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. 2026 Jul 2;16(34):32716–32729. doi: 10.1039/d6ra01978a

Biomimetic synthesis of chitosan-stabilized palladium nanoparticles for biomedical and catalytic applications

Prashant D Sarvalkar a, Aditya B Magdum b, Minseok Nam a, Mrunal N Damle c, Mansingraj S Nimbalkar b, Neeraj R Prasad d, Meghnad G Joshi c,e,✉, Kiran Kumar K Sharma d, Dongcheon Park a,✉, Kwangwoo Wi a,✉
PMCID: PMC13325528  PMID: 42395738

Abstract

We report the biomimetic synthesis of chitosan-stabilized palladium nanoparticles (PdNPs@CS) using Bos taurus indicus (BTI) urine as a benign bioreductant and chitosan as a stability scaffold. Powder X-ray diffraction with Rietveld refinement confirmed the presence of crystalline fcc-Pd, while TEM revealed the presence of quasi-spherical PdNPs uniformly distributed on the chitosan matrix, even after catalysis. Under aqueous and dark conditions, PdNPs@CS catalyzes NaBH4-driven hydride transfer from nitroarenes to aminoarenes via the Langmuir–Hinshelwood pathway. The reaction followed pseudo-first-order kinetics in excess NaBH4 with representative apparent rate constants of 0.0067 s−1 (4-NP) and 0.0087/0.0182/0.0154 s−1 (2-/3-/4-NA). The catalyst showed robust recyclability, offering high conversions till the fifth cycle, with post-reaction XRD/TEM verifying the phase and morphological integrity. Biological assessments demonstrated favorable hemocompatibility, significant antioxidant activity, pro-angiogenic effects, and selective antiproliferative efficacy at appropriate concentrations, indicating a biocompatible interface suitable for biomedical applications. PdNPs@CS integrated the advantages of eco-friendly synthesis, efficient aqueous hydride-transfer catalysis, operational reusability, and promising biological applications, positioning this material as a multifunctional platform for water remediation and allied biomedical applications and aligning with contemporary biopolymer-engineered materials for sustainable pollutant mitigation and cycling stability.

We report the biomimetic synthesis of chitosan-stabilized palladium nanoparticles (PdNPs@CS) using Bos taurus indicus (BTI) urine as a benign bioreductant and chitosan as a stability scaffold.graphic file with name d6ra01978a-ga.jpg

1. Introduction

The rapid growth of global industrialization has led to significant environmental pollution.1 Nitroaromatic compounds are widely used in the preparation of chemicals, pharmaceuticals, and dyes; however, their toxicity makes them hazardous pollutants.2–4 Specifically, 4-nitrophenol (4-NP) causes hepatic, renal, hematological, and neurological damages, with its permissible limit set at 0.43 µmol L−1 in the U.S. Environmental Protection Agency (USEPA) drinking water guidelines. The selective reduction of nitroarene compounds to amines has proven effective in environmental remediation and biomedicine development, where the synthesis of specific amine intermediates is useful in drug development.5,6 Among the various materials used in catalytic processes, palladium (Pd) is highly effective because of its exceptional ability to facilitate hydrogenation, reduction, and oxidation reactions, like those in organic synthesis reactions,7–9 fuel cells,10 and environmental remediation.11,12 However, bulk Pd has limited reactivity owing to its relatively low surface area. To overcome this limitation, palladium nanoparticles (PdNPs) are increasingly used due to their high surface-area-to-volume ratios, which significantly enhance catalytic efficiency, reaction rates, and selectivity, making them ideal for a wide range of applications, including the catalytic degradation of toxic pollutants.13,14

Despite the advantages of biological syntheses, like eco-friendly and large-scale production of NPs, there are challenges to overcome, such as controlling the particle size and aggregation and mitigating poor reproducibility.15,16 This study addresses these issues via a biogenic route, in which Bos taurus indicus (BTI) metabolic waste acts as a reducing agent to convert Pd2+ to Pd0, promoting nucleation and growth, overcoming aggregation, and ensuring colloidal dispersibility, thereby resulting in size-controlled PdNPs with enhanced catalytic efficiency, stability, and reusability. This cost-effective and eco-friendly method provides a scalable solution to improve catalytic efficiency for biomedical applications.

Recent studies have highlighted chitosan–Pd composites for their stability, catalytic efficiency, and biocompatibility.17–20 Chitosan stabilizes PdNPs through electrostatic interactions and coordination between the amine (–NH2) and hydroxyl (–OH) groups of chitosan and Pd, thereby preventing nanoparticle agglomeration. The polymer matrix forms a shell around PdNPs, thereby ensuring uniform dispersion, enhancing stability, and preventing metal leaching during catalytic processes.18,21 Furthermore, Pd-based nanocomposites have shown promise in catalytic and environmental remediation applications, including hydrogenation, Suzuki coupling, and nitroarene reduction reactions.22 Dhanavel et al. reported that chitosan-rGO/Pd composites exhibited a high turnover frequency (TOF) of 19.8 h−1 for the reduction of 4-nitrophenol to 4-aminophenol,23 while cross-linked chitosan/Pd composites demonstrated catalytic efficiency with a maximum rate constant of 4.01 × 10−3 s−1.21 Furthermore, chitosan-reduced graphene oxide/Pd composites loaded with 5-fluorouracil and curcumin showed sustained drug release and therapeutic efficacy against HT-29 colon cancer cells,24 while cross-linked chitosan/Pd composites exhibited broad-spectrum antibacterial activity against Staphylococcus aureus and Escherichia coli.21 In our previous investigations, we demonstrated the green synthesis of various nanomaterials using BTI urine as a reducing agent, including CuO NPs, Co3O4 NPs, Pd NPs, Ag NPs, trimetallic nanostructures, and Ag/rGO composites.25 Although cetyltrimethylammonium bromide (CTAB) is widely used as a capping agent/surfactant, it exhibits inhibitory effects on biological systems at concentrations >0.3 mg L−1 (300 ppb), with inhibition observed in the 8–24 mg L−1 range; accordingly, CTAB-containing nanomaterials are unsuitable for biomedical applications unless residual CTAB is rigorously reduced to <0.3 mg L−1.26 Compared to previous studies, the PdNPs@CS composites developed in this study exhibited enhanced stability and catalytic efficiency. The use of BTI urine as a green reducing agent and the biopolymer chitosan as a stabilizer improved the dispersion of PdNPs, reducing their tendency to aggregate and improving their recyclability, which is crucial for both biomedical and catalytic applications.23,27

