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# Easy synthesis of gold nano-sea-urchins, surfactant-free and seedless, using chitosan as stabilizer

To cite this article: R A Guerra-Santopietro et al 2025 *Mater. Res. Express* **12** 085005

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# Easy synthesis of gold nano-sea-urchins, surfactant-free and seedless, using chitosan as stabilizer

R A Guerra-Santopietro¹,†, S E López-Alvarez²,†, J M Blancas-Flores¹,†, L M Bravo-Anaya³,† and V H Romero-Arellano¹,*‡

¹ Centro Universitario de Tonalá, Universidad de Guadalajara, Tonalá Jalisco, Mexico

² Centro de Investigación en Biomateriales, Departamento de Madera, Celulosa y Papel, Universidad de Guadalajara, Zapopan, Jalisco, Mexico

³ Université de Rennes, CNRS, ISCR (Institut des Science Chimiques de Rennes), UMR 6226, F-35000 Rennes, France

* Author to whom any correspondence should be addressed.

E-mail: hugo.romero@academicos.udg.mx

Keywords: gold nano-sea-urchins, chitosan as stabilizer, one-pot synthesis, growing process

Supplementary material for this article is available online

## Abstract

Gold nano-sea-urchins were synthesized using chitosan as a stabilizer. The growth mechanism occurs by reducing the Au salt (HAuCl₄) followed by an anisotropic nucleation process promoted by the presence of Ag⁺ ions that generate the growth of irregularly shaped nanoparticles (Nps) with an average size of 16 ± 4 nm. In turn, these structures form aggregates to finally give rise to the formation of Au nano-sea-urchins, which are capped superficially by a chitosan layer. The size and aggregation of these structures were studied as a function of pH and chitosan molecular weight. Chitosan acts as a stabilizer, thus avoiding the need of surfactants with a higher degree of cytotoxicity. The obtained Au nano-sea-urchins were characterized by different techniques, including electronic microscopy, dynamic light scattering, FTIR and UV-vis spectroscopy. The results revealed Au nano-sea-urchins nanostructures presenting an average diameter from 59 to 83 nm, formed by aggregates of smaller anisotropic particles (average size 16 ± 4 nm). A redshift of the plasmon was obtained as a function of size, shape and aggregate formation, obtaining favourable results for the sample in which chitosan was used with the conditions of pH 5.5, MMW, and DA 0.22, characteristics that make them viable for biomedical applications.

## 1. Introduction

Metallic nanoparticles have received considerable attention because of their outstanding properties to induce collective oscillation of conduction electrons excited by the electromagnetic field of light, a phenomenon known as localized surface plasmon resonance (LSPR). This effect is highly dependent on the nanoparticle's shape and size [1–3]. In particular, gold nanostructures have been widely studied, and there are currently numerous techniques enabling the production of nearly any desired shape and size, including spheres, triangles, cubes, polyhedrals, rods [2–6]. In particular, gold particles with sharp edges, such as star-shaped [7–9], branched [10], multi-pod [11], and urchin-like particles [12], have attracted considerable attention due to their properties as amplifiers of the electric field [2, 13, 14]. These enhanced, properties render them highly suitable for applications in photonics, electronics and biomedicine [15–18].

Seed-mediated synthesis is a widely used approach for controlling the size and shape of gold nanoparticles, including nanorods, bipyramids, and nanostars [19–21]. This method typically begins with the reduction of HAuCl₄, commonly using citrate, to produce small Au nanoparticles that are subsequently used as seeds during the growth step. Later, during the growth stage, other agents take part to favor anisotropic growth and yield the final morphology. For example, the use of AgNO₃ to introduce silver ions owing to the under-potential deposition on certain crystal facets of Au-seed [22], as well as the same citrate or ascorbic acid as a reducing
agent, play an important role in the growth of anisotropic structures [21, 22]. Similarly, surfactants like cetyltrimethylammonium bromide (CTAB) or zwitterionic lauryl sulfobetaine (LSB) have also been employed as 'shape-inducing' agents [23, 24]. In addition to playing an important role in the growth process, CTAB has also been used as a popular coating agent to stabilize nanoparticles and avoid aggregates; however, its application in biomedical contexts is restricted due to its known cytotoxicity [25, 26]. To overcome this limitation, recent efforts have focused on using biopolymers, particularly chitosan, as alternative stabilizing agents. Chitosan offers several advantages, including low cytotoxicity, excellent biocompatibility, and ease of handling, making it an attractive candidate for the synthesis of nanoparticles for biomedical applications [27–29].

