# Synthesis and characterization of gelatin-hydroxyapatite nanohybrid composites

Alvin Sasia, Lawrence Ochoo, Daniel Bem, Nathalie Lyczko, Martial Sauceau, Ange Nzihou, Arthur Onyuka

► To cite this version:

Alvin Sasia, Lawrence Ochoo, Daniel Bem, Nathalie Lyczko, Martial Sauceau, et al. Synthesis and characterization of gelatin-hydroxyapatite nanohybrid composites. Surfaces and Interfaces, 2025, 73, pp.107533. 10.1016/j.surfin.2025.107533. hal-05264392

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https://imt-mines-albi.hal.science/hal-05264392v1

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# Synthesis and characterization of gelatin-hydroxyapatite nanohybrid composites

Alvin Sasia<sup>a,b,c,†,‡</sup>, Lawrence Ochoo<sup>a</sup>, Daniel Bem<sup>a,D</sup>, Nathalie Lyczko<sup>b</sup>, Martial Sauceau<sup>b,D</sup>, Ange Nzihou<sup>b</sup>, Arthur Onyuka<sup>c</sup>

## ARTICLE INFO

### Keywords:

Nanogelatin

Nano-calcium hydroxyapatite

Colloidal suspension

Interfacial interaction and nanocomposite

fabrication

## ABSTRACT

Gelatin-based nanocomposites have gained significant attention as sustainable and multifunctional biomaterials due to their natural origin, biocompatibility, and tunable properties, making them strong candidates for advanced structural applications, such as matrix fillers and functional coatings in porous substrates like leather. But, pure gelatin suffers from limited mechanical strength, thermal instability, and rapid solubility in aqueous environments, necessitating structural reinforcement. Nano-calcium hydroxyapatite (CaP) is a well-established inorganic additive that enhances the mechanical, thermal, and structural performance of gelatin matrices; however, its tendency to agglomerate in gelatin limits uniform dispersion, and consequently restricts its reinforcement capabilities. This study focuses on the synthesis and characterization of a novel nanohybrid composite, integrating gelatin nano-emulsion as the biopolymer matrix with different weight ratios of nano-CaP colloidal suspension (CaP10 % and CaP20 %) as the reinforcing phase. Various characterization techniques were used to evaluate the influence of nano-CaP on interfacial interaction and effect on microstructure. Fourier transform infrared spectroscopy and X-ray diffraction results show calcium hydroxyapatite as the main phase (75 %) in the nanohybrid composite while scanning electron microscope confirmed its intimate contact and homogenous dispersion. The 10 % CaP loading provided a better physical and thermo response, including a higher glass transition temperature (220 °C), and increased hydrophobicity (45 % increase in water contact angle). These enhancements in properties are attributed to the homogenous dispersion of nano-CaP in the nanogelatin matrix and the strong interfacial interaction between the particle surfaces and the nanogelatin molecule. Overall, the results demonstrate the potential of colloidal nano-CaP suspension as a dual-function agent for cross-linking and thermo-mechanical reinforcement in gelatin-based nanocomposites.

## 1. Introduction

Gelatin nanocomposites have gained significant attention as innovative biomaterials, particularly in applications ranging from water treatment [1], construction material [2], biodegradable packaging [3], and surface modification. Their appeal lies in gelatin's innate versatility, biodegradability, and biocompatibility. In pursuit of sustainable and green materials, gelatin has emerged as a promising alternative to synthetic polymers, particularly as structural fillers in leather substrates [4]. However, the practical use of gelatin in these applications is limited

by its poor mechanical strength, thermal instability, and high aqueous solubility. These functional limitations significantly restrict its uses in the aforementioned applications.

Modification of gelatin's mechanical and functional properties is essential for use in many of the aforementioned applications. The introduction of hybrid or inorganic nanoparticles is recognized as one of the most important strategies for enhancing the mechanical and functional properties of gelatin while preserving biocompatibility [5]. Among the available nanofillers, stoichiometric calcium hydroxyapatite (CaP), with the chemical formula Ca₁₀(PO₄)₆(OH)₂ has attracted much

<footnote><sup>a</sup> Department of Physics, Kenyatta University, Nairobi, Kenya</footnote>

<footnote><sup>b</sup> IMT Mines Albi-Carmaux, CNRS, Centre RAPSODEE, Université de Toulouse, Albi, France</footnote>

<footnote><sup>c</sup> Kenya Industrial Research and Development Institute (KIRDI), Nairobi, Kenya</footnote>

<footnote>* Corresponding author at: IMT Mines Albi-Carmaux, CNRS, Centre RAPSODEE, Université de Toulouse, 81013 Cedex Albi, France.</footnote>

<footnote>E-mail addresses: alvin.sasia@mines-albi.fr, sasialvin25@gmail.com, alvin.sasia@mines-albi.fr (A. Sasia).</footnote>

<footnote>† Present Address: Department of Physics, Kenyatta University. P.O Box 43,844 – 00100. Nairobi, Kenya.</footnote>

<footnote>‡ Permanent Address: Materials Research Centre, Kenya Industrial Research and Development Institute (KIRDI). P.O Box 30,650 - 00100. Nairobi, Kenya.</footnote>
interest due to its chemical similarity to bone and the high thermal stability [6]. In nanoparticulate form, CaP exhibits a high specific surface area and thermal stability, making it a valuable reinforcement agent for improving the thermal, mechanical, and physicochemical properties of gelatin-based nanocomposites [6,7]. However, the incorporation of nano-CaP into the gelatin matrices presents a critical challenge related to poor dispersion and particle agglomeration [8].

Owing to small size and the inherent polar solid surface, nano-CaP particles exhibit a strong tendency to aggregate in concentrated polymer solutions. This aggregation, driven by a thermodynamic tendency to reduce surface energy, significantly reduces the effective surface area available for interaction with the polymer matrix. As a consequence, the physical blending of nano-CaP into gelatin often leads to poor interfacial bonding, compromising the mechanical integrity of the resultant composites. To overcome this challenge, several strategies have been explored, including surface modification of CaP nanoparticles using organic molecules to reduce surface energy [9–12], and the use of dispersing agents or surfactants to achieve steric stabilization and improve dispersion [13].