Here, we report a green, low-cost, and scalable route to synthesize chitosan-stabilized PdNPs (PdNPs@CS), in which BTI urine serves as an intrinsic bioreductant and replaces the use of conventional toxic reductants. In this study, chitosan functioned as a polyelectrolyte matrix that limited NP agglomeration, improved dispersion and stability in water, and preserved access to the Pd active sites. In this study, PdNPs@CS efficiently catalyzed the reduction of representative nitroaromatics in the presence of NaBH4 (as a reducing agent) under aqueous conditions, offering recyclability over five cycles and minimal loss of catalytic performance. Biocompatibility assessments like antioxidant and hemocompatibility assays indicated a benign biointerface that supports biomedical applications. This study advances nanocatalysis using biopolymer supports by mitigating NP aggregation while preserving catalytic activity, positioning PdNPs@CS as a potential candidate for pollutant reduction and in biomedical applications.

2. Experimental section

2.1. Materials

A detailed list of the chemicals and cell lines, along with their sources and purity, is provided in the SI.

2.2. Extraction of chitosan and chitin

Chitin was extracted from the pupae and larval stages of Hermetia illucens (black soldier fly; BSF) using the method reported by Soetemans et al.28 with slight modifications (addition of a decolorization step), followed by conversion to chitosan (CS) via deacetylation. The extraction process involves two main steps: (1) chitin extraction through demineralization, deproteinization, and decolorization, and (2) deacetylation to convert the extracted chitin into chitosan. The detailed reaction steps of the extraction, including the chemical reaction, pH, temperature, and drying conditions, are provided in the SI. Fig. S1 shows a graphical representation of the extraction pathway steps. The decolorization step is a laboratory-scale purification step for improving sample characterization and may be removed or optimized in future large-scale implementations.

2.3. Synthesis of chitosan-stabilized Pd nanoparticles (PdNPs@CS)

A 0.01 M PdCl2 solution was prepared by dissolving 0.35 g of PdCl2 in 200 mL of 0.02 M HCl with continuous stirring. In a separate beaker, a 1% (v/v) acetic acid solution was prepared; 1.0 g of chitosan was added and stirred until completely dissolved. A 200 µL of chitosan solution (1% w/v) was pipetted into the PdCl2 solution with continuous stirring for 30 min. Subsequently, 50 mL of freshly collected BTI urine was added dropwise to the reaction mixture from a burette under constant stirring. Following the complete addition of the BTI urine, the reaction mixture gradually turned blackish. The product was collected by centrifugation and washed repeatedly with double-distilled water until a neutral supernatant pH was achieved, transferred to a Petri dish, and vacuum-dried overnight at 60 °C in a hot air oven. The resulting NPs were stored for further characterization and applications.

2.4. Characterization study

A powder X-ray diffractometer (Bruker D8 Advanced, Germany; Cu Kα) was used to record the XRD patterns of BSF-derived chitin (pupae and larvae), BSF-derived chitosan (pupae and larvae), and the as-synthesized PdNPs@CS. Rietveld refinements and Fourier electron-density maps were performed using the FullProf Suite (version 5.20), employing a pseudo-Voigt profile function. Fourier-transform infrared (FTIR) spectroscopy (ALPHA Bruker) was used to analyze and identify functional groups. Thermogravimetric analysis-differential thermal analysis (TGA-DTA) was performed using an SDT Q600 thermal analyzer in an N2 atmosphere at a heating rate of 10 °C min−1 from 25 °C to 1000 °C to determine the thermal stability. The particle size and surface charge of the as-synthesized materials were measured using an Anton Paar Litesizer 500 particle size analyzer. Scanning electron microscopy (SEM) (Model: TESCAN VEGA3 SEM) and transmission electron microscopy (TEM) (Model: JEM-2100, JEOL), combined with energy-dispersive spectroscopy (EDS), enabled high-resolution imaging and elemental mapping. X-ray photoelectron spectroscopy (XPS) was performed using a JEOL JPS 9030 instrument to determine the binding energies. The reduction of nitroarenes was analyzed using an Agilent Cary-60 UV-vis spectrophotometer. Hemocompatibility, cytocompatibility, and biocompatibility were evaluated using a UV-vis spectrophotometer for hemolysis measurements, a microplate reader for the MTT cytotoxicity assay, and a fluorescence microscope for CAM histology (see the SI S1.4.1–S1.4.3 for detailed protocols).

