Catharanthus roseus Extract-Loaded Zn-Substituted Hydroxyapatite Nanocomposites as a Multifunctional Antioxidant and Anticancer Therapeutic Applications

TL;DR

A new nanocomposite made from fish bone waste and plant extract shows strong antioxidant and anticancer properties.

Contribution

A Zn-HA/CR nanocomposite is developed with enhanced antioxidant and anticancer effects for therapeutic applications.

Findings

The Zn-HA/CR nanocomposite showed improved surface morphology and increased microhardness.

It exhibited strong antibacterial activity against Staphylococcus aureus and Escherichia coli.

The nanocomposite caused significant morphological changes and reduced viability in osteosarcoma cancer cells.

Abstract

During recent decades, bone cancer-related diseases have remained hard to treat because of poor diagnosis, systemic toxicity, and restricted conventional treatments. Hence, the fabrication of functionalised nanoparticles offers a promising alternative by limiting side effects and improving therapeutic outcomes. In this study, zinc-substituted hydroxyapatite (Zn-HA) nanoparticles were fabricated from biogenic tuna fish bone waste via a thermal decomposition method and subsequently functionalised with Catharanthus roseus (CR) flower extract to synthesise a Zn-HA/CR nanocomposite. Structural and compositional characterisations verified Zn ions incorporation into the HA lattice and efficient CR-derived phytochemical functionalisation without altering the hexagonal HA phase. Compared to pure hydroxyapatite, the Zn-HA/CR nanocomposite exhibited improved surface morphology, enhanced swelling behaviour and degradation, and increased microhardness. The nanocomposite demonstrated significantly enhanced antibacterial activity against Staphylococcus aureus and Escherichia coli. The Zn-HA/CR nanocomposite also showed strong, dose-dependent antioxidant activity in DPPH assays. Furthermore, in vitro cytotoxicity studies using MG-63 (HOS) osteosarcoma cancer cells revealed that the proposed nanocomposite leads to pronounced morphological alterations and reduced cell viability. The prepared Zn-HA/CR nanocomposite would be a potential nanocomposite for enhanced antioxidant and anticancer activity, which highlights this composite as a multifunctional biomaterial platform for therapeutic applications.

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1. Introduction

Bone-related ailments, such as osteomyelitis, bone tumours, traumatic injuries, and other orthopaedic disorders, are an important cause of morbidity and mortality around the world [1,2,3]. The goal of the current treatment approach is to encourage the growth of new bone and repair damaged bone tissue [4,5]. However, the intrinsic regenerative capacity of bone is limited, especially when there are major fractures; hence, the implementation of implantable biological materials is required to promote successful rehabilitation [6,7,8]. The development of environmentally friendly and sustainable methods for biomaterial synthesis has attracted greater consideration in the past few decades, particularly in the domains of materials science and biomedical engineering [9,10]. The calcium phosphate mineral hydroxyapatite (HA), which resembles the inorganic element of human hard tissues, has become a popular biocompatible material for dental, tissue engineering, and orthopaedic implants. In addition to their well-known osteoconductive properties, HA nanomaterials have been shown to inhibit the growth of tumour cell lines while doping with bioactive ions or therapeutic agents [11,12]. Because of its exceptional bioactivity, affordability, thermal resistance, and biocompatibility, HA shows great potential, along with its capacity to develop a strong interaction bond with surrounding bone without creating adverse biological reactions [13,14]. Because of these characteristics, HA acts as an excellent candidate for bone reconstruction and regenerative medicine [15].

HA can be derived from natural sources such as fish bones using thermal decomposition methods, offering a sustainable, eco-friendly, and economically viable route for producing bone graft substitute materials [16]. Tuna fish bones are especially attractive among these sources owing to their significant yield and availability [17,18]. However, natural HA has intrinsic drawbacks that restrict its application in high load-bearing applications and demand further functional modifications [19]. These limitations include limited osteoinductivity, weak antibacterial activity, low fracture toughness, and weak mechanical durability [20,21]. In order to overcome these drawbacks, considerable effort has been made to improve HA employing lattice modification techniques to improve osteoconductive and osteoinductive performance, which exhibit desirable biocompatibility and bioresorbability [22,23]. A conventional approach for modifying the physical, biological, and chemical features of the HA crystal structure is elemental substitutions [24,25].

