# Biomimetic CaCO3-chitosan hybrid pigments inspired by sea urchin spines biomineralization: a photostable colored additive for UV-protective self-supported chitosan films

Sandra Castanié, Sylvie Blanc, Mickael Le Béchec, Laurent Billon

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Sandra Castanié, Sylvie Blanc, Mickael Le Béchec, Laurent Billon. Biomimetic CaCO3-chitosan hybrid pigments inspired by sea urchin spines biomineralization: a photostable colored additive for UV-protective self-supported chitosan films. International Journal of Biological Macromolecules, 2026, 336, pp.149277. 10.1016/j.ijbiomac.2025.149277. hal-05445223

HAL Id: hal-05445223

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# Biomimetic CaCO₃-chitosan hybrid pigments inspired by sea urchin spines biomineralization: a photostable colored additive for UV-protective self-supported chitosan films

Sandra Castanié<sup>a,b</sup>, Sylvie Blanc<sup>b</sup>, Mickael Le Béchec<sup>b</sup>, Laurent Billon<sup>a,b,*</sup>

## ARTICLE INFO

**Keywords:**

- Biomineralization
- Calcium carbonate
- Alizarin organic dye
- Hybrid pigment
- Chitosan
- Photostability
- Composite films

## ABSTRACT

To reduce the use of toxic and polluting synthetic pigments and dyes, which are still common in industry, marine biomimicry has inspired the development of functional hybrid materials to enhance the photoresistance of natural pigments. This is a key factor limiting their industrial use. Herein, we propose to develop a new type of photostable hybrid pigment, inspired by the biomineralization process of calcium carbonate (CaCO₃), which traps natural organic dyes within sea urchin spines. Alizarin (polyhydroxyanthraquinone, AZ) was selected to mimic organic chromophores present in sea urchins, i.e. polyhydroxynaphthoquinone. The biomacromolecules chitosan (CS) and α-poly-1-lysine (PLL) were used to imitate the organic matter involved in CaCO₃ biominer-alization in sea urchins, thanks to their abundance of amine groups (–NH₂). Using a straightforward copreci-pitation method under gentle conditions, it was shown that AZ dye molecules became embedded in a CaCO₃ shell, thereby shielding them from photodegradation. It has been demonstrated that chitosan effectively aids in the coprecipitation process by stabilizing the vaterite polymorph, while α-poly-1-lysine has little impact on CaCO₃ formation. To extend the potential of these hybrid pigments for applications such as UV-protective films for food packaging, self-supporting CaCO₃/chitosan (CaCO₃/CS) composite films and tinted hybrid pigment/chitosan (CaCO₃-AZ/CS) films were developed, leveraging the excellent film-forming properties of chitosan. Different quantities of CaCO₃(–AZ) particles were incorporated into the formulations to tailor the films' mechanical and optical characteristics.

## 1. Introduction

Faced with the need to achieve the Sustainable Development Goals (SDGs) outlined in the latest United Nations program 2023, drawing inspiration from nature's efficiency, resilience, and circularity to create new materials is emerging as a promising and ambitious approach. Bioinspiration aims to adapt the principles of natural strategies—such as renewable energy, recycling, and maintaining ecosystem balance—to develop innovative, efficient, and sustainable solutions. Given this, the current study suggests applying these principles to create biomimetic hybrid pigments with UV-protective properties as a greener alternative to synthetic pigments.

In 2025, the global dyes and pigments market is valued at about US $56 billion, and is estimated to reach US$86 billion by 2034 [1]. This

market is dominated by synthetic, cost-effective, and versatile pigments and dyes used in the textile industry, paint, and coatings, even though many of them contain toxic substances harmful to human health and the environment [2–5]. Natural, organic dye sources can offer a greener alternative to synthetic ones because they can be biodegradable and non-toxic, which is particularly attractive to industries such as textiles, cosmetics, food, and pharmaceuticals [6,7]. These dyes accounted for approximately 22 % of the new projects from 2023 to 2025 [1], with an estimated market share of about US$3.5 billion by 2034. Despite a wide range of colors, limitations such as thermal decomposition, photo-degradation, photoisomerization, cross-reactivity, and pH sensitivity restrict their applications [8]. To improve the resistance of organic pigments and dyes, recent research has explored stabilizing them within inorganic matrices [9], as found in nature. This approach combines the

<footnote><sup>a</sup> Bio-Inspired Materials Group: Functionalities & Self-Assembly, 64000, Pau, France</footnote>

<footnote><sup>b</sup> Université de Pau et Pays de l'Adour, CNRS, IPREM UMR 5254, Institut des sciences analytiques et de physico-chimie pour l'environnement et les matériaux, 64000, Pau, France</footnote>

<footnote>* Corresponding author at: Bio-Inspired Materials Group: Functionalities & Self-Assembly, 64000, Pau, France.
E-mail address: laurent.billon@univ-pau.fr (L. Billon).</footnote>
high thermal stability, mechanical strength, and solvent resistance of inorganic materials with the color and functionalities of organic materials [10–12]. For example, hybrid organic-inorganic lakes and pigments made with natural organic dyes can be created by combining organic chromophores with metallic cations in inorganic support materials such as alumina, silica, talc, calcium carbonate, or barium sulfate [13]. Embedding organic chromophores into inorganic matrices stabilizes them and enhances their properties during use [13–16]. In the natural model of sea urchins, spines can display vibrant colors ranging from purple to red, brown, green, and black, thanks to organic chromophores (spinochromes and echinochromes) from the polyhydroxynaphthoquinones family [17,18]. These pigments overlay the inner layers of the shell, which are mainly composed of calcium carbonate, and benefit from mineral support. In return, the chromophores protect against UV damage through their antioxidant properties and UV absorption [19]. To develop our hybrid pigment inspired by sea urchin spines, we selected commercially available anthraquinone alizarin (1,2-dihydroxy-9,10-anthracenedione, AZ), widely used in painting, textiles [21], dye-sensitized solar cells [22], calcium assays [23], and food quality monitoring [24], because it has similar polyhydroxyl groups in the para and meta positions as sea urchin chromophores. No commercial naphthoquinone molecules with similar polyhydroxyl groups were available. Moreover, besides organic chromophores, sea urchin spines contain a small amount (<2 wt%) of intracrystalline organic molecules—proteins, O-glycoproteins and N-glycoproteins mainly linked to serine, threonine, and lysine [20], along with terminal sialic acids—whose carbohydrate parts play roles in biomineralization control mechanisms [20–24], resulting in biogenic calcite with specific structures and mechanical features [23].