2.5. Catalytic study

The catalytic activity of the synthesized PdNPs@CS was evaluated by the reduction of nitroarenes using NaBH4 as the reducing agent. The reduction of nitroarenes was monitored using UV-vis spectroscopy over a wavelength range of 200–800 nm at room temperature, with absorbance measurements recorded at 30 s intervals. In the assay, 1.0 mL of ice-cold NaBH4 solution (12 mM), 1.0 mL of aqueous nitroarene solution (0.15 mM), and 50 µL of PdNPs@CS solution (0.02 mg mL−1) were mixed in a 3.0 mL quartz cuvette, maintaining a molar ratio of 80 : 1 between the NaBH4 and nitro compounds. Detailed protocols for both catalytic assays are provided in the SI section.

2.6. Biomedical evaluation and functional assessment of PdNPs@CS

The biomedical evaluation and functional assessment of PdNPs@CS were conducted using various tests to assess their hemocompatibility, cytocompatibility, biocompatibility, and antioxidant properties. Hemocompatibility was evaluated by conducting a hemolysis study at various concentrations, where the absorbance value at 541 nm indicated minimal or extensive RBC lysis (SI S1.4.1).29 Cytocompatibility was assessed using the MTT assay on MCF-7 cells, and cell viability was determined by measuring absorbance at 570 nm (SI, S1.4.2).30 Cell viability at each PdNPs@CS concentration was normalized to the absorbance of the untreated control cells (negative control: 100% viability). Biocompatibility was further evaluated using a chick chorioallantoic membrane (CAM) assay, in which morphological and histological assessments were performed at different time intervals. Histological analysis included staining with hematoxylin and eosin (H&E) and examination using a fluorescence microscope to assess cellular interactions (SI, S1.4.3).29

The antioxidant properties of PdNPs@CS and CS were evaluated using the ferric reducing antioxidant power (FRAP) assay, ferrous ion chelating activity (FICA) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the results were compared to reference standards (SI, S1.4.4).31–33

3. Results and discussion

3.1. Possible reaction mechanism

The synthesis of PdNPs@CS in this study employed a green and sustainable approach that utilized BTI urine as a reducing agent. BTI urine, which contains organic compounds such as urea and amino acids, facilitates the reduction of Pd2+ ions to metallic Pd (Pd0). During the hydrolysis reaction, urea is transformed into ammonia, which donates electrons, reducing Pd2+ ions to Pd0 ions and forming PdNPs.34,35 The overall reduction of Pd2+ to metallic Pd is driven by BTI urine constituents (urea and amino acids), which generate reducing species under hydrolysis, and can be schematically written as follows:Pd2+ + 2e− → Pd0

This results in the formation of Pd0, as indicated by the color change from brown to blackish. Chitosan may stabilize PdNPs through its –NH2 and –OH functional groups, which interact with the PdNP surface, preventing aggregation. This interaction is primarily driven by weak electrostatic forces and coordinated bonding between chitosan and Pd0.36 The resulting Pd0 nanoclusters are embedded within and stabilized by the chitosan matrix. Along the chitosan backbone, multiple –NH2 and –OH groups act as multidentate coordination sites and hydrogen-bonding donors, anchoring Pd species and suppressing aggregation. PdNPs@CS is a nanocomposite consisting of metallic Pd nanoclusters together with chitosan and adsorbed BTI-urine-derived organic by-products, rather than a single discrete Pd–chitosan complex.

Thus, BTI urine provides a reducing environment for Pd2+ to Pd0, while chitosan functions as a polymeric support that controls the particle size and yields a stable PdNPs@CS nanocomposite. A schematic representation of the possible reaction mechanism is shown in Fig. 1.

Fig. 1. Schematic of the synthesis process of chitosan-stabilized Pd NPs.

Fig. 1

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3.2. Structural and functional analyses

The XRD analysis of PdNPs@CS and CS, shown in Fig. 2a, provides insight into the crystalline structures of these materials. SI Fig. S2a presents the XRD patterns of BSF-derived chitin (pupae and larvae, before deacetylation), while Fig. S2b shows the XRD patterns of the corresponding BSF-derived chitosan (after deacetylation). Chitosan exhibited characteristic 2θ peaks at 10.41° and 22.90°, corresponding to the (020) and (110) hkl planes, respectively.21,37 In the XRD pattern of PdNPs@CS, the peaks observed at 39.92°, 46.43°, 67.76°, 81.64°, and 86.04° correspond to the (111), (200), (220), (311), and (222) crystallographic planes (hkl), respectively. The diffraction pattern of the as-synthesized PdNPs matched well with JCPDS card no. 01-087-0643, confirming their face-centered cubic structure.21,37

Fig. 2. (a) XRD patterns of CS and PdNPs@CS, (b) Rietveld analysis, (c) 3D electron density plots, (d) 2D electron density plots, (e) FTIR spectra of CS and PdNPs@CS, (f) particle size distribution and (g) zeta potential analysis of PdNPs@CS. (h) XPS survey scan spectra of CS and PdNPs@CS. XPS deconvoluted spectra of (i) Pd 3d (PdNPs@CS), (j) C 1s (PdNPs@CS), (k) O 1s (PdNPs@CS), and (l) N 1s (PdNPs@CS).