An alternative and promising approach involves the use of nano-CaP colloidal suspensions to enhance dispersion and improve the homogeneity of gelatin-CaP nanocomposites. Although, stoichiometric calcium hydroxyapatite is generally insoluble, it can release a small amount of calcium ions in acidic conditions (pH 4–6), forming a colloidal suspension that resists its own gravity sedimentation [14]. The treatment with phosphoric acid modifies the surface charge of CaP nanoparticles, increasing their hydrophilicity and dispersion stability while preserving their compatibility and the chemistry of calcium phosphates [15]. The resulting colloidal system enables better integration of CaP into the nanogelatin matrix, promoting enhanced particle-polymer interactions.

Additionally, this colloidal strategy facilitates the penetration of CaP ions throughout the nanogelatin network. Gelatin’s surface chemistry, coupled with the charge and structure complementarity could enable the electrostatic interaction and ionic association of RCOO⁻ and Ca²⁺ ions, resulting in numerous Ca²⁺ rich domains, upon which rapid nucleation of the CaP nanocrystals can occur [16]. This penetrative nucleation and growth of the CaP reprecipitates in the nanogelatin matrix ameliorates interface interactions between the organic and inorganic phases [17]. Thus, CaP can be used not only as a reinforcing agent but also as an endogenous cross-linking agent in composite preparation, and the resulting organic/inorganic hybrid composites are expected to achieve good results in practical application.

In this study, gelatin-CaP nanohybrid composites were prepared via a solution casting method. A gelatin nano-emulsion was prepared in ethanol, while the nano-CaP phase was dispersed as a colloidal suspension. Ultrasonication was employed to promote uniform mixing and ensure homogenous dispersion of the components, vital in optimizing the morphology and mechanical properties of the nanohybrid composite. Morphology, crystal microstructure, molecular interactions, thermal stability, rheological analysis, hydrophilicity, and surface free energy of the nano-calcium hydroxyapatite and the prepared nanohybrid composites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), rheology and contact angle measurements.

# 2. Materials and methods

## 2.1. Materials

Bovine gelatin powder Type B (225 bloom strength, Sigma Aldrich). Nano-calcium hydroxyapatite with physical properties (Table S1) (of particle size diameter ($d_{50}$) 8.1 µm and $S_{BET}$ surface area 135.8 ± 6.9 m²/g), synthesized at industrial scale was employed in this research. Absolute ethanol and ortho-phosphoric acid were of analytical grade, commercially obtained and were used without further modifications.

## 2.2. Methods

### 2.2.1. Nano-CaP suspension

A 10 % mass fraction suspension of nano-CaP was prepared in water. Ortho-phosphoric acid was added by titration until the endpoint (pH 4) was reached with stirring at 250 rpm. The pH evolution of the suspension was monitored over a two-week period until the suspension stabilized at pH 6. The suspension was kept at room temperature for use in the nanohybrid fabrication.

### 2.2.2. Gelatin nano-particles

Gelatin nanoparticles were synthesized by the nanoprecipitation method according to the protocol described by Khan [18], with a few modifications (Table S2). Aqueous gelatin solution (3 % mass fraction) was introduced into the ethanol non-solvent system using a peristaltic pump (0.3 ml/min). The resultant emulsion was strained of ethanol by rotary evaporator under vacuum and 38 °C. The pH of the gelatin nano-emulsion was adjusted to a final pH of between 6–7 by 0.1 M NaOH.

### 2.2.3. Preparation of the nanohybrid composite

The nanohybrid composite was fabricated through the solution casting method, preceded by the preparation of the precursor solutions as illustrated in Fig. 1. The solid content of the nanogelatin aqueous solution with a 3 % mass fraction was determined on a moisture analyser (Mettler Toledo), and this value was used to calculate the weight equivalent of nanogelatin. The nanohybrid composite was fabricated by taking the weight equivalent of nanogelatin as unitary, and the nano-CaP suspension amounts varied relative to the nanogelatin. For instance, a 20 g nano-emulsion of gelatin, having a solid content of 15 % will yield 3 g of the nanogelatin. Thus, in the protocol, we would utilize 10 % and 20 % of the 3 g as the weight equivalent of the as-prepared CaP suspension. The final composite formulations had 10 % and 20 % mass fractions of nano-calcium hydroxyapatite. For purposes of reference, these nanohybrid composites are labelled as NG:CaP10 % and NG:CaP20 % respectively. The nanogelatin emulsion and nano-CaP suspension were mixed (stirring at 250 rpm with a magnetic stirrer) followed by sonication at 50 °C for 15 min. The nanohybrid composite was cast in an aluminum petri dish and lyophilized in a freeze dryer (Virtis SP Scientific) at –40 °C for 16 h to allow, first, for complete freezing then sublimated through thermal treatment steps and held for 54 h until complete drying was realized.