Particularly, the partial substitution of Ca^2+^ using mono-, di-, tri-, or tetravalent ions like Mg^2+^, Ce^3+^/Ce^2+^, Co^2+^, Sr^2+^, Cu^2+^, Ag^2+^, Zn^2+^, Mn^2+^, and Ti^2+^ significantly improves crystallinity, stability, mechanical integrity, antimicrobial effectiveness, and biological activity [26,27,28]. Zinc incorporation is especially desirable among these divalent ions mainly because both calcium and zinc are abundant and required in ecosystems [29,30]. Zinc has been defined as “the calcium of the twenty-first century” due to the increasing support of its vital physiological functions [31]. When compared to other divalent and trivalent ions, the incorporation of Zn induces apoptosis and mitochondrial dysfunction in cancer cell lines. Furthermore, Zn substitution in the HA lattice effectively indicates controlled crystallinity, biodegradation kinetics, and mechanical integrity compared to various ion substitutions. Furthermore, the outstanding biocompatibility and in vivo biodegradation behaviour provide zinc as an attractive candidate for enhancing tissue regeneration and therapeutic efficacy in HA-based biomaterials [32,33].

During recent decades, plant-derived extracts, such as Moringa oleifera, banana peel, Azadirachta indica and Coccinia grandis, Euclea natalensis root, and tamarind extracts combined with HA biocomposites demonstrated potential interest in biomedical applications [33,34]. These plant extract composites provide HA with better bioactive and antimicrobial properties [35]. Even though the wide variety of plant extracts exhibit potential benefits, Catharanthus roseus demonstrates remarkably increased medical benefits compared to other plants, which is because of the presence of unique alkaloid compounds. The flowers of Catharanthus roseus (CR) are particularly interesting among medicinal plants because of their many therapeutic properties, which include neuroprotective, anticancer, antidiabetic, antioxidant, anti-inflammatory, and antimicrobial activities [36]. These therapeutic properties are due to the presence of bioactive alkaloid compounds. Numerous studies have shown that these alkaloids may be useful in cancer treatment because of their extensive medicinal benefits [37,38]. As a result, different nanoparticles and CR flower extracts were combined to create novel composites with improved biological functionality.

In the present investigation, biogenic tuna fish bone residue was incorporated to synthesise zinc-substituted hydroxyapatite (Zn-HA) nanoparticles using a thermal decomposition method with improved physicochemical characteristics. Further, Catharanthus roseus (CR) flower extract was added to create a Zn-HA/CR nanocomposite. Several spectroscopy techniques were utilised to investigate structural, compositional and morphological features. The antibacterial activity of HA, Zn-HA, and Zn-HA/CR was evaluated against Staphylococcus aureus and Escherichia coli using the disc diffusion method. The MG63 human osteosarcoma cancer cell line was used to evaluate in vitro cell viability and cytotoxicity. In aggregate, these findings demonstrate the dual capabilities of Zn-HA/CR nanocomposite as a biocompatible platform for prospective biomedical uses and an efficient antibacterial substance.

2. Results and Discussion

2.1. Structural and Vibrational Characteristics

Figure 1 displays the FTIR spectra of HA, Zn-HA, and Zn-HA/CR nanocomposites. For HA particles, the wide absorption band centred around 3200–3600 cm^−1^ is attributed to O–H stretching vibrations arising from structural hydroxyl groups and adsorbed water, whereas the band near 1630 cm^−1^ is attributed to the H–O–H bending vibrational modes. The characteristic phosphate (PO_4_^3−^) vibrations are clearly observed at ~1030–1090 cm^−1^ (ν_3_ asymmetric stretching), ~960 cm^−1^ (ν_1_ symmetric stretching), and ~560–605 cm^−1^ (ν_4_ bending), confirming the formation of HA. In the FTIR spectrum, the HA particles do not show any additional peaks, which confirms that the as-prepared particles are highly pure without any contamination. In Zn-HA nanoparticles, slight shifts and reduced intensities of the phosphate bands are observed, indicating lattice perturbation due to Zn^2+^ substitution at Ca^2+^ sites. The Zn-HA/CR spectrum shows additional broadening in the O–H and phosphate regions, along with weak bands in the 1400–1500 cm^−1^ range, which can be attributed to organic functional groups from CR phytochemicals interacting with the Zn-HA surface structure. These results indicate that the proposed fabrication techniques are successful synthesis and surface functionalising without interrupting the fundamental HA structure.