Chitosan has been reported to serve a dual-function as both a reducing agent for metal precursors and a stabilizing agent for the resulting nanoparticles [29–31]. In recent years, it has been reported that chitosan can be used in the synthesis of gold nanostructures with pointed branches. However, some of these methods rely on multiple steps processes, such as seed-mediated synthesis, which involves the initial preparation of small gold seed particles by reducing a precursor salt, typically trisodium citrate or sodium borohydride, sometimes requiring solutions at boiling points. These seeds are then used as nucleation centers in a subsequent growth step to produce the final nanostructures [31, 32]. To make this type of structure more attractive, the development of simpler, more efficient synthetic methods is essential. Currently, some reports have explored one-pot or simplified approaches; however, in many cases the growth mechanism are not thoroughly investigated or are explained using different hypotheses. These mechanisms are often highly sensitive to various synthesis parameters such as pH, chitosan concentration, and reducing agent concentration [33, 34]. Vy Phan [35] proposed that the process of formation of gold nanostars (AuNS) involves two stages. In the first stage, Au° nuclei are formed by the reduction of Au (III) ions with ascorbic acid. In the second stage, positively charged chitosan (CS) binds strongly to the nucleus. The negatively charged AuCl⁻ then binds to the positively charged CS, and the tips of the nanostructures grow at the loose contact points between the CS and nuclei. This results in anisotropic growth of the gold nanostructures. Huynh [36] explained that the formation of gold particles occurs through a reversible process at the equilibrium of oxidation and reduction. This process promotes some electrons that reduce Au³⁺ ions to Au⁺ ions which then attach to the previous gold seed formed and are protected by chitosan, resulting in anisotropic growth and the formation of multi-branched particles. Nhung [37] discussed the growth of flower-like structures of gold nanoparticles, influenced mainly by the concentration of chitosan molecules, forming agglomerations of primary gold nanoparticles owing to electrostatic interactions with the amino groups of chitosan. These agglomerations acted as seeds that promoted the growth of flower-shaped Au nanoparticles (AuNPs). All these reports have demonstrated the sensitive interaction between AuNPs and chitosan, since it is dependent on multiple factors, such as the concentration, pH, and characteristics of the biopolymer. Therefore, it is essential to enhance our understanding of the growth process of these structures.

This work presents a one-pot synthesis approach with seedless growth of gold nano-sea-urchins stabilized with chitosan. Our hypothesis regarding the growth mechanism is that it occurs via aggregation of small particles, leading to the formation of anisotropic structures. A similar mechanism has been discussed by other authors, as reported by Zhang [38], who obtained gold-silver nanoflowers owing to a cooperative effect between chitosan and Ag⁺ silver ions. In our report, the growth process of the nanostructures is discussed in terms of the role of pH, degree of acetylation (DA) and molecular weight (MW) of chitosan during synthesis, which are of high importance in biomedical applications [39, 40].