## 2.3. Characterization

### 2.3.1. Crystal microstructure and crystallite size determination

XRD data were collected using a Phillips Panalytical X’pert Pro MPD diffractometer. The radiation source ray used is copper (Cu) whose wavelength $\lambda$ is 1.543 Å. A nickel filter is used to remove the Cu Kβ ray. The apparatus operates at a voltage of 45 kV and an intensity of 40 mA. The diffractogram was recorded from 10 to 80° in 2θ. The step size was 0.0670° in 2θ and the continuous scan step time was 200.02 s. The data was processed using HighScore Plus software for background subtraction, peak search, and peak fitting. Monshi-Scherrer’s equation (Eq. (1)) was used to estimate the crystallite size and peak broadening from the XRD data.

$$ \ln(\beta) = \ln \frac{1}{\cos\theta} + \ln \frac{k\lambda}{D} \quad (1) $$

Where:

* $\beta$ is full width at half maximum (FWHM) in rad,

* $\theta$ is peak position (rad),

* D is crystallite size (nm), k is Scherrer constant (0.9) and

* $\lambda$ the wavelength of the X-ray source (0.1543 nm)

#### 2.3.1.1. Chemical function analysis. FTIR was performed by using
<figure><img src="image_2.png" /><figcaption>Fig. 1. Schematic illustration of the major steps in the fabrication of the nanohybrid composite.</figcaption></figure>

Nicolet Model IN10 MX Thermofisher Spectrophotometer with the attenuated total reflectance mode. Spectra was collected for the infrared region 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹ and 32 scans at room temperature to determine the chemical functions of the nanohybrid composite. Data processing involved background subtraction and peak picking using the peak threshold settings in OMNIC software.

2.3.1.2. *Morphological characterization.* The morphologies of the nano-hybrid composite were examined by SEM (Quattro S). The sample was first deposited on a conductive carbon tape attached on an aluminum mount and sputter coated (Polaron Sputter Coater) with platinum before observation. Both the surface and cross-sectional area were viewed under the microscope.

2.3.1.3. *Thermal stability.* TGA/DSC of the nanohybrid composite was performed on a Labsys Evo 1600 analyser. Approximately 30 mg of the sample was placed in a platinum crucible and heated under air atmosphere from 30–800 °C with a heating rate of 5 °C/min. The TGA/DSC was used to evaluate the thermal stability of the nanohybrid composite.

The data were processed using Calisto software to enable precise identification and quantification of mass loss events. Derivative TG (dTG) curves were generated to resolve overlapping decomposition steps. Baseline subtraction was performed using a blank analysis, followed by smoothing (applied at a value of 200) to reduce signal noise. Mass loss regions were manually defined, and the corresponding temperature ranges and peak thermal decomposition temperatures were extracted.

2.3.1.4. *Estimation of hydrophilicity and surface free energy (SFE).* Pellet discs of the nanohybrid composite were first prepared on pelletizer and used for the determination of contact angles. The contact angle was measured using the sessile drop method on a goniometer (DSA30 Kruss). A probe liquid droplet of 5 µL was deposited on the surface of the prepared pellet discs by a micro syringe and its image on the surface of the sample taken by a digital camera. The tangent lines to the droplet and baseline were drawn and used to measure the equilibrium contact angles. The contact angle in each drop was taken as the average value of the angle measured at the left and right of the droplet image. This procedure was repeated for all the probe liquids. The surface free energy of the nanohybrid composite was estimated from the theta values using the Good-Van-Oss-Chaudhury model (Equation 2) [19].

$$ \sigma_L(1 + \cos\theta) = 2 \left[ (\sigma_S^D \sigma_L^D)^{\frac{1}{2}} + (\sigma_S^+ \sigma_L^-)^{\frac{1}{2}} + (\sigma_S^- \sigma_L^+)^{\frac{1}{2}} \right] \quad (2) $$

Where,

Θ is the contact angle between the liquid and the solid surface

σ_L is the overall surface tension of the test liquid,

σ_S^D the dispersive component of the surface energy of the solid,

σ_L^D the dispersive component of the surface tension of the liquid,

σ_S^- the acid component of the surface energy of the solid, and

σ_S^- the base component of the surface energy of the solid.

### 2.3.2. Rheological behaviour

Information on the viscoelastic behaviour of the nanohybrid composites was obtained through strain-sweep oscillatory tests. The measurements were determined on a Thermo Haake Rheo-stress (MARS 40) rheometer. Plate-plate geometry with a 35 mm diameter and a gap height of 1 mm was used. Dynamic strain-sweep measurements were carried out at constant temperature (25 °C) and frequency (1 Hz). The geometry was sealed with silicon oil to avoid evaporation during measurements. The temperature control was achieved through a Peltier system. The results were analysed using the Rheowin software. The storage modulus (G''), loss modulus (G'''), and phase angle (δ) were taken as plateau values in the linear viscoelastic region (LVER) of the oscillation sweep from 1 to 1000 Pa. The critical strain (γc, %) was defined as strain at the end of LVER (±10 % deviation; according to [20]).

### 2.3.3. Specific surface area and porosity

The surface area, pore volume, and pore diameter of the samples were measured using a Micromeritics 3Flex instrument. Nitrogen adsorption measurements were conducted at 77 K and a relative pressure of up to 1. Prior to analysis, the samples were degassed at 50 °C for 10 h to eliminate any molecules that might obstruct access to the pores. According to [21], the specific surface area was calculated using the Brunauer-Emmett-Teller (BET) model over a pressure range determined by the Rouquerol criteria. Pore volume and pore size distribution were obtained using the Barrett-Joyner-Halenda (BJH) method.

## 2.4. Statistical analysis

Samples were measured in triplicates, and the experimental results expressed as mean value ± standard deviation. The statistical analysis was performed by using two-way analysis of variance (ANOVA) with Origin Software. Significant difference between measurements was
considered at p-value of <0.05 (p < 0.05).

### 3. Results and discussion

In order to understand the mechanical response and interfacial interaction in the nanohybrid composites, the neat biopolymer matrix and precursor materials were analysed and determined as presented (Table S3, S4 and Figure S1, S2).