Figure 2 shows the XRD patterns of HA, Zn-HA, and Zn-HA/CR nanocomposites. The diffraction pattern of HA matches well with the hexagonal phase structure (space group P63/m; JCPDS No. 09-0432), exhibiting characteristic reflections (2θ) at 25.9, 31.7, 32.2, and 32.9°, corresponding to the (002), (211), (112), and (300) planes, respectively. The dominant (211) reflection confirms the formation of phase-pure HA. Further, the XRD pattern confirms that the biogenic waste-derived HA particles are highly phase pure without the formation of any additional phase structure. In Zn substitution, a noticeable reduction in peak intensity and slight broadening are observed, indicating lattice distortion arising from Zn^2+^ incorporation at Ca^2+^ sites. The Zn-HA/CR nanocomposite exhibits further peak broadening and reduced crystallinity, which is attributed to the coordination of phytochemicals from CR with Zn-HA during incorporation, leading to the surface coverage and encapsulation of the nanoparticles, resulting in inhibited crystallinity and nanoscale domain formation. Importantly, no secondary calcium phosphate or Zn-related impurity phases are detected, confirming structural stability of the HA lattice for the nanocomposite. Further, the observed reduction in crystalline structure and peak broadening due to the incorporation of Zn and CR in the HA lattice indicates a distortion and surface interaction effects, which influence the degradation kinetics, ion diffusion, and interfacial biological interactions.

2.2. Morphological Characteristics

Figure 3a–c shows the FE-SEM images of HA, Zn-HA, and Zn-HA/CR, respectively. The HA particles demonstrated in Figure 3a reveal a compact granular morphology composed of irregularly shaped particles with significant agglomeration, which is generally observed in calcium phosphate materials synthesised via thermal decomposition methods. This agglomeration is primarily associated with the high surface energy of HA particles and strong interparticle interactions. Following Zn incorporation in HA, the Zn-HA sample (Figure 3b) exhibits a more uniform and refined microstructure, with a reduced grain size and enhanced surface homogeneity. The presence of Zn ions is likely to modify the crystal growth kinetics by restricting particle coalescence, resulting in a denser and more compact morphology. These features indicate that Zn substitution effectively influences the nucleation and growth behaviour of the HA nanostructure. In contrast, the Zn-HA/CR nanocomposite in Figure 3c demonstrated a distinctly diverse surface morphology, with Zn-HA nanoparticles homogeneously dispersed within an interconnected HA structure. Furthermore, the nanocomposite surface structure appears comparatively rough and porous, reflecting effective integration of the CR component with Zn-HA matrix. Hence, this prepared surface architecture would be beneficial for biomedical applications, because it can facilitate improved surface wettability and enhanced cell–material interactions.