As far as we know, very few literature references address the pho-
tostability of bio-inspired CaCO₃-based hybrid pigments and the effects
of macromolecules on their stability [25]. Although, to further develop
our bio-inspired approach, we studied the effects of macromolecules
that mimic intracrystalline organic molecules on the mineralization of
CaCO₃, on AZ encapsulation, and on the photostability of our hybrid
pigments. Chitosan (CS) appears as a suitable candidate and a common
model for studying the role of macromolecules in CaCO₃ nucleation
[26]. This amino-polysaccharide is a linear macromolecule composed of
randomly distributed β-(1 → 4)-linked D-glucosamine (deacetylated
unit) and N-acetyl-D-glucosamine (acetylated unit). Although a thor-
ough understanding of the mechanistic roles of these macromolecules
and their functional groups in biological crystallization remains com-
plex, it is well known that they play a key role in polymorph selection
and in controlling morphology and crystal orientation [27]. Studies
show that CaCO₃ nucleation with chitosan results in greater poly-
morphic and morphological variability than in sea urchin spines [28],
with this variability depending on the experimental methods. Poly-
Lysine (PL) is another biomacromolecule that mimics amine molecules
involved in CaCO₃ biomineralization. PL is water-soluble, made of
repeated lysine units, possessing many amino groups, and valued in
biomedicine and food preservation for its antibacterial properties,
biocompatibility, and biodegradability [29,30]. A few studies have
referred to the use of poly-1-lysine (PLL) in calcium carbonate synthesis,
with different polymorphs depending on the mineralization process
employed [31–34]. Thus, hybrid pigments of CaCO₃ with AZ (CaCO₃-
AZ) and with AZ plus chitosan or α-poly-1-lysine (CaCO₃-AZ-bio-
macromolecule) were prepared through co-precipitation. Moreover, as
chitosan's suitability in biomedicine and food packaging, owing to its
film-forming, biodegradability, biocompatibility, and antibacterial
properties [35–39], is well-known, CaCO₃/CS and CaCO₃-AZ/CS hybrid
organic/inorganic chitosan-based films were investigated to develop
UV-protective films for medicine and cosmetics, especially given
CaCO₃'s biocompatibility, biodegradability, and low cost. However, the
presence of hydrophilic side-chain groups (-OH) in CS makes it sensitive
to water and acids, leading to structural degradation and poor me-
chanical properties. As reported in the literature, hybrid organic/

inorganic chitosan-based composite films can exhibit enhanced me-
chanical properties thanks to the incorporation of an inorganic compo-
nent [40–44]. Therefore, to enable applications as UV-protective films,
we have elaborated chitosan-based composite films containing CaCO₃ or
CaCO₃-AZ particles (CaCO₃/CS and CaCO₃-AZ/CS), and their mechan-
ical and optical properties have been discussed in relation to the CaCO₃
and CS ratios.

This study demonstrates the potential to develop bio-inspired UV
protection materials that are more environmentally friendly and sus-
tainable than synthetic options derived from petrochemicals. This
approach can support the achievement of Sustainable Development
Goals (SDGs) 9 (Industry, Innovation and Infrastructure) and 12
(Responsible Consumption and Production) by promoting bio-based and
recyclable materials made from plants and marine waste. However,
significant efforts are required to improve the sustainability of raw
material extraction methods and reduce production costs to reach these
goals.

## 2. Materials and methods

2.1. Materials

All the materials were high-quality commercial products were pro-
vided by Sigma-Aldrich, except for α-poly-1-lysine with molecular
weight from 30 to 70 kDa (25104-18-1), which was furnished by BASF.
Acetic acid (CH3COOH) (64-19-7), sodium carbonate (Na2CO3) (497-19-
8), dehydrated calcium chloride (CaCl2) (10043-52-4), sodium chloride
(NaCl) (7647-14-5), potassium chloride (KCl) (7447-40-7), and alizarin
(AZ) (72-48-0) were purchased from Sigma-Aldrich. Sodium hydroxide
(NaOH) pellets (1310-73-2) and anhydrous magnesium sulfate (MgSO4)
powder (7487-88-9), were purchased from ABCR, along with 5-Hy-
droxy-1,4-naphthoquinone (juglone) (481-39-0), 5,8-Dihydroxy-1,4-
naphthoquinone (naphthazarin) (475-38-7), and 1,4-dihydroxyanthra-
quinone (81-64-1) dyes. Magnesium chloride hexahydrate
(MgCl2·6H2O) (7791-18-6) was purchased from Merck. Chitosan pow-
der (9012-76-4) (DDA ≈ 82 %, Mw = 50–150 kDa) was purchased from
GTC Bio.

2.2. Synthesis of hybrid pigment particles

Synthesis of CaCO₃-AZ particles was carried out using a conventional co-precipitation process, with Na₂CO₃ and CaCl₂ as precursors at room temperature, along with the addition of AZ powder to the Na₂CO₃ solution. The Na₂CO₃ to CaCl₂ ratio was 1:1, with a concentration of 5 × 10⁻² M. The concentration of AZ was 2 × 10⁻⁴ M. Solid precursors were first dissolved in MilliQ water by stirring at 500 rpm for 1 h at room temperature. AZ powder was added to the Na₂CO₃ solution during the last 5 min of stirring to ensure complete dissolution. The solutions were then mixed together under magnetic stirring for an additional 30 min (500 rpm). Subsequently, the particles were washed with MilliQ water via vacuum filtration until a clear filtrate was obtained. The precipitate was then oven dried for 12 h at 50 °C. Fig. 1 illustrates the procedure.