## 2. Introduction

### 2.1. Materials

Three chitosan samples were used to study the effect of DA and MW on Au nano-sea-urchins characteristics: two commercial samples supplied by the Sigma-Aldrich Company: medium molecular weight (MMW) chitosan (Ref: 448877) and high molecular weight (HMW) chitosan (Ref: 419419), and one low molecular weight (LMW) chitosan sample obtained from the University of Guadalajara. The complete characterization of chitosan samples from Sigma-Aldrich was previously reported by Bravo-Anaya et al [41]. The MMW chitosan sample has a viscosity-average molecular weight (Mv) of 86 750 $mol^{-1}$ and $DA = 0.22$, and the HMW chitosan sample has an $Mv = 417$ 600 $mol^{-1}$ and $DA = 0.16$. Third chitosan sample was fully characterized through $^1$H NMR, conductivity and viscosity measurements, allowing to determine its DA and MW, i.e. 0.33 and 38 484 $mol^{-1}$, respectively. The reagents used for the synthesis of Au nano-sea-urchins were silver nitrate ($AgNO_3$) (99%), L-ascorbic acid ($C_6H_8O_6$), and gold(III) chloride trihydrate ($HAuCl_4·3H_2O$) (99.9%). All reagents were purchased from Sigma-Aldrich. Distilled water was used for all experimental solutions.
## 2.2. Chitosan solutions preparation

Chitosan solutions were prepared at a final concentration of 3.33 ms ml⁻¹ by initially dissolving 0.05 g of chitosan in 3.75 ml of 0.1 N HCl. The volume of acid was calculated based on the stoichiometric amount required to protonate the amine groups (–NH₂) in chitosan, and a slight excess was added to ensure complete protonation. This step produces a highly viscous and fully protonated chitosan solution. To reduce the viscosity and facilitate handling, the solution was subsequently diluted with 11.25 ml. The mixture was stirred overnight at room temperature to ensure complete dissolution, resulting in a clear solution with a pH of approximately 3.5. The solution was stored at 4 °C. Chitosan samples used for the synthesis of Au nano-sea-urchins were prepared by diluting of the mother solution with water up to 1 mg ml⁻¹. The final pH after dissolution was 3.5. A set of chitosan solutions was also prepared by modifying the protonation degree, that is, the chitosan solutions were adjusted to a pH of 5.5 with a solution of NaOH (0.1 N). The degree of protonation of the chitosan at pH 5.5 was determined through potentiometric titration experiments. Specifically, fully protonated chitosan solution were tritrated by adding small increments of 0.1M NaOH while continuously monitoring the pH. From these titrations, we calculated the degree of protonation (DP) as the ratio of protonated amines groups to the total chitosan concentration using the following equation: $pKa = pH-log(1-DP/DP)$. Using this approach, we confirmed that at pH 5.5, chitosan exhibits approximately 60% protonation of its amino groups. (The potentiometric titration of chitosan samples can be found in the supplementary materials, figure S1). [41, 42].

## 2.3. Synthesis of gold nano-sea-urchins

Gold nano-sea-urchins were prepared using a modified synthesis procedure [43]. In a typical experiment, 250 µl of 0.004 M AgNO₃ and 5 ml of 0.1 × 10⁻³ M HAuCl₄ were added to 5 ml of chitosan solution (for each chitosan stock solution, either at a pH of 3.5 or 5.5). The solution acquires a pale yellow color and is stirred until homogenization of the mixture is achieved. Subsequently, Then, 800 µl of 0.0189 M ascorbic acid was added to the solution, leading to an immediate color change, from pale yellow to dark blue, and the solution was stirred for 6 h at room temperature. The colloidal suspensions were then washed twice, centrifuged at 6000 rpm for 5 min, and dispersed in distilled water to obtain a final pH of 5 for all samples.

## 2.4. Gold nano-sea-urchins nanoparticles characterization

Size measurements using dynamic light scattering (DLS). DLS measurements were performed using a Malvern Zetasizer NanoZS 90 instrument at a temperature of 25 °C and an angle of 90°. The hydrodynamic radius (RH) was calculated using the Stokes Einstein equation [44] proposed for spherical particles. Measurements for each sample were performed in duplicate.