Transmission electron microscopy (TEM) images reveal distinct morphological features for HA, Zn-HA, and Zn-HA/CR nanocomposites, as demonstrated in Figure 3d–f. The HA particles consist of irregularly shaped and moderately agglomerated particles with sizes in the nanometer range, as shown in Figure 3d. The morphology of the HA particles indicates the formation of an agglomerated surface structure, which is due to the biogenic waste preparation, which may lead to enhanced surface charge. Upon Zn incorporation in the HA structure, the Zn-HA nanoparticles become finer and more densely packed, indicating that Zn substitution influences nucleation and growth kinetics, leading to reduced particle size with agglomeration. Further, the Zn incorporation in the HA structure remarkably reduced the particle size and led to the formation of uniform-sized and structured particles (Figure 3e). In contrast, Zn-HA/CR nanocomposite exhibits comparatively well-dispersed spherical nanoparticles with a narrower size distribution, suggesting that phytochemicals from CR act as capping and stabilising agents during synthesis (Figure 3f). Further, the SAED patterns for all samples display concentric diffraction rings indexed to the characteristic planes of hexagonal HA, confirming their crystalline nature (Figure 3g–i). In addition, the incorporation of Zn in the HA structure shows that the discontinued diffraction rings indicate a slight reduction in the HA crystallinity. In addition, the CR incorporation in the Zn-HA structure further reduces the diffraction ring intensity, which confirms the further reduction in crystallinity and the observed SEAD pattern is in good agreement with the XRD analysis.

2.3. In Vitro Swelling and Degradation Behaviours

Figure 4a depicts the swelling behaviour of HA, Zn-HA, and Zn-HA/CR nanocomposites as a function of immersion time (14 days). All samples exhibit a rapid initial increase in swelling, attributed to immediate fluid uptake through surface-accessible pores, followed by a slower swelling stage. Among the materials, pure HA displays the lowest swelling capacity, consistent with its higher crystallinity and reduced porosity. Zn-HA exhibits intermediate swelling behaviour, reflecting the influence of Zn substitution on lattice distortion and surface energy. In contrast, Zn-HA/CR shows the highest swelling ratio, reaching approximately 30% at the final time point, indicating enhanced hydrophilicity and increased surface functionality imparted by plant-derived organic moieties. The observed results highlight the synergistic effect of Zn incorporation and plant extract in improving fluid uptake, which is advantageous for biomedical applications such as tissue scaffolds and drug delivery systems.

Figure 4b shows the in vitro degradation behaviour of HA, Zn-HA, and Zn-HA/CR nanocomposites over the immersion period of 14 days. The as-prepared nanocomposites exhibit a progressive decrease in residual mass, indicating continuous degradation with time. The as-derived HA demonstrates the highest structural stability, retaining approximately 90% of its initial weight after 14 days, which can be attributed to its high crystallinity. Zn-HA displays a moderately increased degradation rate, with residual weight decreasing to around 82%, which reflects the lattice distortion induced by Zn substitution in the HA structure. In contrast, Zn-HA/CR shows the most pronounced degradation behaviour, with nearly 68% weight retention at the end of the immersion period. This accelerated degradation is likely due to the combined effects of Zn incorporation and CR flower extract surface modification, which enhance hydrophilicity and promote fluid penetration in the biological fluid.

2.4. Microhardness Analysis

The microhardness values of HA, Zn-HA, and Zn-HA/CR nanocomposites are summarised in Figure 5. The as-synthesised HA exhibits a microhardness of approximately 1.67 GPa. In contrast, Zn-HA shows a noticeable increase to ~1.79 GPa, indicating that Zn^2+^ incorporation enhances lattice strengthening through substitution at Ca^2+^ sites. The Zn-HA/CR sample displays a comparable hardness value (~1.78 GPa), suggesting that the surface functionalisation of CR flower extract in the HA preserves the mechanical reinforcement induced by Zn. The improvement in hardness for Zn-containing samples can be attributed to lattice distortion, reduced crystallite size, and enhanced intergranular bonding. These results demonstrate that Zn incorporation effectively improves the mechanical performance of hydroxyapatite without compromising structural integrity, which is advantageous for load-bearing biomedical applications.