Fig. 2

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Powder X-ray data for PdNPs@CS were refined by the Rietveld method using FullProf Suite software (Fig. 2b), modeling only the crystalline fcc Pd phase (space group Fm3̄m). The starting structural model and atomic positions were obtained from the crystallographic open database, CIF 9012962 (fcc Pd). The peak profiles were described using a pseudo-Voigt function. The refinement followed the conventional sequence (scale factor, zero shift, lattice parameter, profile U–V–W/η, isotropic B), with the site occupancies fixed to the nominal composition. The refinement converged with Rwp = 12.208%, Rexp = 10.9%, s = Rwp/Rexp = 1.12, χ2 = 1.23, RB = 6.62%, and RF = 18.4% (Table 1), indicating a good fit given the crystalline broadening and amorphous background.38 The unit cell was calculated as a = b = c = 3.9573 Å; no additional crystalline impurity peaks were observed. Fig. 2c (3D-Bode plot) and Fig. 2d (2D-Bode plot) show the electron density computed from the refined model using GFourier (FullProf). The Fourier transform localizes the electron density at the Pd Wyckoff positions expected for Fm3̄m. The refinement statistics, lattice parameters, and Fourier maps indicate that crystalline fcc Pd was preserved in the PdNPs. The sharp Pd reflections and successful Rietveld refinement using an fcc-Pd model confirm that the Pd phase in PdNPs@CS is crystalline, while the chitosan support contributes only a broad amorphous background.

Table 1. Rietveld refinement and structural parameters of the as-prepared PdNPs@CS.

Parameter χ 2 v s R wp R exp R B R F a b c
Value 1.23 61.97 1.12 12.208 10.9 6.62 18.4 3.9573
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Fig. 2e shows the FTIR spectra of CS and PdNPs@CS, revealing their characteristic functional groups and chemical interactions. The broad peak in the range at 3430 cm−1 corresponds to O–H stretching vibrations and N–H (amide A), indicating the presence of hydroxyl groups and hydrogen bonding.36 The peaks at 2850 and 2920 cm−1 are attributed to asymmetric C–H stretching vibrations from chitosan. The sharp peaks at 1663 in CS and slightly shifted in PdNPs@CS at 1635 cm−1 are assigned to the C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching vibration from the amide I group, while the peak at 1559 cm−1 represents the N–H bending vibration of the amide II group.36 Additionally, the presence of amide III groups was confirmed by peaks in the range of 1295–1460 cm−1, which corresponded to C–N and C–O stretching vibrations.21,37 The strong peak at 1080 cm−1 is attributed to the C–O–C stretching vibrations. The peak at 470 cm−1 corresponds to the Pd–OH (hydroxyl) and Pd–NH2 (amine) stretching vibrations, indicating the coordination of Pd0 with chitosan through the –OH and –NH2 functional groups.22,39 The –OH and –NH2 functional groups in chitosan donate lone pairs of electrons to Pd0 ions to form coordinate covalent bonds. This bonding enhanced the stability of the PdNPs and CS, ensuring their dispersibility within the chitosan sheet and preventing PdNP agglomeration.40 The shifts in the peak positions compared to those of CS suggest the formation of a chelated structure of PdNPs in CS. These observations confirm the stabilization, surface modifications, and interactions between chitosan and PdNPs.23,41 Fig. S3 shows that the FTIR spectra of chitin and chitosan extracted from pupae and larvae are provided in the SI, detailing their functional groups.

Particle size analysis graph revealed that PdNPs@CS had an average hydrodynamic diameter of 595 nm, as shown in Fig. 2f, due to the combined size of the PdNPs distributed on the chitosan nanosheets. The larger hydrodynamic diameter was attributed to the presence of PdNPs and chitosan nanosheets, with the polydispersity index indicated a stable colloidal dispersion. Fig. 2g shows the zeta potential analysis, with a surface charge of −17 mV for PdNPs@CS. The observed surface charge indicated good repulsion between the particles to maintain a stable colloidal suspension, demonstrating the role of chitosan in ensuring the enhanced stability of PdNPs@CS.

Fig. 2h shows the XPS survey scan spectra of PdNPs@CS and CS, confirming the presence of C, O, and N as the prominent elements in both samples. These elements are characteristics of the chitosan structure and its functional groups, with additional Pd signals observed in the PdNPs@CS sample, indicating the presence of PdNPs in the composite. The deconvoluted C 1s spectrum of CS (Fig. S4a) exhibited distinct peaks corresponding to the four different carbon bondings in CS. The peak at 285.17 eV is attributed to C–H and/or C–C bonds, representing the aliphatic carbon backbone of the CS. The peak at 286.05 eV corresponds to the C–N bonds associated with amine groups in the glucosamine units.42 Furthermore, the peak at 287.52 eV is assigned to the C–O bonds from the ether linkages (–C–O–). The peak at 289.22 eV indicates C Created by potrace 1.16, written by Peter Selinger 2001-2019 O bonds from carbonyl groups, which may arise from minor acetylation or oxidation of the CS. The deconvoluted O 1s spectrum, as shown in Fig. S4b, includes peaks at 534.27 eV attributed to O–H groups and 533.18 eV, corresponding to C–O bonds present in the hydroxyl and ether groups. The peak at 532.21 eV represents O–C–O bonds associated with ester or ether linkages, while the peak at 531.12 eV is assigned to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O bonds from carbonyl groups.43 The N 1s spectrum, illustrated in Fig. S4c, shows a peak at 399.81 eV, which corresponds to N–C (amine nitrogen) in the glucosamine units of CS.44