#### 3.1. Crystal microstructure and crystallite size determination

Fig. 2 shows the XRD results for the pure stoichiometric CaP and as-prepared CaP. The diffraction patterns, analysed in the 2θ range of 10–80°, confirm that the pure stoichiometric CaP matches the standard CaP phase, with characteristic peaks at specific 2θ values. In contrast, the as-prepared CaP shows peak broadening and slight shifts in peak positions, indicating the presence of low-crystalline calcium hydroxyapatite. Additionally, the as-prepared CaP exhibits two calcium phosphate phases: stoichiometric calcium hydroxyapatite (reference n° 96–901–3628), with main peaks at 2θ values of 25.8°, 31.8°, 32.9°, and 46.6° and brushite (CaHPO₄·2H₂O, reference n° 96–231–0527), with peaks at 13.2°, 26.5°, and 30.5°

The XRD results of the pristine nanogelatin and the corresponding nanohybrid composites are shown in Fig. 3. Nanogelatin is an amorphous organic material, so its contribution to the XRD pattern manifests as a broad and diffuse hump at 2θ values of between 20–22°. Gelatin is a hydrolytic derivative of collagen, whose XRD reflections are related to the partial alpha-helix character of collagen. The XRD diffractogram of the nanohybrid composite showed carbonate hydroxyapatite (reference n° 96–900–3551), with main peaks at 25.8°, 31.6° and 32.7°.

The broadening of the diffraction peaks in the nanohybrid composite suggests a smaller grain size and lower crystallinity of the as-prepared CaP. The crystallite sizes, as determined by Eq. (1), were 17.3 nm, 30.4 nm, 18.8 nm, and 22.05 nm for pure stoichiometric CaP, as-prepared CaP, NG:CaP10 % and NG:CaP20 % respectively. This indicates a strong interaction between the CaP and the nanogelatin. As determined in literature [22], nanogelatin has a strong influence on the crystallite size and crystallinity of the CaP. The carboxylate ions of both the glutamic and aspartic amino acids in gelatin provide sites for electrostatic binding and ionic association with Ca²⁺ ions, which act as structural templates for the nucleation and growth of the nano-CaP within the nanogelatin matrix. Additionally, nanogelatin functions as a surfactant, adsorbing onto the crystal surfaces of the CaP and thereby

influencing crystal growth and morphology.

Semi-quantitative analysis was performed using the reference-intensity ratio (RIR) method [23]. The major crystal phase (55 %) in the as-prepared CaP is calcium hydroxyapatite, and the minor phase (45 %) is the brushite phase confirming the results of the X-ray diffraction analysis. The calculated crystalline phase concentrations in the nanohybrid composite were determined to be 75 % for calcium hydroxyapatite and 25 % for nanogelatin. These results indicate that the crystalline CaP phase dominates the XRD-detectable structure of the nanohybrid composite. Although gelatin is primarily an amorphous biopolymer, it can exhibit detectable XRD peaks, typically at 2θ values between 7–8° and 20–22°, corresponding to its crystalline α-helix domains. Therefore, the 25 % phase composition attributed to nanogelatin by the RIR method is likely associated with the presence of these ordered semi-crystalline regions.

#### 3.2. Chemical function analysis

To further illustrate the chemical interactions between the as-prepared CaP and the nanogelatin matrix, FTIR analysis was carried out. Fig. 4 shows the absorption spectra of the mineralized nanogelatin with various nano-CaP loading. From the spectra of the nanogelatin, we see the characteristic absorption bands associated with the neat biopolymer: typical amide bands at 3307 cm⁻¹ corresponding to amide A (N–H stretching and O–H stretching due to intermolecular hydrogen bonding) and 2973 cm⁻¹ which is the amide B band for Fermi resonance overtone of the amide II vibration [24]. Other notable vibrations are 1648 cm⁻¹ corresponding to amide I (carbonyl group C=O and weak C–N stretching coupled with in-phase N- bending); 1535 cm⁻¹ attributed to amide II (N–H in-plane bending and C–N stretching vibrations); 1241 cm⁻¹ corresponding to amide III (the in-plane vibration of C–N and N–H groups of bound amides); and 1449 cm⁻¹ attributed to CH₂ vibrations of glycine [16,25].

Above the isoelectric point (IEP), gelatin bears a net negative charge, because of the ionic forms of the carboxylate ions. Thus, gelatin's RCOO⁻ groups provide binding sites for ionic interaction with the Ca²⁺ ions of the CaP [26]. The asymmetric stretching of the carboxylates caused by the weakening of the C=O bond in the peptide chain leads to a redshift [27]. From the spectra of the nanohybrid composite, we note a redshift of the amide I band by up to 10 cm⁻¹. This confirms the chemical bond formation between the carboxyl ions on nanogelatin and the stoichiometric CaP phase. The strength of the redshift is determined by factors such as pH, temperature, concentration, and aging /time of contact [28].

<figure><img src="image_3.png" /><figcaption>Fig. 2. XRD patterns of (a) pure stoichiometric CaP and (b) as-prepared CaP.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3. XRD patterns of (a) nanogelatin, (b) NG:CaP10 % and (c) NG:CaP20 % nanohybrid composites.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 4. FTIR spectra of (a) nanogelatin; (b) NG:CaP20 %; (c) NG:CaP10 % and (d) as-prepared CaP.</figcaption></figure>

The fundamental vibrational mode of the PO₄³⁻ species was discerned at 963 cm⁻¹ and 1030 cm⁻¹ in the as-prepared CaP which correspond to the γ₁ and γ₃ stretching modes respectively [29,30]. The characteristic absorption peak corresponding to the asymmetric P-O stretching vibration predominantly appeared at 1030 cm⁻¹ in the nanohybrid composite with its relative intensity increasing with an increase in stoichiometric CaP loading. The γ₁ stretching mode was red-shifted by up to 25 cm⁻¹ in the nanohybrid composite as a factor of stoichiometric

CaP loading and is attributed to the strong H-bonding between the oxygen anion on the phosphate and the amide hydrogens of nanogelatin. This implies the existence of the organic-inorganic interaction between the as-prepared CaP and the nanogelatin matrix.