2.5. Antibacterial Activity

The antibacterial activity of HA, Zn-HA, and Zn-HA/CR nanocomposites was systematically assessed against S. aureus and E. coli using the agar well diffusion method, as depicted in Figure 6. The diameter of the inhibition zone formed around the disc was used as a quantitative measure of antibacterial efficacy at various loading concentrations, such as 30, 40, 50 and 60 µg/mL. In this antibacterial analysis, HA exhibited relatively low antibacterial activity against both bacterial strains, with inhibition zones in the range of approximately 8–12 mm for S. aureus and 6–10 mm for E. coli against various concentrations. This limited activity is expected, as HA is inherently bioinert and does not possess strong antimicrobial functionality. The slight inhibition observed may arise from weak surface interactions and local ionic effects. In contrast, Zn-HA showed a remarkable enhancement in antibacterial efficacy. The inhibition zones increased to approximately 11–16 mm against S. aureus and 9–14 mm against E. coli, confirming the effective contribution of Zn ions to bacterial growth suppression. Generally, Zn is known to disrupt bacterial cell membranes, interfere with enzymatic activity, and alter intracellular metabolic pathways, leading to reduced bacterial viability [39]. Further, the higher concentration of the nanoparticles shows a maximum zone of inhibition for all the nanocomposites. The gradual increase in the inhibition zone with concentration further indicates a dose-dependent antibacterial response.

Notably, Zn-HA/CR demonstrated the highest antibacterial activity, and the inhibition zones increased from approximately 14 to 20 mm for S. aureus and 12 to 18 mm for E. coli as the sample concentration increased. This excellent performance is assigned to the synergistic effect of Zn incorporation and phytochemicals derived from CR in the HA structure. These phytochemicals exhibit potential antibacterial activity and also act as surface-bound functional groups that enhance bacterial membrane interaction, facilitate Zn ion release, and promote oxidative stress-induced damage. Furthermore, S. aureus displayed larger inhibition zones compared to E. coli, which can be attributed to structural differences in their cell envelopes. Overall, the antibacterial effectiveness demonstrates that Zn-HA/CR nanocomposite offers enhanced and broad-spectrum antibacterial activity. The results of the antibacterial investigations, which were performed in triplicate, are displayed as mean ± SD. The significant impacts of material type and concentration on the zone of inhibition measurements were identified using two-way ANOVA (p < 0.05). At identical concentrations, Zn-HA and Zn-HA/CR demonstrated considerably greater antibacterial activity than HA.

2.6. Antioxidant Activity

The antioxidant activity of the Zn-HA/CR nanocomposite was assessed using the DPPH radical scavenging assay (Figure 7). As evident from the bar graph, the Zn-HA/CR nanocomposite exhibited a clear concentration-dependent increase in radical scavenging activity across the tested range (0.1–500 µg/mL). At lower concentrations, the scavenging efficiency was relatively modest, while a pronounced enhancement was observed at higher concentrations, indicating effective interaction between the nanocomposite and DPPH radicals. The Zn-HA/CR nanocomposite demonstrated a clear dose-dependent increase in radical scavenging activity across a wide concentration range (0.1, 0.5, 1, 5, 10, 50, 100, and 500 µg/mL). At lower concentrations, the scavenging efficiency was relatively modest, while a pronounced enhancement was observed at higher concentrations, indicating effective interaction between the nanocomposite and DPPH radicals. The antioxidant activity of the Zn-HA/CR nanocomposite was slightly lower than that of the standard, which remains a substantially concentration-dependent trend. For comparison, the radical scavenging activity of ascorbic acid was assessed at concentrations ranging from 2 to 10 µg/mL. In this, ascorbic acid demonstrated strong radical scavenging activity even at low doses. Although the scavenging efficiency of the Zn-HA/CR nanocomposite was lower than that of ascorbic acid at comparable concentrations, the overall trend and magnitude of activity were comparable at higher doses, highlighting the strong antioxidant potential of the nanocomposite. The enhanced antioxidant performance of Zn-HA/CR can be attributed to the presence of bioactive constituents derived from the CR flower component. Because the flower extract containing phenolic and other redox-active secondary metabolites can donate electrons or hydrogen atoms to neutralise free radicals. These functional groups contribute to the improvement of the radical scavenging capability of the Zn-HA/CR nanocomposite. Additionally, the synergistic combination of HA, the incorporation of Zn, and the CR extract exhibited remarkably higher antioxidant activity.