The Pd 3d spectrum in Fig. 2i displays characteristic peaks for Pd 3d5/2 (335.13 eV) and Pd 3d3/2 (340.67 eV), confirming the presence of metallic Pd. Deconvoluted peaks at 336.68 eV and 341.92 eV correspond to Pd–N (amine) and/or Pd–O (hydroxyl) interactions,45 respectively, indicating the coordination of Pd with nitrogen and oxygen functional groups in the chitosan matrix. Similar to chitosan, the C 1s spectrum in PdNPs@CS, as shown in Fig. 2j, retains peaks at 284.88 eV for C–C/C–H bonds, 285.93 eV for C–N bonds, 287.69 eV for C–O bonds, and 289.36 eV for C Created by potrace 1.16, written by Peter Selinger 2001-2019 O bonds. The O 1s spectrum illustrated in Fig. 2k shows peaks at 533.29 eV, corresponding to C–O bonds; 531.28 eV, corresponding to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O bonds; and 530.16 eV, indicating Pd–O (hydroxyl) interactions.18,22 The N 1s spectrum presented in Fig. 2l shows a peak at 399.93 eV for N–C bonds, with a slight shift to 401.02 eV attributed to Pd–N (amine) coordination, confirming the interaction of palladium with the nitrogen groups in chitosan. XPS analysis confirmed the presence of the characteristic functional groups of chitosan and validated the successful incorporation of Pd into the chitosan matrix, thereby ensuring the structural stability of the PdNPs@CS heterostructure.

TGA-DSC analysis (Fig. S5a–d) revealed two-step decomposition of pupae and larvae-derived chitin or chitosan [initial moisture loss (25–150 °C), followed by saccharide backbone degradation (150–600 °C)] with distinct exothermic/endothermic transitions, leaving 24–52% residues, which indicates overall stability of up to 300 °C. The morphological analysis of chitin and chitosan extracted from BSF using SEM is shown in the SI (Fig. S6a–h). The TEM images of PdNPs@CS shown in Fig. 3a and b confirm the controlled, relatively spherical morphology of PdNPs. Particle size analysis revealed an average diameter of 6.7 nm, as indicated by the particle size distribution shown in Fig. 3a and b. Additionally, the Pd NPs were uniformly distributed on the chitosan nanosheets, with chitosan effectively controlling particle size and serving as a stabilizing agent throughout the synthesis process, ensuring the formation of stable, well-dispersed nanoparticles.20,27 TEM micrographs revealed that the incorporated PdNPs on the nanosheet-like structure of CS facilitated their uniform distribution. CS nanosheets offer an interlinked structured network that prevents particle agglomeration and supports the dispersion of NPs.21 This highlights the stabilizing role of CS in controlling PdNP size and dispersion compared with previously reported BTI urine-assisted synthesis using CTAB as the stabilizer.34 The ability of chitosan to stabilize NPs is attributed to its unique structural features, including the presence of amino and hydroxyl groups, which facilitate strong interactions with Pd NPs.19

Fig. 3. (a and b) TEM images of PdNPs@CS (inset: particle size distribution histogram of PdNPs), (c) EDS spectrum of PdNPs@CS, (c–g) TEM/EDS elemental mapping of PdNPs@CS, including (c) PdNPs@CS map, (d) Pd map, (e) N map, (f) C map, and (g) O map.

Fig. 3

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Fig. 3c shows the EDS analysis of PdNPs@CS, which confirms the presence of Pd, C, O, and N as the primary constituents of the nanocomposite. The EDS analysis indicated the following weight percentages of these elements: Pd (32.43%), C (53.24%), N (1.33%), and O (1.89%). Fig. 3d–g shows the elemental mapping of PdNPs@CS, illustrating the uniform distribution of Pd (Fig. 3d), nitrogen (Fig. 3e), carbon (Fig. 3f), and oxygen (Fig. 3g). The even distribution of Pd highlights its successful deposition onto the chitosan nanosheets, while nitrogen mapping confirms the presence of chitosan as a stabilizing matrix. The significant presence of Pd and N is particularly noteworthy, as it confirms the successful incorporation of PdNPs into chitosan nanosheets.34

3.3. Nitroarene reduction and mechanism

As a model nitroarene transformation, 4-NP was cleanly reduced to 4-aminophenol (4-AP) using NaBH4 as the hydride source under PdNPs@CS catalysis. The reaction followed pseudo-first-order kinetics, as monitored by UV-vis spectroscopy (Fig. S7a–d). In the presence of PdNPs@CS, the 400 nm band of 4-nitrophenolate decayed monotonically, and well-defined isosbestic points at 232 and 298 nm emerged, indicating a clean interconversion to 4-AP. The reduction was completed within 480 s with a rate constant (k) of 0.0067 s−1. Fig. 4a and b illustrate the kinetic model and nitroarene reduction percentage, respectively.34 Further analysis was conducted on nitroaniline derivatives: 2-NA, 3-NA, and 4-NA. These nitroaniline compounds exhibited maximum absorbance peaks at 410, 360, and 380 nm, respectively, and were transformed into their corresponding amines (2-aminoaniline, 3-aminoaniline, and 4-aminoaniline, respectively) within 210 s, 150 s, and 135 s, respectively. The rate constants are 0.0087, 0.0182, and 0.0154 s−1. Fig. 4c shows the recyclability study of PdNPs@CS over 5 cycles. The catalyst maintained reduction efficiency over five cycles, with conversion efficiencies of 90.87% for 4-NP, 83.10% for 2-NA, 76.16% for 3-NA, and 86.23% for 4-NA. This recyclability study is attributed to Pd-mediated electron transfer synergizing with the chitosan support, which stabilizes the NPs and preserves accessible active sites.23