The band at 875 cm⁻¹ can be ascribed to the presence of hydrogen phosphate (HPO₄²⁻) ions, present from the dissolution-precipitation reactions with phosphoric acid. However, this band is known to overlap with the carbonate band, making a straightforward conclusion difficult.
Nevertheless, the presence of peaks at 1458 cm⁻¹ and 1419 cm⁻¹ known in the literature as the substitution of PO₄³⁻ ions with CO₂³⁻ helps us attribute the former to carbonate substitution [30]. Moreover, the facile isomorphic replacement of the HPO₄ anions by CO₂³⁻ ions has already been reported in the literature [31]. The CO₂³⁻ ions are probably incorporated into the solution from the atmosphere during the dissolution-precipitation reactions, thus, leading to the formation of B-type apatite [32]. There was no discernible change in peak positions and intensity of this band in the nanohybrid composite.

### 3.3. Morphological analysis

Micrographs of the initial pure stoichiometric CaP are presented in Fig. 5a. The micrograph depicts needle-like crystalline formations of calcium hydroxyapatite, which form a compact network of aggregated nanoparticle structures. SEM micrographs of the as-prepared CaP reveal the presence of two calcium phosphate phases formed during the dissolution-precipitation reaction, corroborating the results of XRD. Fig. 5b exhibits a granular and spherical particle structure, which appears to form large clusters of rounded nanoparticles. The particles are densely packed indicating strong aggregation. The individual particles appear larger compared to the particles in the pure stoichiometric CaP, though clusters of smaller particles attached to the aggregates are also visible (Figure S3c). On the other hand, the micrograph (Fig. 5c), depicts the lamellar form which indicates the presence of a brushite phase alongside stoichiometric CaP. The precipitation of the brushite phase underscores not only the kinetic control but also the importance of pH control in the crystallization process. Literature on calcium apatite dissolution in acidic media indicates that dissolution begins with direct chemical interaction between solid apatite and acidic species, breaking lattice bonds and releasing ions into solution [33]. These ions can then recombine, leading to precipitation of either calcium-deficient or stoichiometric hydroxyapatite through metastable phases like brushite (Figure S3). Thus, in addition to pH, controlling crystal morphology depends on the solubility of calcium phosphate, which directly affects supersaturation and subsequent nucleation and growth [31].

SEM micrographs at different magnifications of the selected solid samples are illustrated in Fig. 6a-f. The nanogelatin micrograph (Fig. 6a-b) depicts spherical particles with diameters (measured on ImageJ software) in the range from 50 to 300 nm. The nanoparticles are polydisperse, with an average size of 166.1 ± 61 nm. From the nanohybrid composite micrographs (Fig. 6c-f), we note the homogenous dispersion of the as-prepared CaP in the nanogelatin matrix, which indicates interfacial bonding and intimate interaction between the components in the nanohybrid composite. While the presence of brushite phase (shown by the blue circle in the micrograph) in the nanohybrid composite suggests that the nanogelatin matrix exerts significant control over the strong hydrolysis of the stoichiometric calcium phosphate, we could also attribute the retention of this brushite phase to the presence of carbonate ions in the as-prepared CaP, leading to formation of carbonated CaP (C—CaP), which is known to slow down brushite hydrolysis (Equation

S1), thereby stabilizing it [34]. The nanohybrid composites also exhibit porosity with interconnected and irregular porous networks and contain low-crystalline CaP nanoparticles which agglomerate in the composite with CaP loading. This finding is consistent with similar reports in the literature [35].

### 3.4. Thermal stability

The thermal gravimetric analysis (TGA) for the as-prepared CaP, pristine nanogelatin, and nanohybrid composite are presented in Fig. 7, and their differential thermal gravimetric curves (dTG) in Fig. 8. The as-prepared CaP depicts 4 stages of mass loss, which accounts for a combined 8.11 % mass loss, indicating the high thermal stability of the as-prepared CaP. From 70 to 130 °C, 1.48 % mass loss occurs due to the removal of surface moisture. The 3.7 % mass loss at 192 °C, is attributed to the dehydration of brushite leading to the formation of monetite, while at 225 °C, interstitial water is eliminated from the crystal lattice leading to a mass loss of < 1 % [36].

Thermogravimetric analysis of nanogelatin revealed a total mass loss of 100 %, occurring over three distinct stages. An initial mass loss of 9.5 % between 33 °C and 161 °C is attributed to the evaporation of moisture and loosely bound volatiles. The primary decomposition of the nanogelatin chains occurs over a broad temperature range from 161 °C to 412 °C, accounting for a 54.4 % mass loss. Complete degradation of nanogelatin is observed between 412 °C and 578 °C, corresponding to a final 36.1 % mass loss.

TGA of the nanohybrid composite shows a typical mass loss pattern with a significant decrease in mass occurring between 200 and 600 °C. The first mass loss event (11.77 %) that occurs below 164 °C can be attributed to the loss of bound moisture and low molecular weight fractions [32]. A second mass loss regime, (40.4 % loss), occurred in a large temperature range starting at 164 °C up to 373 °C, and the event was more pronounced between 164 °C and 301 °C, peaking at 284 °C. This temperature represents a 13 °C increase in the peak thermal decomposition temperature of the cross-linked nanogelatin. This mass loss represents a 14 % decrease in the mass lost compared to the pristine nanogelatin form. These increases in the peak thermal decomposition temperatures and mass loss decrease are indicative of the thermal stability conferred by the cross-linking of the nanogelatin chain with the calcium hydroxyapatite. The mass loss event is attributed to the degradation of the polymer chain, depolymerization, and decomposition of the constituent amino acids of the monomer chain [37]. The final mass loss event that occurs between 373 °C to 616 °C, and peaks at 516 °C is associated with the carbonization of the remaining polymeric material.