2.7. Anticancer Activity

The anticancer potential of Zn-HA/CR was investigated using MG-63 (HOS) osteosarcoma cells over a concentration range of 6.25–200 µg/mL through qualitative morphological assessment, as shown in Figure 8. At lower concentrations such as 6.25 and 12.5 µg/mL, MG-63 cells maintained their typical elongated spindle-shaped morphology and strong adhesion to the culture surface, indicating minimal cytotoxic stress at low therapeutic doses. As the concentration increased to 25 and 50 µg/mL, distinct morphological alterations were observed, including partial rounding, reduced spreading, and weakened adhesion, accompanied by a visible decrease in cell density. These features are characteristic of early-stage cytotoxic responses in cancer cells and suggest disruption of cytoskeletal integrity, metabolic activity, and proliferative capacity. The gradual nature of these changes indicates a concentration-dependent anticancer effect rather than abrupt nonspecific toxicity. At higher concentrations like 100 and 200 µg/mL, Zn-HA/CR induced pronounced cytotoxicity in MG-63 cells, as evidenced by extensive cell rounding, shrinkage, and detachment from the culture surface, along with a substantial reduction in viable cell population. IC 50 values were calculated and demonstrated in Figure 8. In this, the IC 50 value for the nanocomposite was 33.7 μg/mL, and the Zn-HA/CR nanocomposite has better cytotoxicity against MG63 cells at minimal concentrations. Similarly, the cell viability dropped drastically, confirming severe loss of metabolic activity at the higher sample concentrations. Such morphological hallmarks are consistent with apoptotic or late-stage cytotoxic processes frequently observed in osteosarcoma cells exposed to metal-based or ion-releasing therapeutic systems. The enhanced anticancer activity at elevated concentrations is plausibly associated with excessive intracellular Zn accumulation, which can trigger oxidative stress, mitochondrial dysfunction, and cell-cycle arrest in malignant cells. Overall, Zn-HA/CR demonstrates a clear dose-dependent anticancer effect against MG-63 osteosarcoma cells, with minimal impact at low concentrations and substantial cytotoxicity at higher doses. These results highlight the potential of Zn-HA/CR as a concentration-tunable anticancer material and provide a morphological basis for further mechanistic and quantitative investigations. In this, the phenolic and alkaloid components of the CR extract are primarily responsible for the radical scavenging behaviour seen in the DPPH assay, while Zn release and phytochemical–metal interactions that encourage intracellular oxidative stress at higher concentrations may mediate the anticancer activity in MG-63 cells.

3. Materials and Methods

3.1. Preparation of HA and Zn-HA Particles

Tuna fish bones were collected from a local fish market in Chennai, Tamil Nadu, India, and thoroughly cleaned prior to use. After removing decaying tissue and residual proteins by boiling the bones in distilled water at 100 °C for 6 h, they were soaked in a 1% sodium hydroxide (NaOH, Merck, Darmstadt, Germany) solution for a total of 12 h. After being cleaned with distilled water, the cleaned bones were dried for 24 h at 100 °C in an oven. The calcination process was performed in a furnace at 900 °C for 6 h. To obtain HA particles, the calcined material was crushed mechanically for 3 h at 400 rpm. Finally, the extraction of tuna fish bone demonstrates the formation of HA with a percentage yield of ≈50 nm. Afterward, 0.1 M zinc nitrate hexahydrate (Zn(NO_3_)2·6H_2_O, ≥99%, Sigma-Aldrich, St. Louis, MO, USA) of 15.8 mL was added dropwise to the freshly prepared 3.0 g of HA particles, which were liquified in 200 mL of distilled (DI) water under constant magnetic agitation for 2 h to ensure the synthesis of Zn-substituted HA (Zn-HA). The suspension was stirred for 2 h at 25 °C after the pH was brought to 9 by adding 0.1 M NaOH [30]. After filtering and repeatedly washing with DI water to eliminate remaining contaminants, the resultant Zn-HA nanocomposite was then dried for 24 h at 100 °C, monitored by the calcination at 600 °C for 4 h.

