysis percentage was detected upon increasing the Bi₂O₃ NRs in a concentration-dependent manner, the maximal hemolysis percentage was determined to be 1.68% after interaction of 3 mg/mL Bi₂O₃ NRs with erythrocytes for 60 min (Figure 2a).

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Figure 2.

(a) Hemolysis assay of prepared Bi₂O₃ NRs with different concentrations (0.1-3 mg/mL) after 60 min at 37 °C. (b) Hb binding of different concentrations of Bi₂O₃ NRs (0.1-3 mg/mL) was assessed by reading the absorbance of the supernatant at 540 nm. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

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As a result, the prepared Bi₂O₃ NRs demonstrated negligible hemolytic activity based on the ASTM F 756-00 (2000). These data are in good agreement with those reported by Lu et al. [27], which indicated that metal oxide NPs (Ag/ZnO composite) had negligible hemolytic properties (2.81%) and were therefore considered non-hemolytic nanomaterials.

Negligible hemolytic activity suggests a reduced likelihood of acute cytotoxicity following accidental or continuous exposure, thus supporting the consideration of Bi₂O₃ NRs as relatively safe candidates for biomedical applications.

However, while the lack of hemolysis is encouraging, it does not eliminate concerns related to chronic exposure, bioaccumulation, or interactions with non-erythroid cells. Therefore, further studies are warranted to assess long-term biocompatibility.

As there was no significant amount of hemolysis induced by Bi₂O₃ NRs, it inspired us to explore the interaction between Hb in erythrocyte lysate and Bi₂O₃ NRs, as already reported previously for titanium oxide NPs [28]. Importantly, from a biosafety perspective, the concentration-dependent binding could raise concerns about the potential for nanoparticle bioaccumulation and aggregation effects in biological systems. Thus, understanding the dose-dependent nature of these interactions is critical for evaluating the toxicological risks associated with nanoparticle utilization.

It was detected that as the concentration of Bi₂O₃ NRs increased, the Hb binding percentage increased (Figure 2b), notably at 0.5 mg/mL and above. This phenomenon could likely be driven by several key factors related to the surface area, available binding sites, and interactions between the NRs and Hb. Additionally, as the concentration of Bi₂O₃ NRs increases, the number of available NR particles in the solution also increases. Also, as the concentration of NRs increases, more binding sites are available for interaction with Hb. At higher concentrations, the local concentration of Hb in the vicinity of the NRs increases, which may also promote stronger protein-nanoparticle interactions.

These data were in line with Ghosh et al. [28], who reported that metal oxide NPs can bind Hb in a concentration-dependent manner.

3.3. Fluorescence spectroscopy studies

The intrinsic fluorescence intensity of Hb originates mainly from β-Trp37 [29]. The fluorescence intensity of Hb shows a λ_max at 339, which is in line with a previous study [29], when the λ_ex was fixed at 295 nm (Figure 3a). During the titration of Bi₂O₃ NRs, a progressive decrease in the fluorescence intensity of Hb was observed, accompanied by a red shift in the fluorescence maxima. This behavior suggests a strong interaction between Bi₂O₃ NRs and Hb, likely inducing conformational alterations in the protein structure [4,29]. Such nanoparticle-protein interactions may have important implications for the biological behavior of nanomaterials.

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Figure 3.

(a) Fluorescence quenching measurement of Hb after interaction with Bi₂O₃ NRs, (b) Stern-Volmer plots of Hb-Bi₂O₃ NR bioconjugates, (c) modified SV plots of Hb-Bi₂O₃ NR bioconjugates, (d) Van ‘t Hoff plot of Hb-Bi₂O₃ NR bioconjugates.

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3.4. Conformational change studies

3.4.1. Synchronous fluorescence spectroscopy measurement

NPs can bind to different proteins and trigger conformational changes. Such changes can be evaluated by SFS. This technique provides useful insights into the molecular environment surrounding fluorophore moieties of Hb. By fixing Δλ at 60 nm or 15 nm, information about the microenvironment of Trp or Tyr residues, respectively, can be obtained. Figure 4 exhibits the SFS measurement of Hb with increasing Bi₂O₃ NR concentration. The addition of Bi₂O₃ NR resulted in a decrease in SFS intensity, along with a red shift in λ_em for both Trp (Figure 4a) and Tyr (Figure 4b) residues.

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Figure 4.

Synchronous fluorescence measurement of Hb after interaction with Bi₂O₃ NRs at Δλ values of (a) 60 nm or (b) 15 nm.

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Such data disclose that the interactions between Bi₂O₃ NRs and Hb could occur in the vicinity of Tyr/Trp residues. It is also obvious that the structure of Hb could be changed in a way that the polarity surrounding the Tyr and Trp residues increased, and concurrently, the hydrophobic index decreased.