The same co-precipitation process was used to produce the CaCO3-AZ-biomacromolecule hybrid pigments. CS powder was solubilized in 2 wt% acetic acid (2 g of acetic acid in 98 g of MilliQ water) to prepare a 2 wt% solution. For PLL, the biomacromolecule was directly dissolved in MilliQ water to obtain a 2 wt% solution. The solutions were stirred at room temperature for 48 h until complete dissolution. To simulate the composition of natural organisms like sea urchins, the biomacromolecules were initially added at a concentration of 1 wt%. However, to investigate their effect on the precipitation of CaCO3, proportions of 5 and 10 wt% were also tested. The results (illustrations and discussions) for these systems are provided in the Supporting information. Thus, the appropriate amount of the biomacromolecule solution was added to the CaCl2 precursor solution to achieve final CaCO3: biomacromolecule or (CaCO3-AZ):biomacromolecule ratios of 99:1,
<figure><img src="image_4.png" /><figcaption>Fig. 1. Schematic illustration of the hybrid CaCO₃-AZ particles co-precipitation process from Na₂CO₃ and CaCl₂ precursors. (Inspired from [34]).</figcaption></figure>

95:5, and 90:10 wt%. After stirring at 500 rpm for 15 min, the solutions were mixed together under magnetic stirring for an additional 30 min (500 rpm). The particles were then washed with MilliQ water via vacuum filtration and oven dried for 12 h at 50 °C as previously described.

For comparison, pure CaCO₃ powder was prepared following the same procedure.

## 2.3. Hybrid pigments characterization methods

### 2.3.1. Fourier-transform infrared (FTIR) spectroscopy

Qualitative analysis of the hybrid pigment particles was performed using FTIR with a Thermo Fisher Nicolet IS50 spectrometer in Attenuated Total Reflection (ATR) mode, at a resolution of 4 cm⁻¹, within the range of 400–4000 cm⁻¹; 64 scans were accumulated.

### 2.3.2. Powder X-ray diffraction (XRD)

The crystal phases of the hybrid dyes were analyzed using a Bruker D2 Phaser powder diffractometer equipped with a Cu Kα radiation source. XRD patterns were recorded over a range of 2θ° to 50° 2θ with a 0.02° step and a 0.2 s counting time per step. The identification of the crystallized phases was done using the DIFFRAC.EVA software.

### 2.3.3. Scanning electron microscopy

The morphology of the CaCO₃, CaCO₃-AZ, and CaCO₃-AZ-biomacromolecule particles was characterized by SEM using a Hirox SH-3000 microscope with an accelerating voltage of 25 kV. All samples were sputtered with gold (30 mA, 90 s) prior to analysis.

### 2.3.4. Thermogravimetric analysis (TGA)

TGA was carried out using a Mettler Toledo TGA 2 Star® System apparatus to determine the amount of biomacromolecule involved in the co-precipitation process, within a temperature range of 30–850 °C at a scan rate of 10 °C/min in air.

### 2.3.5. Liquid UV-visible spectroscopy

Liquid UV-visible spectroscopy was used to determine the concentration of AZ retained in the hybrid pigment. UV-visible absorbance spectra were recorded using a Shimadzu UV-21021PC spectrometer in the range of 300–800 nm, at a resolution of 0.5 nm, using a 1 cm path length quartz cuvette.

### 2.3.6. pH sensing of the hybrid pigments

The pH-sensing ability of the hybrid pigments was evaluated by immersing a fraction of powder in basic solutions at a concentration of 5 mg/ml (buffer pH 8, NaOH pH 10.6, and NaOH pH 13.4). After 1 and 15 days, the appearance of the particles and solutions was compared with free alizarin in solution at different pH levels. The pH of the solutions was measured using a pH meter.

### 2.3.7. Photo-stability tests and characterization

Photo-stability tests were performed on the hybrid pigments (CaCO₃-AZ and CaCO₃-AZ-biomacromolecule) to assess the protective effect of the CaCO₃ matrix and the biomacromolecule. Tests were conducted both in solution and on powders, in comparison with pure alizarin. In solutions, 5 mg of powder was added to either 20 ml of artificial seawater or a NaOH solution (pH 12). The artificial seawater was prepared according to the Roscoff station protocol (Table S1). For powder experiments, the samples were placed into glass tubes and compared with alizarin precipitated at pH 12 (NaOH solution). All samples were placed in a solar simulator, the Atlas Suntest XLS+ from Ametek, equipped with a Xenon lamp, and irradiated for 10 h at 65 W/m² (2340 kJ/m²). After the tests, the hybrid pigments were washed with MilliQ water by vacuum filtration and oven-dried for 12 h at 50 °C. The photo-fastness of the hybrid pigments was evaluated by diffuse reflectance measurements using a PerkinElmer 860 spectrophotometer equipped with a 15 cm diameter integrating sphere and a sample holder in the bottom horizontal position. Spectra of powder samples, both before and after irradiation, were recorded at room temperature in 1 nm steps, within the range of 250–800 nm, with a bandwidth of 2 nm. The instrument was calibrated with a certified Spectralon white standard (Labsphere, North Sutton, USA).

## 2.4. Preparation of chitosan-based composite films incorporating CaCO₃ or CaCO₃-AZ particles

Chitosan-based films incorporating CaCO₃ or CaCO₃-AZ particles were prepared by simple deposition on a substrate and dried at room temperature. A 4 wt% chitosan solution in acetic acid was first prepared to achieve a viscosity sufficient to prevent the sedimentation of the CaCO₃ or CaCO₃-AZ particles. The particles were then added to the chitosan solution, and the mixture was subjected to ultrasonication for 5 min to prevent aggregation of the CaCO₃ particles, followed by magnetic stirring for 15 min at 500 rpm until homogenized. Five different CS: CaCO₃ ratios, ranging from 50:50 to 90:10, were elaborated to study the influence of the particles on the film properties. The mixtures were deposited onto polycarbonate plates and placed under fume hood to dry out for 24 h. After drying, the films were stored in petri boxes.