3.2. Extraction of Catharanthus Roseus (CR) Flower

CR flowers were collected locally from Namakkal, Tamil Nadu, India. After being shade-dried, the dried flowers were ground into a fine powder using an electric blender. To obtain the flower extraction, 20 g of the powder was suspended in an ethanol/water solution (80:20, v/v) and agitated for 2 h at the ambient temperature. Then, the extract solution was ultrasonically agitated for 30 min at 35 kHz. The resulting solution was filtered using fine filter paper, and the collected filtrate was used directly for the preparation of the nanocomposite [40].

3.3. Fabrication of Zn-HA/CR Nanocomposites

To facilitate for preparing the Zn-HA/CR nanocomposite, 10 mL of CR flower extract and 1 g of the prepared Zn-HA nanoparticles were combined with 30 mL of DI water. After adjusting the solution pH to 10, the mixture was ultrasonicated for 1 h to ensure an even dispersion and successful composite fabrication. To obtain a fine powder, the solution was aged for 24 h, filtered, and then dried for 12 h at 80 °C in a microwave oven.

3.4. Characterisation Studies

X-ray powder diffraction (XRD, Bruker D8 Advance, Bruker, Billerica, MA, USA) was used to investigate the crystalline structures of HA, Zn-HA, and Zn-HA/CR nanocomposites with Cu Kα radiation (λ = 1.5406 Å). The Fourier transform infrared (FTIR, Spectrum 100; Perkin Elmer, USA) spectroscopy, functional groups and chemical interactions were determined in the wavenumber range of 4000–500 cm^−1^. Field-emission scanning electron microscopy (FE-SEM, JEOL JSM-7600F, Tokyo, Japan) was utilised to determine the surface characteristics of the as-prepared materials. Particle morphology and surface topography were inspected further using high-resolution transmission electron microscopy (HR-TEM, JEOL JEM-2100, Tokyo, Japan). Vickers microhardness (Hv) measurement was performed to examine the mechanical characteristics of HA, Zn-HA, and Zn-HA/CR nanocomposites using a Shimadzu HMV-2T microhardness tester, Shimadzu Corporation, Kyoto, Japan. The as-prepared HA, Zn-HA, and Zn-HA/CR composite were pressed using a hydraulic press under a load of 5 tonnes for 5 min to obtain cylindrical pellets. Indentations were carried out at an indentation rate of about 100 mm min^−1^ with a weight of 100 g and an indentation duration of 30 s.

3.5. Swelling and Degradation Behaviours in SBF

The swelling behaviour of HA, Zn-HA, and Zn-HA/CR nanocomposites was systematically evaluated by immersing each specimen in 10 mL of simulated body fluid (SBF) practiced in accordance with Kokubo’s protocol [41]. Over the duration of a 14-day immersion time, the samples were incubated in physiological conditions and swelling evaluations were carried out every 24 h. Before immersion, the initial dry weight of each sample was recorded. The samples were collected from the SBF after the scheduled time period, and extra surface-bound fluid was removed by gently air drying at 37 °C. After the samples were weighed again, the percentage rise in sample weight after the immersion procedure was used to determine the swelling ratio (%). In order to get rid of loosely connected ions, the specimens were removed from the SBF and thoroughly washed with distilled water at each specified time period. Using a conventional procedure, excess surface-bound fluid was eliminated through tapping the specimen surface with pre-weighed filter paper for 30 s, then carefully air-drying the material for 5 min at 37 °C. After that, the wet weight was immediately measured, and the same procedure was continued for all the samples to measure the swelling ratio.

The in vitro degradation behaviour of the nanocomposites was assessed by immersing the samples in SBF for time periods ranging from 0 to 14 days. Before immersion, the initial dry weight for each sample was determined, and then the samples were placed in the SBF solution. The samples were separated from the solution at scheduled intervals, dried under similar conditions, and analysed again. Based on the variations between the initial and final sample weights, the degradation characteristic was expressed as a weight loss (%).