3.4.2. CD and FTIR studies

To evaluate the structural modifications of Hb in the presence of Bi₂O₃ NRs, circular dichroism (CD) spectra of native Hb and Hb-Bi₂O₃ NR bioconjugates were recorded (Figure 5a), and the secondary structure fractions have been summarized in Table 4. The CD spectrum of native Hb displayed two minima at ∼208 and 222 nm, characteristic of the α-helix conformation due to n → π^* transition in the peptide bond of α-helix. Upon formation of Hb-Bi₂O₃ NR bioconjugates, a decrease in α-helix and β-sheet content and a corresponding increase in random coil fractions were observed. These changes were concentration-dependent.

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Figure 5.

(a) CD and (b) FTIR spectra of Hb after interaction with Bi₂O₃ NRs.

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Table 4. Secondary structure fractions of Hb after interaction with Bi₂O₃ NRs.

Concentration of Bi2O3 NR	%α-helix	%β-sheet	%Random coil
0	71.16	5.21	14.12
0.1	65.12	4.58	22.97
1	59.91	4.09	24.68
10	55.31	3.54	35.98

This analysis confirms that Bi₂O₃ NRs interact with the Hb polypeptide backbone, leading to protein unfolding.

To further study the conformational changes in FTIR spectra of Hb and Hb-Bi₂O₃ NR bioconjugates, were recorded. The FTIR spectrum of Hb showed amide I and II bands near 1650 cm⁻¹ and 1540 cm⁻¹, respectively (Figure 5b). After binding with Bi₂O₃ NRs, an increase in the intensity of both bands and a shift in the amide I band from 1650 to 1625 cm⁻¹ were observed. The amide I band is particularly sensitive to secondary structure changes, including alterations in α-helices, β-sheets, and aggregate formation [29]. These spectral changes confirm that Bi₂O₃ NRs interact with the protein backbone, causing unfolding and conformational modifications [29]. The FTIR findings are in strong agreement with CD results, reinforcing the conclusion that Bi₂O₃ NRs compromise Hb’s structural integrity. From a biosafety standpoint, such nanoparticle-induced protein denaturation could have broader biological consequences. Disruption of vital proteins, for example, could impair essential physiological processes such as oxygen transport, metabolism, and stress responses, ultimately imposing health risks.

3.4.3. UV-visible study

The UV-Vis spectra of native Hb and Hb–Bi₂O₃ NR bioconjugates have been presented in Figure 6. Native Hb displays two main absorption bands: ∼278 nm, associated with aromatic residues (Figure 6a), and ∼405 nm, corresponding to the heme Soret band (Figure 6b) [29]. Upon Bi₂O₃ NR addition, increased absorbance was observed at both 278 and 405 nm. A red shift in λ_max at 278 nm was also detected.

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Figure 6.

UV-visible spectra of Hb after interaction with Bi₂O₃ NRs at (a) wavelength ranges 230 -320 nm and (b) 380-430 nm.

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Similar behavior has been reported for silver [31] and gold NPs [29]. In the current study, the interaction of Bi₂O₃ NRs with Hb also led to noticeable changes in the microenvironment surrounding aromatic residues and the heme group, indicating partial protein unfolding. This is significant because nanoparticle-induced protein conformational changes could impair vital biological functions, including oxygen transport and redox balance, in exposed organisms.

Proteins, such as hemopexins, OmpF, OmpA, Flagellin, and chaperones in bacteria, could similarly undergo structural alterations, leading to compromised physiological functions. Thus, the observed Hb unfolding behavior highlights the need to assess not only human biosafety but also the broader toxicological impacts of Bi₂O₃ NR exposure.

3.5. Molecular docking study

Molecular docking was employed to examine the interaction between Bi₂O₃ NRs (Figure 7a) and Hb, as well as the structure of the resulting Hb-Bi₂O₃ NR bioconjugate (Figure 7b). The calculated binding energy was –512.50 E-value, which is more favorable than Hb–Hb-zero-valent iron (–311.80) [10], Hb-nickel oxide (−389.42) [8], and Hb-alumina NPs (−374.41) [7], but less favorable than Hb–silica (–813.27) [9] and Hb-cerium oxide NPs (−651.85). These results demonstrate that the type and properties of the NPs significantly influence binding affinity.

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Figure 7.

(a) Bi₂O₃ NR cluster, (b) Hb-Bi₂O₃ NR bioconjugate complex, and (c) the amino acid residues in the binding pocket from (i, ii) two rotational views.

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The binding site analysis identified residues such as ASP94, PRO95, VAL96, LYS99, SER133, ALA130, ASP126, ALA123, ASP99, GLU101, LEU105, LYS139, THR137, TPR37, ARG40, TYR35, VAL33, and PRO36 [Figure 7c (i, ii)]. Notably, TRP37, located at the α₁β₂ interface and a key contributor to Hb fluorescence and structural stability, appears to be a primary binding site. Binding at this location likely accounts for the observed fluorescence quenching and potential Hb destabilization [29]. The presence of TYR35 in the binding pocket supports the SFS findings, suggesting changes in the aromatic residue microenvironment.