The DSC thermograms of the neat nanogelatin, as-prepared CaP and the nanohybrid composites are shown in Fig. 9. Nanogelatin shows a wide endothermic peak centred at 90 °C, which is attributed to the evaporation of adsorbed water. The DSC profile of nanogelatin exhibits broad transitions without a sharp melting endotherm, indicating its predominantly amorphous nature. Subtle endothermic events at approximately 90 °C and 211 °C corresponds to the loss of moisture and

<figure><img src="image_6.png" /><figcaption>Fig. 5. SEM micrograph of (a) pure stoichiometric CaP, (b) as-prepared CaP showing the crystal morphology of stoichiometric CaP, and (c) as-prepared CaP depicting the lamellar structure indicative of brushite phase.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 6. SEM micrographs at various magnifications of (a) and (b) nanogelatin matrix; (c) and (d) NG:CaP10 %; (e) and (f) NG:CaP20 % corresponding to the nanohybrid composites at various CaP loading at different magnifications.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 7. Thermogravimetric analysis of (a) as-prepared CaP, (b) NG:CaP20 % nanohybrid composite, and (c) nanogelatin. In general, the nanohybrid composites exhibit better thermal stability compared to the neat nanogelatin precursor.</figcaption></figure>

glass transition temperature (Tg) of nanogelatin; however, this signal is obscured due to the sensitivity limit of the equipment (0.4–10 µW) to detect small thermal changes. The endothermic process in gelatin is associated with the rupture of hydrogen bonds and the transformation of the triple-helix structure into random coil configurations. Above 250 °C, the observed exothermic events are attributed to the thermal degradation of the gelatin chains, as N-terminal residues are released before complete separation as free amino acids [38].

Calcium hydroxyapatite (CaP), on the other hand, shows excellent thermal stability across the tested temperature range. A minor endothermic dip at approximately 190 °C is linked to the loss of interstitial water from the CaP structure. The DSC analysis of the nanohybrid composite presents a similar behaviour to the neat nanogelatin for scans

below 250 °C. The glass transition temperatures of the as-prepared CaP, nanogelatin, NG:CaP10 %, and NG:CaP20 % nanohybrid composites are presented in Table 1. Upon incorporation of 10 % CaP, the Tg increased markedly to 220 °C, indicating enhanced molecular restriction due to the interfacial interactions. However, a further increase in the CaP loading to 20 % resulted in a Tg that reverted closer to nanogelatin’s Tg at 212 °C, suggesting a possible saturation of interaction sites or phase separation caused by agglomeration at higher CaP loading. This observation is supported by SEM micrographs, which reveal aggregation of CaP particles, indicating reduced dispersion and weaker interfacial bonding at high CaP loading.

The incorporation of calcium hydroxyapatite into nanogelatin enhances the thermal stability of the resulting nanohybrid composites.
<figure><img src="image_9.png" /><figcaption>Fig. 8. dTG curves of the (a) as-prepared CaP, (b) nanogelatin and the (c) NG:CaP20 % nanohybrid composite.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 9. Differential scanning calorimetry (DSC) analysis of the (a) as-prepared CaP, (b) NG:CaP20 % and (c) NG:CaP10 % nanohybrid composites, and (d) nanogelatin.</figcaption></figure>

<table><caption>**Table 1**

Glass transition temperature of the as-prepared CaP, nanogelatin, NG:CaP10 %, and NG:CaP20 % nanohybrid composites.</caption><thead><tr><th>Material</th><th>Loss of moisture (°C)</th><th>Glass transition temperature (°C)</th></tr></thead><tbody><tr><td>CaP10 %</td><td>190.7</td><td>-</td></tr><tr><td>Nanogelatin</td><td>90.0</td><td>211.0</td></tr><tr><td>NG:CaP10 %</td><td>96.0</td><td>220.0</td></tr><tr><td>NG:CaP20 %</td><td>76.8</td><td>212.0</td></tr></tbody></table>

This is also evident from the shift of thermal transitions, occurring above 250 °C, which correspond to strong exothermic peaks. Similar observations have been recorded in literature [39]. Additionally, the denaturation temperature of the nanohybrid composites increases with CaP loading. The reduced intensity of the exothermic features suggests

restricted molecular chain mobility, likely due to the interfacial interactions such as hydrogen bonding and ionic associations. These interactions contribute to improved thermal resistance and mechanical strength of the nanohybrid system.

### 3.5. Hydrophilicity and surface free energy approximations

Table 2 presents the contact angles of various probe liquids on both the neat precursor materials and the nanohybrid composite surfaces. The water contact angle (WCA) of the neat nanogelatin is significantly lower than previously reported in the literature [40,41]. This difference is attributed to the increased density of hydrophilic groups and the nanoscale dimensions of the prepared nanogelatin. Gelatin, as a linear polymer with random spatial arrangements, exhibits variations in WCA based on amino acid conformations and the preferred orientation of hydrophobic moieties, often resulting in a higher WCA [41].
<table><caption>**Table 2**
Contact angle measurements.</caption><thead><tr><th rowspan="2">Probe liquid</th><th rowspan="2">Pristine nanogelatin (°)</th><th rowspan="2">CaP (°)</th><th colspan="2">Nanohybrid composite</th></tr><tr><th>NG: CaP10 % (°)</th><th>NG: CaP20 % (°)</th></tr></thead><tbody><tr><td>Diiodomethane</td><td>26.5 ± 1.50</td><td>10.87 ± 2.00</td><td>49.83 ± 7.26</td><td>39.26 ± 3.45</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>27.16 ± 2.52</td><td>11.37 ± 1.61</td><td>33.36 ± 1.96</td><td>22.12 ± 1.00</td></tr><tr><td>Water</td><td>55.91 ± 5.21</td><td>13.31 ± 3.15</td><td>81.05 ± 7.00</td><td>78.57 ± 3.25</td></tr></tbody></table>

The data indicate that the addition of as-prepared CaP to nanogelatin reduced the overall hydrophilicity of the nanohybrid composites relative to the precursor materials. Notably, apart from water, significant differences ($p > 0.05$) were observed in the effects of different CaP loadings on the hydrophilicity of the nanohybrid composites for each of the probe liquids. According to Golubevas [38], surface characteristics, including porosity (pore diameter, interconnectedness, and orientation), roughness, topography, and surface chemistry (composition, polarity, and acid-base properties), substantially influence hydrophilicity. From Table 2, we see a 45 % increase in water contact angle for NG:CaP10 % compared to a 40 % increase for NG:CaP20 %, indicating stronger interfacial interactions and a greater reduction in surface hydrophilic groups at the lower CaP loading. At this loading, the nanohybrid composite exhibits more pronounced changes in the surface chemistry and morphology, likely due to better dispersion and less agglomeration of CaP.