3.6. Analysis of Antioxidant Activity

A modified procedure for a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay was employed to methodically determine the antioxidant properties of the Zn-HA/CR nanocomposite. A freshly developed 500 μM DPPH mixture in methanol was utilised as the source of stable radicals. Zn-HA/CR nanocomposite at different concentrations (0.1, 0.5, 1, 5, 10, 50, 100, and 500 μg/mL) was incorporated with 100 μL of the DPPH solution to produce the reaction mixtures to conduct scavenging investigations and ethanol was then employed to adjust the final volume to 1.0 mL. To enable sufficient interaction among the nanocomposite and DPPH radicals, the mixtures were incubated for 7 h at ambient temperature in the absence of light. Employing a UV-visible spectrophotometer to measure the absorbance values at 517 nm, antioxidant activity was assessed by observing the decrease in the distinctive purple colour of DPPH.

3.7. Analysis of Antibacterial Activity

The agar disc diffusion method was utilised to establish the antibacterial activity of the as-prepared HA, Zn-HA, and Zn-HA/CR nanocomposites against Staphylococcus aureus and Escherichia coli. These bacterial strains are used to evaluate the antibacterial effectiveness of the nanocomposites because they are frequently associated with diseases associated with biomaterials. The strains were placed in a solution of tryptic soy broth enhanced with 0.6% fermented yeast at 37 °C to produce fresh overnight bacterial cultures.

After that, the bacterial suspensions were uniformly distributed over Müller–Hinton agar discs (agar thickness ≈ 4 mm) for inoculating the bacterial strains. HA, Zn-HA, and Zn-HA/CR nanocomposite suspensions in different volumes (20, 30, 40, 50, and 60 µL) were infused into sterile discs (4 mm diameter) made from Whatman No. 3 filter paper. The discs were positioned equally distributed on the prepared agar plates and then incubated for 24 h at 37 °C. The zone of inhibition (mm) that developed around each disc after incubation was determined and evaluated. The diameter of the inhibition zones for the nanocomposites was used to determine the antibacterial activity against both bacterial strains.

3.8. Cell Viability

The MTT assay was used to measure the cell viability towards HOS MG63 cells in order to evaluate the biocompatible characteristics of the as-synthesised Zn-HA/CR nanocomposite. HOS MG63 cells were sourced from NCCS, Pune, and cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic–antimycotic solution at 37 °C with 5% CO_2_. In order to measure the cell viability as a function of concentrations such as 6.25, 12.5, 25, 50 and 100 µg/mL at the incubation time of 24 h, the cells were seeded in the composite sample in 24-well plates at an average density of 1.5 × 104 cells m/L. For control, each well was investigated without the sample specimen with an identical quantity of cells. The culture medium was removed and substituted with a fresh medium containing MTT solution (0.5 mg/mL) during the prescribed incubation time with the Zn-HA/CR nanocomposite. After that, the cells were incubated at 37 °C for 4 h to enable the formation of formazan crystals. After incubation, each well was loaded with dimethyl sulfoxide (DMSO) and gently shaken for 10 min to dissolve the insoluble formazan crystals and then the resulting solution was carefully removed. A UV spectrophotometer was employed to measure the absorption at 570 nm after the nanocomposite samples were taken out of the well plates and sterilised with phosphate-buffered saline.

4. Conclusions

A HA, Zn-HA, and Zn-HA/CR nanocomposite was successfully developed through a sustainable synthesis from biogenic waste using tuna fish bone. Structural and physicochemical analyses confirmed effective Zn incorporation and CR extract integration without disturbing the hexagonal HA framework, while also improving surface morphology, swelling behaviour, degradation, and mechanical strength. Biologically, the Zn-HA/CR nanocomposite exhibited significantly enhanced antibacterial activity against both S. aureus and E. coli, along with strong, dose-dependent antioxidant performance. In vitro cytotoxicity studies using MG-63 (HOS) osteosarcoma cells further demonstrated a clear concentration-dependent anticancer effect, which clearly demonstrates the morphological changes with reduced cell viability. From these, the obtained results highlighted that the prepared Zn-HA/CR nanocomposite is a sustainable and multifunctional biomaterial with promising potential for therapeutic applications.