Transmission electron microscopy (TEM) measurements. TEM images were obtained using a JEOL 1400 TEM operating at 120 kV and equipped with a GATAN Orius 1000 camera. Samples were prepared by the direct deposition of a gold nano-sea-urchin nanoparticle suspension droplet on copper grids to dry at room temperature. The particle size distribution was determined one-by-one from TEM micrographs using Image-J software. Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) studies of gold nano-sea-urchin nanoparticles were also performed using a JEOL 2100 instrument operating at 200 kV and equipped with a Gatan Orius 200D camera.

Scanning electron microscope (SEM) measurements. Scanning electron microscope (SEM) observations were performed using a JEOL JSM 7100F microscope. The copper grids used for TEM measurements were metallized with Au/Pd for 40 s using a Leica EM ACE 200 metallizer. Surface topography images were obtained using a secondary electron image detector. The particle size distribution was determined using the Image-J software. Scanning transmission electron microscopy (STEM) images were obtained using a JEOL JSM-7800F instrument.

UV-Visible measurements. UV-visible absorption spectra were recorded in the 350–800 nm range using an Agilent Technologies Cary Series UV-Visible-NIR spectrophotometer (Cary 5000).

Fourier-transform infrared spectroscopy (FTIR) measurements. Fourier transform infrared (FTIR) measurements were performed using an Alpha FT-IR spectrometer (Bruker). The samples were prepared by drying the colloidal suspension at room temperature and directly placing the obtained powder into the equipment.

# 3. Results and discussion

Gold nano-sea-urchins were synthesized via a simple one-pot, seedless method assisted by silver ions. The addition of silver nitrate was selected since it's a widely used strategy for preparing gold nanostars. Ag⁺ ions have been proposed as catalysts in the growth process, as shape-directing agents, and as anisotropic promoters [45]. Our results show that the morphology of gold nano-sea-urchins was obtained by the aggregation and
<figure><img src="image_9.png" /><figcaption>**Figure 1.** Schematic representation of Au nano-sea-urchins formation process. (a) HAuCl₄ and AgNO₃ were reduced by ascorbic acid in a chitosan solution, in an early reaction, small gold nanoparticles are formed that later form aggregates to give rise to the formation of Au nano-sea-urchins covered on the surface by a layer of chitosan; (b) Characteristic blue color of Au nano-sea-urchins colloid; (c) TEM micrographs, showing Au nano-sea-urchin structures, and small Au nanoparticles in the surroundings.</figcaption></figure>

coalescence of chitosan-coated Au-anisotropic nanoparticles. According to the growth scheme illustrated in figure 1, the synthesis began with the preparation of a mixture of chitosan solution (LMW, MMW, or HMW), gold (HAuCl₄), and silver (AgNO₃) precursors under constant stirring to ensure a homogeneous mixture. Owing to the protonated –NH₂ groups of chitosan at pH of 3.5 and 5.5 (i.e. –NH₃⁺), AuCl₄⁻ ions interact with the positively charged polysaccharide chains through electrostatic interactions [46]. The electron-rich nitrogen atoms in chitosan coordinate with the Au surface, helping to control particle growth and stabilize the urchin-like morphology. When ascorbic acid was added, both the gold and silver salts were reduced to Au° and Ag⁺ respectively, leading to the beginning of the anisotropic nucleation process that occurs due to the deposition of silver ions on the gold structures during the nucleation process. This implies the formation of active sites owing to the catalytic effect of the silver ions, thus favoring anisotropic growth [43]. This results in the formation of Au nanoparticles with irregular shape and an average size of 16 ± 4 nm. These asymmetric nanoparticles tend to aggregate and form Au nano-sea-urchin nanoparticles, stabilized by a chitosan layer that is preferentially bound by the Au° orbitals and the electron-rich nitrogen present in the –NH₂ groups of chitosan through the lone pair, as stated in the literature [47]. The reduction, asymmetric growth, and aggregation processes occur immediately in just a few seconds and are dependent on multiple parameters, such as DA and MW, of chitosan and pH of chitosan solution, giving rise to Au nano-sea-urchins with average sizes dependent on these parameters.