## 2.5. Composite films characterization methods

### 2.5.1. Mechanical properties

Tensile strength (TS; MPa) and elongation at break (EAB; %) of the hybrid films were measured at room temperature using a universal materials tester, Alliance RT/50 (Material Testing Systems), equipped with pneumatic jaws. Measurements were conducted at an acquisition speed of 50 Hz and a test speed of 20 mm/min, on samples cut into rectangular shapes of 80 × 10 mm. The sample thickness was measured randomly at least 5 times using an electronic digital micrometer with a resolution of 0.0001 mm. TS and EAB were calculated using Eqs. (1) and (2), respectively. A minimum of 5 specimens were tested for each composition, accounting for the possible presence of aggregates or bubbles in the films. The results presented are the average and standard deviation of the different tests performed on the same composition.

$$TS(\text{MPa}) = F_{\text{max}}/S \qquad (1)$$

where Fmax is the maximum load, and S is the cross-sectional area of the film.

$$EAB(\%) = (L - L_0)/L_0 \times 100 \qquad (2)$$

where L is the length at which the film breaks, and L₀ is the initial length of the sample.

### 2.5.2. Stability in aqueous solutions and water uptake

The stability of the self-supported CaCO₃/CS and CaCO₃-AZ/CS
composite films was studied in two different aqueous solvents. MilliQ water (pH = 6.4) was compared with artificial seawater. The samples were immersed in the different solvents for 48 h to evaluate their stability.

Water uptake tests were conducted on dried films, with the initial weight (W1) measured after samples were dried overnight in an oven at 40 °C. The samples were then immersed in MilliQ water (test 1) and in artificial seawater (test 2) at room temperature for 1 h to reach equilibrium. Wet weight (W2) was measured after removing excess water from the swollen saturated films using filter paper. The samples were subsequently placed back into the solvent for 1 h to repeat the test. Water uptake (W) of the hybrids was calculated using the following equation (Eq. (3)):

$$W(\%) = \frac{w_2 - w_1}{w_1} \times 100\% \qquad (3)$$

The results are presented with standard deviation bars derived from 5 tests to account for potential imprecision in water removal.

### 2.5.3. Contact angle measurements

Contact angle measurements were conducted using the optical device GBX contact angle meter, DGD Fast/60, which enables the determination of the contact angle of a droplet with a surface. A drop of MilliQ water or artificial seawater was deposited on the surface of the composite films elaborated on glass microscope slides. Final values are the average of four replicates obtained at different spots.

### 2.5.4. Optical properties of the composite films

The UV-visible spectra of the self-supported films were recorded using a Shimadzu UV-21021PC spectrometer in transmittance mode within the range of 200–800 nm, at a resolution of 0.5 nm.

## 3. Results and discussion

### 3.1. Characterization of the hybrid pigments obtained by simple co-precipitation process

In this section, we compare three different types of hybrid pigments: calcium carbonate co-precipitated with alizarin (CaCO₃-AZ), calcium carbonate co-precipitated with alizarin and chitosan (CaCO₃-AZ-CS), and calcium carbonate co-precipitated with alizarin and α-poly-L-lysine (CaCO₃-AZ-PLL). As mentioned in the Materials and methods section, in the biological model of sea urchin spines, organic molecules constitute less than 2 wt% of the matter. For comparison, the following section focuses on compositions containing a maximum of 1 wt% biomacromolecule. However, to assess the influence of the biomacromolecules on the different particles, ratios of up to 10 wt% have been examined to support our observations and results. Particles without alizarin have also been synthesized as a reference. The results (illustrations and discussion) related to these different systems are presented in the Supporting information.

#### 3.1.1. Color analysis of the hybrid pigments and dye retention

AZ is a natural pH-sensitive dye that exhibits a broad range of colors,

<figure><img src="image_5.png" /><figcaption>Fig. 2. Photos of A) the sea urchin model, and B) CaCO₃-AZ particles.</figcaption></figure>

from orange at pH below 5 to dark bluish-purple pH above 13 (Fig. 2B). When incorporated into the hybrid pigments, a dark purple powder similar to the sea urchin model was obtained (Fig. 2A and B), regardless of the presence, nature, or quantity of biomacromolecules. This color reflects the basicity of the Na₂CO₃ precursor solution in which AZ was incorporated (pH = 11.4). Table S2 provides details on the pH of the different precursor solutions, and the pH after 30 min of reaction.

After precipitation, the filtered solutions of each precipitate appeared slightly colored, indicating that a small amount of AZ was not retained in the co-precipitate (Fig. 2D). However, the difference in color intensity between the precursor solution Na₂CO₃-AZ (Fig. S1C) and the filtered solution post-reaction suggests that the majority of the dye was trapped in the calcium carbonate during co-precipitation. The orange/red color observed for CaCO₃-AZ aligns with the solution's pH. When co-precipitated with 1 wt% of biomacromolecule, the filtered solutions showed a faint orange/red tint with a purple hue. This purple shade does not match AZ's color at pH below 9, indicating that, in the presence of the biomacromolecule, there is a competition for the protonation of chitosan and alizarin, favoring chitosan and keeping AZ in its basic form.

After filtration, the precipitates were washed through successive steps using 50 ml of MilliQ water until a clear washing solution was obtained, to evaluate the release of alizarin from the hybrid pigments. 100 ml of MilliQ water were sufficient to wash the CaCO₃-AZ precipitate (5.0045 g theoretical mass), while the washing solutions remained slightly pink after 300 ml for the other precipitates. However, washing was ceased at this stage due to low UV absorbance (close to the absorbance threshold) associated with solutions containing very low AZ concentrations (see photos of the last 50 ml of the solutions in Fig. S2D, and spectra of the entire washing solution in Fig. S2C). The very low absorbance signal intensity compared to the UV-spectra of free AZ in solution at 2.10⁻⁴ M (Fig. S2A) confirms that this co-precipitation method is suitable for synthesizing hybrid pigments of calcium carbonate trapping alizarin.