Altogether, the structural alterations of Hb identified via spectroscopic analyses are strongly supported by molecular docking results. These findings raise broader biosafety concerns, as they imply that Bi₂O₃ NRs can target and perturb conserved structural domains of globular proteins. While molecular docking provides valuable static insights into binding modes and interaction sites, future studies incorporating molecular dynamics simulations would allow us to explore the dynamic behavior, stability, and conformational flexibility of the Bi₂O₃ NR–Hb complex under physiological conditions. Such analyses would provide a more comprehensive understanding of the potential toxicological impacts of these NRs.

Having established the significant interaction between Bi₂O₃ NRs and Hb, including evidence of nanoparticle-induced conformational changes, it is essential to extend the investigation to another critical biomedical application of nanomaterials: their antibacterial activity. While the interaction with proteins such as Hb provides valuable insight into biosafety and molecular-level behavior in physiological environments, the probable therapeutic utility of Bi₂O₃ NRs also depends on their functional efficacy, particularly in combating bacterial infections.

3.6. Antibacterial activity of Bi₂O₃ NRs

Bacterial resistance to conventional antibiotics has become an escalating clinical concern, necessitating the development of alternative agents capable of circumventing traditional resistance mechanisms. Metal oxide nanoparticles, including Bi₂O₃, have shown potential in this area due to their physicochemical properties, which allow for unique modes of antibacterial action, ranging from membrane disruption to interference with intracellular metabolic processes. Among various nanostructures, rod-shaped particles are of particular interest because their high aspect ratio may enhance surface interactions with bacterial membranes, thereby increasing antibacterial potency. Given the strong affinity between Bi₂O₃ NRs and Hb, it was essential to evaluate whether complexation alters antibacterial efficacy. In this context, the current section focuses on evaluating the antibacterial activity of Bi₂O₃ NRs and Bi₂O₃ NR–Hb complexes. By assessing their effects on model bacterial strains, we aim to determine the practical relevance of these NRs as potential antimicrobial agents in the absence and presence of macromolecules and provide a broader understanding of their biomedical applicability.

The MIC and MBC values obtained in this study (Table 5) clearly indicate that the synthesized Bi₂O₃ NRs exhibit notable antibacterial effects against both Gram-positive (S. aureus) and Gram-negative (E. coli, P. aeruginosa) bacteria. Specifically, the MIC values ranged from 25  (E. coli) to 100  (P. aeruginosa), with corresponding MBC values confirming strong bactericidal activity.

However, the antibacterial activity of the Bi₂O₃ NR–Hb complexes was significantly lower than that of the bare Bi₂O₃ NRs (Table 5). For all bacterial strains, both MIC and MBC values increased markedly following Hb complexation. For instance, the MIC of Bi₂O₃ NRs against S. aureus increased from 50 µg/mL to 150 µg/mL upon Hb binding, while E. coli showed an increase from 25 µg/mL to 100 µg/mL. Similar trends were observed for P. aeruginosa, the most resistant strain tested. These results suggest that Hb adsorption forms a protein corona that partially masks reactive surface sites of the NRs and promotes aggregation, thereby decreasing the effective surface area available for bacterial interaction and reducing ROS generation or ion release. Consequently, the antibacterial potency of Bi₂O₃ NRs is substantially attenuated when complexed with Hb.

We indicated that protein corona formation is an important consideration for biomedical applications involving nanomaterials. Therefore, our study focused on investigating the antibacterial behavior of Bi₂O₃ NRs and Bi₂O₃ NR–Hb complexes to fully assess their applicability in such systems.

Previous reports have shown that gold NRs (with length, width, and aspect ratio of ∼50 nm, ∼12 nm, and ∼4 nm, respectively) functionalized with different moieties show MIC values against S. aureus in the range of 0.461-0.117 nM, depending on their purity and surface groups [32]. Moreover, Guglielmelli et al. [33] and Ban et al. [34] indicated that protein corona on nanomaterials induces structural change of proteins and reduces their bactericidal potential. Bai et al. suggested that the antibacterial activity of hierarchical titanium oxide NRs is derived from the direct interaction of NRs with bacterial cell walls and subsequent localized piercing mediated by the sharp outward spikes presented on rod nanostructures [35]. In addition, the growth inhibition of E. coli and S. aureus by gold nanoparticles (NPs) revealed that the strongest antibacterial effect was exhibited by gold nanostars, followed by NRs and nanospheres. Interestingly, it was reported that ion release alone did not directly correspond to the observed antibacterial trends. Although the ion concentrations released from NRs and nanospheres were higher than those from nanostars, the superior antimicrobial activity was associated with the nanostars. This phenomenon was attributed to factors beyond simple ion release, including particle shape and surface reactivity, probably with biomacromolecules. The higher ion release from NRs and nanospheres was linked to their larger surface areas due to their small particle sizes. Therefore, it can be inferred that variations in the antimicrobial potency of nanoparticles are influenced not only by quantity or surface area but also by intrinsic physicochemical characteristics and protein binding. A key limitation of the present study is the lack of mechanistic insights into the antibacterial actions of Bi₂O₃ NRs, such as contributions from ion release, ROS generation, or membrane disruption. Future investigations should aim to elucidate these mechanisms, particularly under biologically relevant conditions, to better assess the biological fate associated with Bi₂O₃ nanomaterials.