It can be observed in figure 1(c)) that some primitive nanoparticles remain dispersed, likely neutralized by chitosan prior to the aggregation and coalescence process. This effect may be attributed to the rapid nature of the reaction and growth process. Additionally, in the supplementary material (figure S3) shows some primitive nanoparticles covered by chitosan.

To confirm the aggregate formation and coalescence of asymmetrical Au nanoparticles, different electron microscopy techniques were used. Figure 2(a)) shows a typical TEM micrograph in which a small group of Au nano-sea-urchins is observed, as well as several irregularly shaped nanoparticles in the surroundings (indicated by arrows). An enlargement of the obtained nanostructures is shown in figures 2(b) to (f), in which it can be seen that the Au nano-sea-urchins are formed by the aggregates and coalescence of smaller asymmetric nanoparticles. This statement was verified by the STEM image shown in figure 2(g)), in which the contrast confirms that the nanostructures are the result of the aggregation of nanoparticles. Furthermore, the HRTEM micrographs in figures 2(h) and (i) indicate that the tips of the Au nano-sea-urchins originate from asymmetrical shaped nanoparticles located in the superficial edges of the nanostructure.

Owing to its biocompatibility, chitosan has stood out as a good candidate stabilizer in the synthesis of nanoparticles for biomedical applications [27–29]. In the HRTEM micrograph presented in figure 3(a)), it can be seen that this chitosan-stabilizing layer is approximately 4 nm thick. However, in the TEM and SEM images shown in figures 3(b) and (c), respectively, it was observed that chitosan tends to trap small groups of nanoparticles forming agglomerates of Au nano-sea-urchins. The size distributions of the nano-sea-urchins as a function of MW and pH of the chitosan solution are shown in figure 4. From the TEM micrographs, it was observed that the samples presented a shift in the average nanoparticle size, depending on the pH of the chitosan solution and the MW of the chain. For samples synthesized using chitosan solution at pH = 3.5 (corresponding to 100% protonated chitosan), the nanostructures exhibited uniform average size ranging from 75 to 79 nm.
<figure><img src="image_10.png" /><figcaption>**Figure 2.** (a) to (f) TEM micrographs, (g) STEM micrographs, (h) and (j) HRTEM micrographs: of typical Au nano-sea-urchins. Red arrows point primitive irregularly shaped nanoparticles. As a representative sample, all micrographs correspond to the sample synthesized with LMW chitosan, pH 3.5. The scale bar of (c)–(f). it is 50 nm.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 3.** (a) The HRTEM image shows the surface layer of chitosan as a stabilizing agent, with a thickness of approximately 4 nm. (b) TEM and (c) SEM images showing a group of agglomerates of nanoparticles surrounded by chitosan. All micrographs correspond to the sample synthesized with MMW chitosan, pH 5.5. No significant changes were found in the chitosan layer of the other samples. See figure S3 in Supplementary Material.</figcaption></figure>

However, when chitosan solutions prepared at pH = 5.5 (corresponding to 60% protonated chitosan) were used, a trend of increasing size was observed with decreasing molecular weight.

On the other hand, in table 1, we observe that the average sizes obtained by DLS measurements are higher than those obtained from TEM images. This difference is due to the fact that DLS measurements analyze the aggregates of particles in solution, determining their hydrodynamic diameter including the thickness of the chitosan layer, as well as the groups of agglomerates discussed above [48]. However, the same size trend was maintained, that is, relatively constant hydrodynamic diameters for samples prepared using chitosan solutions at pH 3.5, and an increase in size with the decrease in chitosan MW for samples prepared using chitosan solutions at pH 5.5, obtaining a PDI value below 0.3 for all samples. (The size distribution in terms of the scattered intensity is presented in the supplementary materials, figure S4).