WCA measurements were performed to reveal the interfacial interactions on the surface of the nanohybrid composite (Figure S4). Interactions between nano-CaP and nanogelatin chains affect surface hydrophilicity by altering the proportion and orientation of exposed hydrophilic groups on the nanohybrid composite surface. Hydroxyl groups in nano-CaP can form hydrogen bonds with amide hydrogens in nanogelatin, increasing cohesion and thereby reducing surface hydrophilicity. Literature suggests that calcium hydroxyapatite interaction with amino acids influences interaction patterns, amino acid conformations, and interaction strength, further impacting surface characteristics [42].

The Good-van-Oss-Chaudhury model (Eq. (2)), is the tool that allows for the determination of the surface free energy (SFE) by calculation of the Lewis acid or base properties of the nanohybrid composite surface from the contact angles and is a good approximation, to consider for the results of the interfacial interactions. The SFE of the nanohybrid composite was calculated by employing the surface tension parameters of the different probe liquids (Table S5) obtained from the literature [43, 44]. SFE values are given in Table 3. Although the dispersive components of the precursor materials are similar, the basic polar components differ significantly. The overall increase in surface free energy of the nanohybrid composite is mainly driven by the dispersive components, reflecting the stronger non-polar interactions on the nanohybrid composite surface as CaP loading increases. According to literature,

<table><caption>**Table 3**
Surface free energy approximations.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Pristine nanogelatin</th><th rowspan="2">CaP</th><th colspan="2">Nanohybrid composite</th></tr><tr><th>NG: CaP10 %</th><th>NG: CaP20 %</th></tr></thead><tbody><tr><td>Dispersive component (mJ/m²)</td><td>37.87</td><td>36.72</td><td>31.92</td><td>37.40</td></tr><tr><td>Acidic component (mJ/m²)</td><td>0.04</td><td>0.06</td><td>0.85</td><td>0.62</td></tr><tr><td>Basic component (mJ/m²)</td><td>25.36</td><td>68.05</td><td>3.18</td><td>3.04</td></tr><tr><td>Overall SFE (mJ/m²)</td><td>63.26</td><td>104.84</td><td>35.96</td><td>41.06</td></tr></tbody></table>

dispersion forces contribute significantly to SFE due to the high number of neighbouring molecules that generate conflicting local fields, which greatly diminish mean dipole interactions [44].

### 3.6. Rheological behaviour

Rheological analysis provides insight into the dispersion of the as-prepared CaP, its interactions with the nanogelatin matrix, and the resulting microstructure of the nanohybrid composite. Strain sweep tests (Fig. 10) reveal a linear viscoelastic (LVE) regime where the storage modulus (G') remains higher than the loss modulus (G''), indicating a solid-like response for all the samples. At higher applied strain, both G' and G'' decrease and reach a crossover point, corresponding to the threshold strain, above which G'' dominates and flow occurs, denoting material breakdown. The neat nanogelatin exhibits significantly higher modulus values with variations in pH value (Figure S6a). In relation with the described evolution of the modulus, phase angle remains relatively stable at low strains, but increases sharply at higher strains (Fig. 11). This is typical viscoelastic behaviour of gelatin gels. The behaviour of the non-linear viscoelastic domain (at high strain amplitude) is dominated by an increase in G'' near the critical strain, resulting from the complex structure resisting deformation against external strain. This behaviour is described as type III weak strain overshoot and has been attributed to the balance between the formation and destruction of network junctions formed by weak molecular associations such as hydrogen bonding. At large deformations over the critical strain, the complex structure is destroyed, and the polymer chains align with the flow field resulting in a G'' decrease [45,46].

Contrary to reported trends in the literature, a reduction in modulus is observed for all the nanohybrid composites with increased CaP loading (Fig. 11), making the nanohybrid composite less elastic in the LVE region (Fig. 11) [47–49]. The values of the critical strain $γ_c$, at which the nanogelatin phase angle increase and structure breakdown onset begins to occur are illustrated in Fig. 12 as a function of CaP. The critical strain was recorded at around 40 % for the pure nanogelatin and increased linearly for the nanohybrid composites with CaP loading up to 10 %, and above this loading, a diminutive reduction is observed. This suggests a reduction in the interaction between nanogelatin molecules on addition of the as-prepared CaP. A plausible explanation could be the as-prepared CaP agglomeration at high loading and the brushite phase contamination, both evidenced by XRD and SEM analyses (Figure S5), which are responsible for the high heterogeneity in particle sizes in the matrix. This leads to poor dispersion, interfacial slippage and nanogelatin matrix embrittlement, thus the materials' inability to stretch under load. Similar observations have been recorded with multi-walled carbon nanotube-reinforced gelatin nanohybrid composites. The larger dimensions of the aforementioned serve as stress concentration points and thus hinder chain mobility and disrupt the polymer matrix continuity resulting in attenuation in the modulus [49].

In this study, the addition of the as-prepared CaP shifts this response to lower moduli and higher strain amplitudes, suggesting that, while calcium hydroxyapatite initially reinforces the nanohybrid composites, its resistance to deformation diminishes at larger strains. This indicates that the nanogelatin's mechanical properties are negatively impacted at higher calcium hydroxyapatite loading.