#### 3.1.2. Structural analysis of the hybrid pigments

It is known that CaCO₃ has three crystalline polymorphs, each with distinct forms: calcite, aragonite, and vaterite. While calcite is the most stable phase, the different polymorphs can be obtained by adjusting the synthesis procedure and parameters, such as pH, concentration, and reaction time [34,45,46]. Infrared spectroscopy is a powerful tool for identifying these polymorphs, as each phase exhibits characteristic absorption bands in its infrared spectrum [47–51]. The fundamental vibrational modes of carbonate ions (CO₂⁻) can be assigned to the following regions: 1100–1000 cm⁻¹ (ν₁ symmetric stretching of aragonite, and vaterite), 900–800 cm⁻¹ (ν₂ out-of-plane bending of calcite, aragonite and vaterite), 1500–1400 cm⁻¹ (ν₃ in-plane bending and symmetric out-of-plane bending of calcite, aragonite, and vaterite), and 750–700 cm⁻¹ (ν₄ in-plane bending of calcite, vaterite and aragonite). ATR-FTIR spectra of alizarin, CaCO₃, and CaCO₃-AZ-biomacromolecule (chitosan or α-poly-L-lysine at 1 wt%) are presented in Fig. 3A. The presence of the aforementioned specific regions of the carbonate ion confirms the formation of CaCO₃ in all cases, indicating that the introduction of alizarin and biomacromolecules into the reactive media does not prevent CaCO₃ formation. For the CaCO₃ precipitate (Fig. 3A, black line), ν₄, ν₂ and ν₃ absorption peaks of calcite are observed at 711 cm⁻¹, 847 cm⁻¹, and 870 cm⁻¹, and at 1389 cm⁻¹, respectively. In the presence of alizarin and biomacromolecules, the band characteristic of the ν₃ vibrational mode appears shifted from 1389 cm⁻¹ to 1393 cm⁻¹, and broadened to around 1530 cm⁻¹ (grey area in Fig. 3A). This broadened band was assigned to a mixture of different polymorphs. Moreover, in the presence of biomacromolecules, new band around 745 cm⁻¹, was also assigned to the ν₄ mode and symmetric vibrations of vaterite [49]. The AZ spectrum is also presented in Fig. 3A for comparison of its characteristic peak positions with those of the CaCO₃-AZ and CaCO₃-AZ-biomacromolecule precipitates. Although the peak intensities cannot be directly compared, the superposition of the spectra confirmed that the
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** A) ATR-FTIR spectra of (a) CaCO₃, (b) CaCO₃-AZ, (c) CaCO₃-AZ-CS (1 wt%), (d) CaCO₃-AZ-PLL (1 wt%), and (e) AZ. B) XRD patterns of (a) CaCO₃, (b) CaCO₃-AZ, (c) CaCO₃-AZ-CS (1 wt%), and (d) CaCO₃-AZ-PLL (1 wt%). (v: vaterite phase, c: calcite phase). C) Quantitative analysis of the crystal structure and phase of CaCO₃-AZ-CS, and D) CaCO₃-AZ-PLL hybrid pigment.</figcaption></figure>

peaks identified in the precipitates are due to CaCO₃ calcite and vaterite polymorphs rather than AZ.

Similar results were observed for CaCO₃ precipitated in the presence of chitosan without the addition of alizarin. However, with PLL, only the characteristic peaks of calcite were observed (Fig. S3). This indicates that the presence of alizarin and/or chitosan in the reaction media is responsible for the formation of the vaterite polymorph, while PLL does not affect the formation of calcite under these synthetic conditions. The stabilization of vaterite by organic molecules has been documented in the literature [52], particularly by molecules containing a wide variety of functional groups such as C=O, HO-, N-H, and C-N. The nucleation and growth of calcium carbonate particles can be affected by electrostatic interactions between the calcium ion Ca²⁺ and these functional groups. In the presence of AZ, hydroxyl (-OH) and carbonyl groups (-C=O) can chelate metallic cations, Ca²⁺, to form a complex [53], slowing down CaCO₃'s nucleation and growth. AZ can also be adsorbed onto CaCO₃'s surface by H-bonding and direct Ca—O coordination [54],

stabilizing the vaterite polymorph. Due to the free electronic doublet of nitrogen, depending on the pH conditions, molecules possessing amine groups can also easily coordinate with metal ions such as Ca²⁺. In our conditions, the pH value of co-precipitation with chitosan ranged from 7.8 to 8.7, depending on the amount of chitosan (Table S2). At this pH level, the amine function of chitosan was not protonated, allowing the free electronic doublet of nitrogen to interact with Ca²⁺ (Whang et al. determined the pKa of chitosan around 6.5 [55]). In the case of PLL, the pH of the co-precipitation reaction was about 9.1, and the pKa of the amino side chains of PLL is around 9.0. Therefore, PLL could be partially positively charged under these conditions due to the protonation of primary amino groups, which decreases its reactivity with Ca²⁺.

FTIR results were further confirmed through XRD analysis (Fig. 3B). The CaCO₃ pattern showed characteristic peaks of pure calcite (marked “C”), while peaks of calcite and vaterite phases (marked “V”) appeared in all other precipitates, confirming the coexistence of both phases. Polymorphic composition calculation from the XRD patterns also indicates that the proportion of vaterite polymorph is higher when CaCO₃-AZ is precipitated alone or with chitosan (4.5 % vaterite) than with PLL (2 % vaterite) (Fig. 3C). Moreover, in the presence of PLL and AZ, a lower amount of vaterite was detected compared to CaCO₃-AZ alone. This may be due to a lower amount of AZ retained in the precipitate in the presence of PLL compared to CaCO₃-AZ; however, this hypothesis should be confirmed by a specific study on AZ and macromolecules specific interactions, responsible for AZ release from the precipitates (Fig. S2).

Thus, the presence of biomacromolecules in small amounts (up to 1 wt%) does not affect the calcite formation. These findings align with the biological model where less than 2 wt% of organic molecules participate in the mineralization process, minimizing the amount of material while promoting the most stable CaCO₃ phase.

In SEM images, calcite was clearly observed in the pure CaCO₂ sample (Fig. 4A), exhibiting a rhombohedral form and an average size of 5 µm. For CaCO₃-AZ (Fig. 4B) and CaCO₃-AZ-biomacromolecule precipitates (Fig. 4C and D), both irregular rhombohedral calcite and spherical and irregular ellipsoidal vaterite particles were observed [34,45,47,52], confirming the results of FTIR and XRD measurements. Particle sizes were about 3 to 8 µm for CaCO₃-AZ precipitates, while larger particles between 4 and up to 13 µm in diameter were observed in the presence of biomacromolecules. The increase in particle size can be explained by the interaction between AZ and macromolecules with Ca²⁺ or CaCO₃ during the co-precipitation process, which slows the nucleation step, favoring growth [52]. With PLL (Fig. 4D), a bigger population of irregular rhombohedral calcite was observed compared to CaCO₃-AZ and CaCO₃-AZ-CS precipitates, confirming that PLL does not help vaterite stabilization.