The size trend for pH 5.5 can be explained by the decrease in protonation of the –NH₂ groups compared to that of the acid solution. This decreases the electrostatic repulsion, favoring the aggregation of the asymmetric Au-Nps and giving rise to larger structures compared to those synthesized with the chitosan solution at pH 3.5. Similarly, at pH 5.5, the particle size increased as the molecular weight decreased. This is attributed to the decreased in amino groups due to the respective degree of acetylation (DA) of each chitosan solution used in the synthesis.
<figure><img src="image_12.png" /><figcaption>**Table 1.** Average size of Au nano-sea-urchins from TEM and DLS analysis as a function of MW and pH of chitosan solutions.</figcaption></figure>

<table><thead><tr><th>pH and chitosan MW used in the synthesis</th><th>Average size from TEM (nm)</th><th>Hydrodynamic diameters from DLS (nm)</th><th>PDI</th></tr></thead><tbody><tr><td>pH. 3.5, High MW</td><td>78 ± 23</td><td>126 ± 1</td><td>0.250 ± 0.015</td></tr><tr><td>pH. 3.5, Medium MW</td><td>75 ± 14</td><td>109 ± 1</td><td>0.144 ± 0.010</td></tr><tr><td>pH. 3.5, Low MW</td><td>79 ± 20</td><td>109 ± 2</td><td>0.178 ± 0.018</td></tr><tr><td>pH. 5.5, High MW</td><td>59 ± 12</td><td>201 ± 1</td><td>0.156 ± 0.042</td></tr><tr><td>pH. 5.5, Medium MW</td><td>71 ± 16</td><td>212 ± 3</td><td>0.143 ± 0.029</td></tr><tr><td>pH. 5.5, Low MW</td><td>83 ± 15</td><td>224 ± 3</td><td>0.202 ± 0.001</td></tr></tbody></table>

Furthermore, to confirm the formation of chitosan-coated Au nano-sea-urchins, FTIR measurements were performed. Figure 5 shows the spectra of chitosan as a reference and a representative Au nano-sea-urchin sample. In the reference spectrum, the typical bands of the functional groups of chitosan were observed; the broad band between 3 238 and 3 430 cm⁻¹ corresponds to the overlap bands due to elongation and tension of the O–H and N–H groups; the C–H stretching bands were also observed at 2870 cm⁻¹; then, C=O carbonyl bonds (amide I) of C=O-NHR were localized at 1 636 cm⁻¹, and the amine groups N–H bonds (amide II) (NH₂) at 1 540 cm⁻¹, as well as bands at 1 408 cm⁻¹ (CH₃ wagging), 1 367 (C–C stretching of glucosamine group), and 1 013 cm⁻¹ (C–O stretching) [49]. For the Au/chitosan curve, the same bands can be seen with some differences, such as a slight shift of the band from 1 540 cm⁻¹ to 1 521 cm⁻¹, attributed to the reduction in the vibration intensity of the N–H bond due to the increase in molecular weight by the bond to the metal particle. Other shifts in some other peaks were also identified, in good agreement with previous reports [50], including O–H stretching and C=O, attributed to a weak interaction between oxygen and Au, with nitrogen atoms being the main sites for complexation of Au/chitosan.
<figure><img src="image_13.png" /><figcaption>**Figure 5.** FTIR spectra of chitosan as a reference and representative Au nano-sea-urchin sample. (Conditions of chitosan used: pH 5.5, HMW).</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 6.** Absorption spectra of Au nano-sea-urchin samples synthesized with chitosan solutions at different pH and MW.</figcaption></figure>