Fig. 4. (a) Kinetic analysis of the catalytic reduction of nitroarene compounds (4-NP, 2-NA, 3-NA, and 4-NA); (b) conversion percentages of nitroarene compounds; (c) recyclability study for the catalytic reduction of nitroarene compounds using PdNPs@CS; (d) possible nitroarene reduction reaction pathway; (e) XRD pattern of the PdNPs@CS nanocomposite recovered after the catalytic reduction reaction; and (f) TEM micrograph of the PdNPs@CS nanocomposite recovered after the catalytic reduction reaction.

Fig. 4

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The PdNPs@CS-catalyzed reduction of nitroarenes followed the Langmuir–Hinshelwood pathway, with four elementary steps: adsorption, activation, reduction, and desorption (Fig. 4d).34,46 First, 4-NP and BH4− were co-adsorbed on PdNPs@CS through electrostatic and coordinative interactions; the chitosan shell enriched the local substrate concentration via H-bonding/ionic sites and suppressed nanoparticle aggregation. On the Pd NP surface, BH4− is activated to form surface hydrides (Pd–Hx), generating an electron/hydride reservoir that drives reduction.47 Electron/hydride transfer to the adsorbed nitro group proceeds stepwise (–NO2 to –NHOH to –NH2), with 4-nitrophenolate (formed in the alkaline BH4− medium) serving as the kinetically important intermediate that promotes interfacial charge transfer and mitigates repulsion between anionic BH4− and 4-NP. Similar reduction mechanisms have been proposed for other nitroarene (2-NA, 3-NA, and 4-NA) reduction reactions.48

Fig. 4d (pathway): (i) co-adsorption of 4-NP and BH4− on PdNPs@CS; (ii) BH4− to Pd–Hx; (iii) 4-NP to 4-nitrophenolate to p-hydroxylamine to 4-AP via sequential e−/H+ transfers and –OH dehydration; (iv) desorption of 4-AP; and (v) net stoichiometry:3.3.

Compared to other nanocatalysts (as summarized in Table 2), PdNPs@CS exhibited enhanced catalytic activity and eco-compatibility, effectively reducing nitroarene pollutants in aqueous systems.34,49Fig. 4e shows that PdNPs@CS recovered after catalysis retained the fcc Pd reflections at 2θ = 39.9°, 46.4°, 67.8°, 81.6°, and 86.0° indexed to the (111), (200), (220), (311), and (222) planes, respectively (JCPDS 01-087-0643). The peak positions and relative intensities were essentially unchanged from those before the addition of the catalyst, indicating phase retention and crystallographic stability during the reaction. No additional impurities are observed in the XRD spectra. This confirms that the pristine Pd phase remained intact after the recyclability study. As shown in Fig. 4f, the TEM micrograph of the recovered PdNPs@CS reveals quasi-spherical NPs uniformly dispersed on the CS matrix (particle size less than 10 nm), confirming the retained morphology after the reaction.

Table 2. Comparative catalytic performance of various palladium-based nanocomposites for the reduction of nitroarenesa.

Sr. no. Nano-catalyst Aromatic nitroarene chemical Nitroarene conc. in mM (vol. in mL) NaBH4 conc. in mM (vol. in mL) Catalyst conc. in mg mL−1 (vol. in µL) Molar ratio (nitroarene : NaBH4) Rate constant (s−1) Reference
1 PdNPs 4-NA 30 (1.0) 100 (1.5) 0.1 (30) 1 : 500 0.006 34
2 PdNPs 4-NP 0.1 (1.5) 50 (1.0) 0.05 (500) 1 : 333 0.013 49
3 PdNPs@CS 4-NP 0.15 (1.5) 12 (1.5) 0.05 (25) 1 : 80 0.006 This work
4-NA 0.15 (1.5) 12 (1.5) 0.05 (25) 0.016
3-NA 0.15 (1.5) 12 (1.5) 0.05 (25) 0.081
2-NA 0.15 (1.5) 12 (1.5) 0.05 (25) 0.008
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a

Molar ratios (nitroarene : NaBH4) were calculated based on the concentrations and volumes provided in the respective experimental protocols.

3.4. Biomedical applications of PdNPs@CS

3.4.1. Hemocompatibility study

Hemolysis assays evaluate the viability of red blood cells (RBCs) upon exposure to the tested as-prepared samples.50 Hemolysis assays for PdNPs@CS were performed at various concentrations to assess their compatibility with the blood, as shown in Fig. 5a. The hemolysis percentage for PdNPs@CS exhibited a concentration-dependent increase, with values of 0.14% at 0.625 mg, 0.83% at 1.25 mg, 0.94% at 2.5 mg, 1.59% at 5 mg, and 5.59% at 10 mg. According to the hemolysis classification criteria, samples with hemolysis values below 2% were considered non-hemolytic, those between 2% and 5% were classified as slightly hemolytic, and values exceeding 5% were categorized as hemolytic.51 Based on these criteria, PdNPs@CS exhibited non-hemolytic behavior at concentrations ranging from 0.625 to 5 mg, while a 10 mg concentration showed hemolytic activity, with a hemolysis percentage of 5.59%. At low PdNPs@CS concentrations, their interactions with RBCs were insufficient to cause significant damage. However, at higher concentrations, increased oxidative stress may compromise membrane integrity, resulting in hemolysis.50 Additionally, owing to the cationic nature of chitosan-stabilized PdNPs@CS, at elevated concentrations, the interaction between charged PdNPs@CS and negatively charged RBC membranes may lead to electrostatic destabilization, promoting hemolysis. Hemolysis studies revealed a dose-dependent analysis, with PdNPs@CS showing no hemolysis at lower doses but exhibiting hemolytic activity at higher doses, due to oxidative effects and cumulative membrane interactions.52