Moreover, the comparison with alizarin raw powder, which is composed of fragmented or agglomerated needles (Fig. 4), does not indicate any recrystallization of the dye on the surface of the hybrid pigments.

TGA analysis was performed to determine the amount of biomacromolecule co-precipitated with the particles. Residues at 600 °C were selected for the calculation, considering the approximation that alizarin and biomacromolecules were almost completely degraded at this temperature, while the main process of CO₂ removal from CaCO₃ powder had not yet started (TGA analysis of the raw materials alizarin, chitosan, and α-poly-L-lysine, and synthesized calcium carbonate are shown in Fig. S8A).

With 1 wt% of biomacromolecule, the TGA curves of CaCO₃-AZ-CS and CaCO₃-AZ-PLL (Fig. 5) appear to show a slightly higher amount of CS degraded than PLL. With PLL, the TGA curve overlaps with that of CaCO₃-AZ particles, indicating that the interaction between this biomacromolecule and CaCO₃ is weaker than that of CS with CaCO₃. These results have been confirmed using high amounts of biomacromolecule in the co-precipitation (Fig. S8).

Details of the values concerning the theoretical amount of organic
<figure><img src="image_7.png" /><figcaption>Fig. 4. SEM images of A) rhombohedral calcite CaCO₃, and irregular rhombohedral calcite, plus spherical and irregular ellipsoidal vaterite particles of B) CaCO₃-AZ, C) CaCO₃-AZ-CS (1 wt%), and D) CaCO₃-AZ-PLL (1 wt%).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. TGA analysis of CaCO₃ co-precipitated with alizarin and chitosan or α-poly-L-lysine at 1 wt%.</figcaption></figure>

<caption>**Table 1**</caption>

Residue at 600 °C and calculated wt% of organic matter retained in the precipitates as a function of theoretical wt% organic matter added in the synthesis.

<table><thead><tr><th></th><th>Wt% biomacromolecule (theory)</th><th>Wt% organic (experimental)</th><th>Residue @ 600 °C</th><th>Wt% organic matter retained</th></tr></thead><tbody><tr><td>CaCO<sub>3</sub>-AZ</td><td>0</td><td>1.9</td><td>96.6</td><td>3.4</td></tr><tr><td>CaCO<sub>3</sub>-AZ-CS</td><td>1</td><td>2.9</td><td>95.9</td><td>4.1</td></tr><tr><td>CaCO<sub>3</sub>-AZ-PLL</td><td>1</td><td>2.9</td><td>97.4</td><td>2.6</td></tr></tbody></table>

matter incorporated and residues at 600 °C are provided in Table 1.

The overestimation of the wt% of remaining organic matter is assumed, considering the approximations made regarding the level of degradation of the different materials at 600 °C, as well as the contribution of water molecules in the calculation. Nevertheless, these results are consistent with the FTIR, XRD, and SEM analyses, indicating that PLL does not seem to interact with calcium carbonate under these synthetic conditions, while CS co-precipitates into the particles.

For comparison, the calculated wt% of organic matter retained in the precipitates for the hybrid pigment containing 5 and 10 wt% of biomacromolecule is plotted in Fig. S9.

### 3.1.3. pH sensing of alizarin in the hybrid pigments

In light of the previous results, and to discuss the impact of the biomacromolecule on the stability of the hybrid pigments, the following analyses focus on CaCO₃-AZ and CaCO₃-AZ-CS 1 wt%.

The pH sensing of alizarin hybrid pigments was studied by immersing a fraction of hybrid CaCO₃ powder in basic solutions at pH 8.0, pH 10.6, and pH 13.4 (Fig. S11). After 1 day in solution, the solution color at pH 8.0 suggests partial AZ solubilization, probably due to partial precipitate dissolution. At pH levels of 10.6 and 13.4, the particles seem unaffected by the solution, and no change in the hybrid pigment color was observed. However, at pH levels higher than 13, a blue color of alizarin was expected (Fig. S1B). This may indicate that alizarin is protected within a CaCO₃ shell and is not accessible or sensitive to the solution unlike free AZ. The purple color of the precipitate also suggests that AZ is embedded in a basic environment inside the CaCO₃ shell.

After 15 days in solution, no differences in color or solution were observed, indicating good stability of the particles. Therefore, these hybrid pigments could be used in areas such as clothing, painting, and others that need color stability rather than pH-sensing. These results are in good agreement with the literature, which reports that urchin spines' color is not directly affected by water acidification, which impacts the skeleton chemistry [53,54], whereas pigments, when extracted, are pH-
sensitive [56]. Hybrid pigment photo-stability.

Alizarin's lightfastness is known to depend on its molecular structure [27]. Jiang and coworkers studied the influence of pH on AZ photo-stability in solution, showing that the phenolic hydroxyl group affected by the pH value has a significant impact on the dye's photostability. They demonstrated that a higher pH of AZ solution is associated with poorer photostability [57]. Before studying the photostability of our hybrid pigment, we analyzed the behavior of alizarin in a NaOH solution at pH 12, exposed to solar simulator light for 10 h. At this pH, the color of free AZ in solution matches that of AZ incorporated in the hybrid dye, ensuring that free AZ remains in the same ionization state as in the hybrid dyes. After exposure, the solution's initial purple color faded completely (about 97% fading) and turned light orange. This result was consistent with the literature [57], validating our test protocol (Fig. S12).

Fig. 6A compares the reflectance spectra of the samples (expressed in F(R), the Kubelka-Munk remission function) before and after the

irradiation of the dried particles. Only the peak positions are considered, as the signal intensity can be influenced by the grain size and sample preparation. AZ spectra precipitated at pH 12 (red lines in Fig. 6A) show a shift of the main peak centered around 580 nm to 640 nm. This shift was not observed when AZ was co-precipitated with CaCO₃ or CaCO₃-CS, demonstrating better light fastness. In any case, the precipitates appeared purple both before and after irradiation (Fig. S13), and the fading observed for alizarin in solution was not seen in dry conditions.