For biomedical applications, it is important that the plasmons of metallic nanoparticles present a shift toward the IR region. Typically, Au-nanoparticles are characterized by a plasmon centered at approximately 525 nm. Therefore, in different reported methodologies, this shift towards the IR region has been achieved by manipulating the size, morphology, and formation of aggregates [51, 52]. Figure 6 shows the absorption spectra of Au Nano-sea urchin samples that were prepared with chitosan solutions at pH of 3.5 and pH 5.5. First, one can observe that for the samples synthesized with chitosan solutions at pH 3.5, the absorption spectrum was quite stable, presenting curves centered at around 652 nm. However, at pH 5.5, different trends were observed depending on the chitosan MW. Broad band was obtained for the sample prepared with high MW chitosan, going from 450 to 800 nm and centered at 597 nm. Later, as the molecular weight decreased, the fall of the curve extended beyond 800 nm, and the positions of the plasmon were red-shifted to 614 nm and 624 nm for the molecular weights of medium and low chitosan, respectively. This trend is consistent with the hydrodynamic size and interplasmon coupling effect due to the formation of nanoparticles aggregates. As previously discussed, the formation of aggregates is favored by a reduction in electrostatic repulsion, which occurs because of the decrease in amino groups resulting from the pH and DA of each sample.
## 4. Conclusions

AuNPs with a sea urchin-like shape were synthesized using chitosan as a stabilizing agent at varying pH levels (3.5, 5.5) and different molecular weights (low, medium, and high). Evidence from various spectroscopy techniques and electron microscopy images suggests that the formation of Au nano-sea-urchin structures is the result of the coalescence and aggregation of anisotropic gold nanoparticles. The size of the Au nano-sea-urchin structures was influenced by the pH, degree of acetylation, and molecular weight of the chitosan solution used during synthesis. Larger structures were obtained when synthesizing samples with chitosan solutions at pH 5.5, owing to the reduction in electrostatic repulsion caused by the decrease in amino groups, while the size range was more stable for samples synthesized with chitosan solutions at pH 3.5. Given the above, MMW chitosan with a DA of 0.22 and a pH of 5.5 provides sufficient colloidal stability for nanoparticle formation without contributing to excessive charge density that could compromise biocompatibility. Furthermore, under these conditions, the sample presents interesting optical properties, due to the red shift of the plasmon. This balance makes MMW chitosan a promising candidate for further development of biocompatible gold nanostructures for biomedical applications. In summary, the document presents a straightforward method for synthesizing chitosan-stabilized nano-sea-urchin gold structures and demonstrates that their morphological properties and size can be controlled by adjusting the pH and molecular weight of the chitosan solution.

## Acknowledgments

The authors of this work acknowledge Loïc Joanny from the ScanMat platform CMEBA, Agnès Burel from Microscopy Rennes Imaging Center (MRic) of the UMS Biosit (FBI member (ANR-10-INBS-04))- University of Rennes, and L. Rault for assistance with TEM experiments performed on the THEMIS platform (ScanMAT, UMS 2011 University of Rennes 1-CNRS; CPER-FEDER 2007–2014). Lourdes Mónica Bravo-Anaya acknowledges the IEA P²NanoBio founded by the CNRS.

## Conflict of interest

The authors declare that they have no conflicts of interest.

## Data availability statement

All data that support the findings of this study are included within the article (and any supplementary files).

## Author contributions

R A Guerra-Santopietro ID 0009-0001-7997-9308
Investigation (supporting), Methodology (supporting)

S E López-Alvarez ID 0009-0000-0379-3526
Formal analysis (supporting), Investigation (supporting), Methodology (supporting)

J M Blancas-Flores ID 0000-0003-0679-3948
Formal analysis (equal), Investigation (equal)

L M Bravo-Anaya ID 0000-0002-8121-6887
Formal analysis (equal), Investigation (equal), Methodology (equal), Supervision (equal), Writing – original draft (equal)

V H Romero-Arellano ID 0000-0002-9211-5862
Conceptualization (equal), Formal analysis (equal), Investigation (equal), Methodology (equal), Project administration (equal), Writing – original draft (equal)

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