Fig. 5. Hemolysis percentage induced by (a) PdNPs@CS evaluated based on haemoglobin release from red blood cells. Data are presented as mean ± SD, including positive and negative controls for hemolysis (P < 0.05). For each PdNPs@CS concentration, hemolysis measurements were carried out in triplicate (n = 3), and the data are reported as mean ± SD. (b) Cytotoxic effects of PdNPs@CS on MCF-7 breast cancer cells, as assessed using the MTT assay. Data are presented as mean ± SD, with statistical significance set at P < 0.05.

Fig. 5

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These results reveal that PdNPs@CS shows good biocompatibility at low doses. However, PdNPs@CS applicability at higher doses requires more detailed analysis. Hemocompatibility analysis confirms PdNPs@CS applicability for biomedical applications, at doses below 5 mg, where hemolysis levels remained within the non-hemolytic limit, suggesting biocompatibility.53 This analysis provides the mechanistic basis of PdNPs@CS hemolytic behavior.

3.4.2. Cytotoxicity study

Fig. 5b presents the MTT assay results for PdNPs@CS, revealing distinct cytotoxicity profiles on cell viability. MCF-7 breast cancer cells were treated with PdNPs@CS at doses of 6.25, 12.5, 25, 50, and 100 µg. For the MCF-7 breast cancer cell line, the PdNPs@CS sample showed moderate and constant cytotoxicity across the optimized concentration range from 6.25 to 100 µg, with cell viability ranging from 29.91% to 32.88%. These results suggest that PdNPs@CS possess cytotoxicity against cancer cells while maintaining overall biocompatibility. This selective cytotoxicity is due to PdNP-mediated mitochondrial disruption in cancer cells, which are inherently more susceptible to oxidative stress than normal cells. Additionally, PdNPs may interfere with cellular signaling pathways in cancer cells, leading to apoptosis.54 Similar trends have been observed in previously reported cytotoxicity studies of other compounds, such as valproic acid55 and quercetin-loaded solid lipid NPs,56 which exhibited minimal variation in cell viability across a broad dose range, indicating a controlled cytotoxic effect.

To increase the therapeutic potential of PdNPs@CS while minimizing cytotoxicity, future studies should investigate surface modifications/functionalization to stabilize PdNPs, limit ROS overproduction, and enable targeted cancer cell delivery.57,58 The integration of PdNPs with CS provides a novel material for the development of controlled cytotoxicity for targeted cancer therapy.

3.4.3. Angiogenesis study

The angiogenic potential of PdNPs@CS and CS was evaluated using a CAM assay, in which as-prepared materials demonstrated vascularization and formation of new vessels. Fig. 6a and b shows enhanced angiogenesis around PdNPs@CS and CS, with increased vascular density compared to the control at 24, 48, and 72 h. The formation of newly developed vessels extended towards and interacted with the PdNPs@CS and CS samples, indicating effective vascularization, angiogenesis, and interaction between the nanomaterial and tissue. Histological analysis further confirmed the absence of necrosis and the presence of a well-formed vascular network, as depicted in Fig. 6a–c. Notably, PdNPs@CS, which adhered to the CAM membrane, appeared blackish-brown, while CS exhibited a pinkish-orange stain, distinguishing it from the as-prepared material. The endothelial lining of the newly formed vessels remained intact, suggesting the preservation of vascular integrity and functionality.59 These findings demonstrate that both PdNPs@CS and CS actively supported vascularization and tissue growth, reinforcing their potential applications in tissue engineering and regenerative medicine.

Fig. 6. Photographic images obtained from the CAM assay comparing the control with varying concentrations of (a) PdNPs@CS and (b) CS at 24, 48, and 72 hours.

Fig. 6

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The mechanism underlying angiogenic activity can be attributed to several key factors. Chitosan enhances extracellular matrix interactions, providing a hydrophilic and biocompatible scaffold that promotes cell adhesion, proliferation, and migration, which are essential for angiogenesis.60 The positive charge of chitosan enables electrostatic interactions with negatively charged cell membranes, stimulating endothelial cell activation and migration, which is crucial for the formation of new blood vessel.61 CS promotes the secretion of vascular endothelial growth factor (VEGF) and stimulates endothelial cell proliferation and migration. PdNPs may further enhance angiogenesis through catalytic redox reactions, which affect endothelial function.62 PdNPs@CS effectively facilitated the release of localized oxide, a key mediator of vasodilation and endothelial proliferation. PdNPs can trigger intracellular pathways that stimulate endothelial sprouting and neovascularization by acting as catalysts for ROS-mediated signaling. The synergistic effect of PdNPs and CS in PdNPs@CS ensures a balanced redox environment, preventing excessive oxidative stress while promoting controlled angiogenesis.