The results from the tests repeated in solutions are shown in Fig. 6B and C for CaCO₃-AZ, and CaCO₃-AZ-CS (1 wt%), respectively. Regardless of the sample, the F(R) curves are similar, and the peak positions after irradiation resemble those of the reference sample before testing. Nevertheless, in artificial seawater, the irradiated samples exhibited the appearance of a new peak around 690 nm. All precipitates appeared purple before and after irradiation (Fig. S13), and the fading observed for AZ in solution was not noted, indicating that the dye was not as sensitive as free alizarin when incorporated in the CaCO₃ hybrid pigment, regardless of the presence or absence of biomacromolecule in the particles. Therefore, the incorporation of up to 1 wt% of CS does not affect the stability of the hybrid dye, as for the biological model.

The samples irradiated in artificial seawater appear slightly darker than the others, which may explain the appearance of a new peak around 690 nm (Fig. 6B and C). The chemical reason for this color change is currently under investigation. The effects of treating the particles by immersing them in solution, and their irradiation, were also studied using SEM and XRD analyses (Figs. S14 and S15, respectively). No modifications were noticed except for the CaCO₃-AZ sample. XRD analyses revealed that the amount of vaterite polymorph before treatment was around 5%, while only 1% was found in the sample after treatment in solution. Since this trend was not observed for the other samples, it indicates that vaterite polymorph is not stable in solution and recrystallizes in calcite form in the CaCO₃-AZ sample. It also showed that adding chitosan helped stabilize vaterite.

## 3.2. Chitosan-based composite films containing CaCO₃ or CaCO₃-AZ particles

In this part of our study, only CaCO₃ and CaCO₃-AZ particles were used to prepare chitosan-based composite films. The "/" mark was used to refer to the hybrid films (i.e. CaCO₃(–AZ)/CS).

Up to 50 wt% of particles, the films remain self-supported, easily handled and flexible.

### 3.2.1. Mechanical properties

Very few studies have examined the mechanical properties of chitosan-based films or hydrogels incorporating CaCO₃ particles [58–60]. Results indicate that CaCO₃ presence affects the mechanical properties of the hybrids, but in different ways depending on the load factor. For instance, Le Goué et al. [59] have shown that the tensile strength of their material increases with increasing CaCO₃ loading, while Zheng et al. [60] noticed an increase in Young's modulus and a decrease in tensile strength.

Herein, the mechanical properties of the CaCO₃/CS composite films were studied through Young's modulus (YM) (Fig. 7A), tensile strength (TS) (Fig. 7B), and elongation at break (EAB) (Fig. 7C), vs. CaCO₃ loading. Films of pure chitosan had a Young's modulus of around 1450 MPa (Fig. 7A), a tensile strength of about 46 MPa (Fig. 7B), and an elongation at break of 0.35% (Fig. 7C). Generally, the incorporation of CaCO₃ particles from 10 to 50 wt% showed an impact on the mechanical properties of the films by increasing the Young's modulus while decreasing the tensile strength and the elongation at break. YM increased from 23% to 74% with the incorporation of CaCO₃ at ratios of 20 wt% and 50 wt%, respectively. In the case of tensile strength, a decrease of 4% to 31% was observed with the incorporation of 10 to 50 wt% of CaCO₃ particles (TS = 45 MPa and 32 MPa, respectively). Finally, EAB decreased from 1% to 86% with the incorporation of 10 to

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Fig. 6. F(R) spectra of A) AZ precipitated at pH 12 and hybrid pigments before and after 10 h of irradiation in a solar simulator: (a) AZ pH 12, (b) AZ pH12, Irr, (c) CaCO₃-AZ, (d) CaCO₃-AZ, Irr, (e) CaCO₃-AZ-CS (1%), (f) CaCO₃-AZ-CS (1) %, Irr, B) CaCO₃-AZ hybrid pigment irradiated in solution, C) CaCO₃-AZ-CS (1 %) hybrid pigment irradiated in solution.</figcaption></figure>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>Fig. 7. A) Young Modulus, B) Tensile strength and C) Elongation at break of CaCO₃/CS composite films with 0, 10, 20, 30, 40 and 50 wt% of CaCO₃ particles.</figcaption></figure>

50 wt% of CaCO₃ particles (EAB = 0.324 and 0.05 %, respectively). Thus, the incorporation of CaCO₃ particles stiffens the composite films, making them increasingly brittle, as no interaction between CaCO₃ and chitosan is expected. Therefore, regarding the targeted applications, the amount of particles to be incorporated into the hybrid films will need to

<figure><img src="image_15.png" /><figcaption>Fig. 8. (A) Water uptake and (B) Contact angle measurements of CaCO₃/CS composite films containing 0, 10, 20, 30, 40 and 50 wt% of CaCO₃, using MilliQ water and artificial seawater.</figcaption></figure>

be adjusted to achieve the corresponding strength-to-flexibility balance.

### 3.2.2. Stability in aqueous solutions and water uptake

The stability and water uptake tests involved immersing the films in MilliQ (pH 6.4) and artificial seawater (pH 8.2). These two different solvents were selected to evaluate the effect of pH on the solubility of the films, without being too acidic to avoid a rapid dissolution of chitosan.

Fig. 8A presents the average of seven measurements taken at 1-hour intervals. In MilliQ water, chitosan films containing less than 30 wt% CaCO₃ were partially dissolved before the first measurement, while all other films remained stable and manipulable until the end of the test. As described in the literature [61,62], amino groups of chitosan were probably protonated in acidic conditions, leading to the formation of positively charged groups -NH³⁺. Electrostatic repulsion between these groups may cause the network to expand until the films dissolve. With 30 wt% CaCO₃ and higher, the presence of the inorganic particles helped to prevent the dissolution of the films in acidic conditions. The results showed that increasing the amount of CaCO₃ in the films decreased the water uptake (W) from 156 % with 30 wt% CaCO₃ to 43 % with 50 wt% CaCO₃.