It can be noted that the neat nanogelatin exhibits significantly higher modulus values with variations in pH value (Figure S6). Similarly, at higher pH, the addition of the as-prepared CaP does not alter the modulus significantly (Figure S7a), but shifts the critical strain to lower values (Figure S7b). This could be due to the deprotonation of functional groups on nanogelatin, which promotes intra H-bonding interactions and enhances structural rigidity. Consequently, pH induced structural rearrangements offset mechanical effects of CaP addition.
<figure><img src="image_11.png" /><figcaption>Fig. 10. Strain-sweep measurements for nanogelatin, NG:CaP5 %, NG:CaP10 %, and NG:CaP20 %.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Fig. 11. Phase angle measurements for nanogelatin, NG:CaP5 %, NG:CaP10 %, and NG:CaP20 %.</figcaption></figure>

### 3.7. Specific surface area and porosity

Fig. 13 shows the nitrogen adsorption-desorption isotherms for the nanogelatin, as-prepared CaP, NG:CaP10 %, and NG:CaP20 %. These isotherms are characteristic of type IV behaviour, typically associated with mesoporous materials, and indicate relatively weak adsorbent-adsorbent interactions. Type IV isotherms suggest multi-layer adsorption followed by capillary condensation within mesopores. For all samples except as-prepared CaP, the adsorption volume remains below 50 cm³/g for relative pressures (P/Po < 1.0). This highlights limited interaction between nitrogen molecules and the nanohybrid composite surface. In contrast, the as-prepared CaP shows a sharp increase in adsorption beyond P/Po equal to 0.8, reflecting stronger adsorbent-adsorbent interactions.

The surface area and pore volume of the precursor materials and the nanohybrid composite are summarized in Table 4. The nanohybrid composite exhibit low mesoporosity, with mesopore volumes under 0.05

cm³/g. A clear trend is observed in which both pore volume and surface area decrease with increasing contents of nanogelatin (low CaP loading). Similar trends have been recorded in literature [50,51]. This decline could be attributed to nanogelatin blocking the pores of the CaP, thus, reducing the accessible surface area. Moreover, the decrease in porosity may be a result of network densification associated with chemical cross-linking.

From the SEM micrographs of the nanohybrid composite, structural densification can be confirmed (Figure S5). This observation correlates well with the observed reduction in surface area and mesopore volume. This densification is likely driven by strong interfacial interactions, such as hydrogen bonding and ionic associations between the nanogelatin matrix and CaP particles, which further compromises access to the internal surfaces of CaP.
<figure><img src="image_13.png" /><figcaption>Fig. 12. Variation of critical strain as a function of CaP loading for nanogelatin, NG:CaP5 %, NG:CaP10 %, and NG:CaP20 % samples respectively.</figcaption></figure>

## 4. Conclusion

In this study, nanogelatin-CaP nanohybrid composites were synthesized at different weight ratios of CaP (5 %, 10 %, 20 %). FTIR and XRD analyses show calcium hydroxyapatite as the main phase (75 %), while SEM revealed its homogenous dispersion in the nanohybrid composite. The nanohybrid composite fabricated in this study successfully preserves the structural and morphological characteristics typical of organic-inorganic nanohybrid composites. Thermogravimetric analysis showed a 14 % reduction in mass loss and a 13 °C increase in the peak thermal decomposition temperature relative to the pristine nanogelatin form, indicating effective cross-linking and enhanced thermomechanical stability. The distinctive surface characteristics of the precursor materials and the interfaces produced by scaling their nano dimensions are vital for increasing interfacial interactions. The dissolution-precipitation of the nano-calcium hydroxyapatite into a colloidal CaP suspension facilitated improved dispersion of the nano-CaP in the nanogelatin matrix, enhancing both component homogeneity and mineralization. The 10 % CaP loading presented the best

physical and thermo response showing a higher glass transition temperature and improved hydrophobicity. These characteristics establish it as the optimal loading of CaP in the preparation of gelatin-based nanocomposites. Thus, NG:CaP10 % composite fabrication could be vital for the incorporation of this material into highly reactive porous substrates like leather for structural reinforcement as matrix fillers.

## Author agreement statement

I declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. I confirm that the manuscript has been read and approved by all the co-authors and that there are no other persons who satisfied the criteria for authorship but are not listed. I further confirm that the order of authors listed in the manuscript has been approved by all the co-authors. I understand that the corresponding author is the sole contact for the Editorial process. S/He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs.

## CRediT authorship contribution statement

**Alvin Sasia:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Lawrence Ochoo:** Writing – review & editing, Supervision, Project administration. **Daniel Bem:** Writing – review & editing, Supervision, Project administration. **Nathalie Lyczko:** Writing – review & editing, Supervision, Project administration, Methodology. **Martial Sauceau:** Writing – review & editing, Supervision, Project administration, Methodology. **Ange Nzihou:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Conceptualization. **Arthur Onyuka:**

<caption>Table 4</caption>

BET Surface area and pore volume of as-prepared CaP, nanogelatin, NG:CaP10 %, and NG:CaP20 %.

<table><thead><tr><td>Material</td><td>BET surface area (m²/g)</td><td>Total pore volume (cm³/g)</td></tr></thead><tbody><tr><td>As-prepared CaP</td><td>71.76</td><td>0.329</td></tr><tr><td>Nanogelatin</td><td>17.62</td><td>Non-porous</td></tr><tr><td>NG:CaP10 %</td><td>10.09</td><td>0.026</td></tr><tr><td>NG:CaP20 %</td><td>16.11</td><td>0.045</td></tr></tbody></table>

<figure><img src="image_14.png" /><figcaption>Fig. 13. Shows the nitrogen adsorption-desorption isotherms for (a) as-prepared CaP, (b) NG:CaP20 % and (c) NG:CaP10 %, and (d) nanogelatin.</figcaption></figure>
Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Partnership for Applied Skills in Sciences, Engineering and Technology- Regional Scholarship Innovation Fund (PASET-RSIF), French Embassy in Nairobi and Centre RAPSODEE, IMT Mines Albi of France.

Supplementary materials

Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.surfin.2025.107533.

Data availability

Data will be made available on request.

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