The intact endothelial structure along the newly developed vasculature, as shown in Fig. 7a–c, further indicates that neither material elicited inflammatory reactions in the CAM microenvironment. The biodegradable nature of chitosan prevents its long-term accumulation, reducing the risk of chronic inflammation, while PdNPs, when stabilized within a chitosan matrix, minimize toxic ion leaching. The ability of PdNPs@CS and CS to promote vascularization highlights their suitability for biomedical applications requiring enhanced tissue perfusion, such as wound healing, scaffold-based tissue engineering, and drug delivery.63 The observed proangiogenic properties suggest their potential role in stimulating vascular integration in implanted biomaterials.64 In addition, the controlled redox activity of PdNPs provides a promising avenue for engineering nanomaterials with precise angiogenic modulation. Future research should explore PdNPs@CS-mediated angiogenesis pathways, optimize pro-angiogenic activity, minimize cytotoxicity, and validate in vivo tissue repair, enhancing their potential in regenerative medicine.

Fig. 7. Histological images of the CAM assay comparing the control with varying concentrations of (a) PdNPs@CS, (b) CS, and (c) control at 24, 48, and 72 hours.

Fig. 7

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3.4.4. Anti-oxidant study

Fig. 8a–d demonstrates a concentration-dependent increase in antioxidant activity in the FRAP, FICA, DPPH, and ABTS assays. In the FRAP assay (Fig. 8a), the reducing power of PdNPs@CS increased from 0.51 O.D. at 20 µg mL−1 to 1.22 O.D. at 100 µg mL−1, indicating its electron-donating potential, although it is lower than that of ascorbic acid as a standard (2.10 O.D. at 100 µg mL−1). The FICA assay (Fig. 8b) revealed moderate Fe3+ ion-chelating activity, with PdNPs@CS achieving 63.50% activity at 100 µg mL−1 compared with 93.33% for EDTA, which was attributed to the presence of functional groups in the chitosan matrix. In the DPPH assay (Fig. 8c), PdNPs@CS achieved 72.23% scavenging activity at 100 µg mL−1, demonstrating its capability to capture free radicals albeit slightly lower than that of ascorbic acid (89.94%). Similarly, the ABTS assay (Fig. 8d) showed a scavenging efficiency of 70.50% for PdNPs@CS at 100 µg mL−1 compared to 94.11% for ascorbic acid, confirming its efficacy in both hydrophilic and lipophilic environments. DPPH and ABTS assays rely on the single-electron transfer-proton transfer (SPLET) mechanism in aqueous solutions, highlighting the interaction between radicals and antioxidants through the ionization of phenolic groups.65 The DPPH assay measures activity via the reduction of the violet DPPH radical, while the ABTS assay evaluates activity in both hydrophilic and lipophilic environments by generating a green/blue ABTS˙+ radical cation.66 These results highlight the potential of PdNPs@CS as multifunctional antioxidants. Its activity is attributed to the synergistic effect of the PdNPs and chitosan matrix, which facilitates electron transfer and interaction with radicals.65 The increasing trend in antioxidant activity with increasing concentrations further supports its dose-dependent effectiveness.67

Fig. 8. Antioxidant activity of PdNPs@CS assessed through the (a) FRAP assay, (b) FICA assay, (c) DPPH assay, and (d) ABTS assay.

Fig. 8

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4. Conclusion

In summary, we developed a green, BTI-urine-assisted route to synthesize chitosan-stabilized palladium nanoparticles (PdNPs@CS) with crystalline fcc-Pd domains uniformly dispersed on an amorphous chitosan matrix, as confirmed by Rietveld-refined XRD and TEM (the average PdNP size is 6.7 nm). In aqueous NaBH4, PdNPs@CS catalyzed the reduction of 4-nitrophenol to 4-aminophenol with a pseudo-first-order rate constant k = 0.0067 s−1, and 2-, 3-, and 4-nitroaniline with k = 0.0087, 0.0182, and 0.0154 s−1, respectively, achieving complete conversion within 135–480 s under 80 : 1 NaBH4 : nitroarene conditions. The catalyst retained high activity over five cycles (conversion after cycle 5 : 90.87% for 4-NP, 83.10% for 2-NA, 76.16% for 3-NA, and 86.23% for 4-NA), with XRD and TEM confirming preservation of the fcc-Pd phase and nanoparticle dispersion. Hemocompatibility tests showed hemolysis below 2% for PdNPs@CS doses up to 5 mg (non-hemolytic), with 5.59% hemolysis only at 10 mg, while MCF-7 viability remained in the 29.9–32.9% range across 6.25–100 µg mL−1, indicating selective but controlled cytotoxicity. CAM assays demonstrated enhanced vascularization without necrosis, and antioxidant measurements revealed concentration-dependent activity (e.g., FRAP absorbance increasing from 0.51 to 1.22 and DPPH scavenging up to 72.23% at 100 µg mL−1), positioning PdNPs@CS as a recyclable aqueous nanocatalyst with quantifiable catalytic performance and promising, dose-dependent biological behavior.

Conflicts of interest

There is no conflicts of interest for the publication of the article.

Supplementary Material

RA-016-D6RA01978A-s001

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (RS-2025-00564062 and RS-2025-02303676).

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra01978a.

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Associated Data

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Supplementary Materials

RA-016-D6RA01978A-s001
RA-016-D6RA01978A-s001.pdf (2.3MB, pdf)

Data Availability Statement

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d6ra01978a.

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