In artificial seawater, all the films remained stable until the end of the test, confirming the hypothesis that acidic media on chitosan. A water uptake of about 155 % was measured for pure chitosan films. Then, an increase in water uptake was observed with increasing CaCO₃ content up to 20 wt% (W = 179 %). This phenomenon could be due to the adsorption of water or salts at the surface of the CaCO₃ particles, which helps to retain water in the film. With more than 20 wt% CaCO₃ in the film, the water uptake decreased with the addition of CaCO₃, i.e., decreasing chitosan.

### 3.2.3. Contact angle

Contact angle (CA) measurements (Fig. 8B) were performed using MilliQ water and artificial seawater to minimize the influence of water absorption. In MilliQ water, pure CS films displayed the highest contact angle among all the films. The incorporation of CaCO₃ particles resulted in a general decrease in CA. This can be attributed to the high hydrophilicity of CaCO₃, which enhances the film's wettability [63]. These results also indicate that CaCO₃ particles are located at or near the surface of the films. With the incorporation of CaCO₃, the contact angles remained almost constant (~61–64°) from 10 wt% to 40 wt% CaCO₃. Compared to pure CS films, CA was lower by approximately 25 % in these cases. With 50 wt% CaCO₃, the decrease in CA was less pronounced, measuring 68° instead of 76° for pure chitosan films.

The same trend was observed with artificial seawater. The contact angle was highest for pure CS films (79°). Subsequently, the films showed about a 25 % improvement in wettability with the incorporation of 10 wt% to 20 wt% CaCO₃. For 30 wt% CaCO₃ and higher, contact angles of 73° to 70° were obtained. The variability in the results could be due to inhomogeneities in the distribution of CaCO₃ particles within the film and the presence of domains that are more or less rich in particles or

<figure><img src="image_16.png" /></figure>
in chitosan.

For all compositions, the contact angle was higher with artificial seawater than with MilliQ water. This tendency can be explained by the presence of salts in the artificial seawater, which induced electrostatic repulsions with chitosan and CaCO₃.

### 3.2.4. Optical properties of the films

The optical properties of CaCO₃/CS composite films were studied using UV-visible spectroscopy in transmittance mode. Fig. 9 compares pure CS film and chitosan-based films incorporating 30 wt% CaCO₃ or CaCO₃-AZ. Spectra of chitosan-based films incorporating 10 and 50 wt% CaCO₃(-AZ) are presented in Fig. S16.

Pure CS films exhibit good transparency to visible light up to 600 nm (Fig. 9). Beyond this point, the transmittance slightly decreases until 450 nm, and the films become almost opaque to UV light at wavelengths of 300 nm and below (T% ≤ 5%). A detailed comparison between the spectra was not possible due to differences in the films' thicknesses (Table S5). Nevertheless, the pure CS film was thicker than the others. Consequently, these results provide evidence of the influence of adding CaCO₃ or CaCO₃-AZ particles to the films' compositions on their transmittance properties. Films containing CaCO₃ particles had poor transparency to visible and UV light across all measured wavelength ranges.

## 4. Conclusion

This study demonstrates the value of a biomimetic approach to enhance the photostability of natural dyes, a key factor for their industrial use. Hybrid organic-inorganic pigments inspired by sea urchin spines were prepared using a simple co-precipitation method. This straightforward procedure shows good results in trapping AZ dye, used to replace the natural chromophores (spinochromes and echinochromes), in CaCO₃ particles, providing protection against photofading.

To evaluate the effect of biomacromolecules on the properties of the hybrid pigments, chitosan and α-poly-L-lysine were selected to mimic the functions of natural biomacromolecules involved in the biomimicization process of CaCO₃ in the spines. It was shown that chitosan interacts with CaCO₃ during co-precipitation reaction, influencing its morphology and phases by stabilizing the vaterite polymorph. Conversely, α-poly-L-lysine does not exhibit any interaction with CaCO₃. Regardless of the type and proportion of biomacromolecule used in the synthesis, we achieved good results in mimicking the intense color of urchin spines by using alizarin dye. The resulting hybrid pigments showed high photostability under UV light compared to free alizarin molecules. Moreover, it was demonstrated that the presence of CS also stabilized vaterite during irradiation, whereas this polymorph tends to evolve into calcite in its absence, confirming the interest of using this biomimetic approach. A detailed chromatic analysis and longer stability tests are considered to complete this preliminary study.

Thanks to the hybrid pigments ability to absorb and resist UV light, UV-protective films were then developed using chitosan. We demonstrated that self-supported, cohesive films could be obtained by varying the amount of CaCO₃ from 0 to 50 wt%, allowing for designing their mechanical and optical properties to meet the intended application. However, since chitosan is soluble in acidic media, a minimum of 30 wt % of CaCO₃ particles was necessary to maintain the film's cohesiveness in MilliQ water. Further physical tests, such as dye release quantification and oxygen permeability, are now planned to complete this study regarding a specific application. Furthermore, other types of bio-sourced quinones, specifically anthra- and naphthoquinone dyes with single or double hydroxyl groups have been envisaged to replace alizarin in the co-precipitation process. Results have shown a dependence on the number of hydroxyl groups in the dye. Indeed, regardless of their position, it is notable that the ability to be trapped in co-precipitated CaCO₃ is higher for dyes with two hydroxyl groups (Fig. S17). This preliminary study must be completed with a full characterization of the structure, morphology, and size of the CaCO₃ co-precipitated particles.

<figure><img src="image_17.png" /><figcaption>**Fig. 9.** UV-vis spectra of self-supported CaCO₃/CS and CaCO₃-AZ/CS composite films with 0 %, and 30 wt% CaCO₃, presented in transmittance mode. The insert in the figure represents a zoomed view of the UV-vis graph in the wavelength range of 300-200 nm.</figcaption></figure>

## CRediT authorship contribution statement

**Sandra Castanié:** Writing – original draft, Validation, Methodology, Data curation. **Sylvie Blanc:** Validation, Methodology. **Mickael Le Béchec:** Validation, Methodology. **Laurent Billon:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Formal analysis, Conceptualization.

## Declaration of competing interest

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

## Acknowledgment

The authors thank E2S UPPA and Rectorat Nouvelle Aquitaine for the financial support of the “DSG2 Biomimétisme” project, and the S.C. fellowship especially.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2025.149277.

## Data availability

Data will be made available on request.

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