# Investigating of the physico-chemistry dhiolated dextran derivatives

Cléa Chesneau, Andréa Pelletier, Angélique Goffin, Loren Jørgensen, Madara

Dias Wickralanayaka, Alexandre Hinzpeter, Sabrina Belbekhouche

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Cléa Chesneau, Andréa Pelletier, Angélique Goffin, Loren Jørgensen, Madara Dias Wickralanayaka, et al.. Investigating of the physico-chemistry dhiolated dextran derivatives. Colloids and Surfaces B: Biointerfaces, 2025, 241, pp.114603. 10.1016/j.col surfb.2025.114603. hal-04973426

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# Investigating of the physico-chemistry thiolated dextran derivatives

Cléa Chesneau<sup>a</sup>, Andréa Pelletier<sup>a</sup>, Angélique Goffin<sup>b</sup>, Loren Jørgensen<sup>c</sup>,
Madara Dias Wickramanayaka<sup>d</sup>, Alexandre Hinzpeter<sup>d</sup>, Sabrina Belbekhouche<sup>a,∗</sup>

## ARTICLE INFO

Keywords:

Thiol derivative

Modification dextran

Redox-responsive

Particles

Hydrophobic loading

## ABSTRACT

The study aimed to create redox-responsive dextran carriers for controlled hydrophobic molecule release using glutathione, a natural cellular reducing agent, by modifying dextran with a thiol derivative. Investigating the impact of different hydrophobic length on the molecular self-organization of polysaccharide derivatives into nanoparticles helped to understand their roles in this process. The study demonstrated that thiolated dextran particles can be used as emulsifier and can effectively encapsulated hydrophobic molecules like Nile red dye, with the disulfide linkage being cleaved by glutathione under physiological conditions for rapid release. Additionally, the dextran-based particles were found to be non-toxic to living cells.

## 1. Introduction

The body's mononuclear phagocyte system and filtration organs like the spleen, liver, and kidneys pose obstacles to utilizing drug-loaded particles for biomedical purposes within living organisms [1]. The addition of polyethylene glycol or polysaccharides to particle surfaces has been observed to create stealth compounds that can affect circulation times and cellular uptake, potentially reducing interaction with plasma proteins [2–6]. In contrast, there is only minimal research coverage on coating with polysaccharides [5,6].

We chose to concentrate on dextran in this study, as it is hydrophilic, biocompatible polysaccharide that can be readily altered chemically and has found extensive applications in biomedicine, including drug delivery and gene transfection. Dextran, an extracellular bacterial homopolysaccharide, consists of a main chain composed of contiguous α-(1,6)-linked d-glucopyranose residues and branches, stemming mostly side-chains of α-(1,6) glucose units linked by α-(1,3) branch linkages to α-(1,6)-linked chains [7]. Some dextrans have α-(1,2) or α-(1,4) branch linkages, according to the bacterial source.

Dextran's abundant hydroxyl groups in its anhydroglucose unit make it structurally suitable for chemical modification to create new properties, specifically amphiphilic copolymers with self-organizing abilities in aqueous media, which is of great interest for efficient drug delivery purposes [8,9]. Research indicates that dextran can be altered by

attaching alkyl chains or long polymers using ester or acetal linkages [10–12] for biomedical applications [13–15]. Many of the documented altered dextrans were observed to self-assemble into particles or micelles [11,16–19].

Redox-responsive systems are capable of changing in response to redox reactions in their environment, impacting various properties such as chemical links, material shape, permeability, and sol-gel transition depending on the specific redox-responsive material and the type of redox stimulus applied, whether it's induced electrochemically or through oxidative or reducing molecules [20]. The disulfide bond is commonly employed as a redox-sensitive bond for creating materials that are responsive to redox. Specifically, disulfide bonds can be created under oxidative conditions and can be reversibly reduced to thiols under reductive conditions.

The literature contains numerous examples of interest in utilizing disulfide bonds for drug delivery, as this type of bond exhibits high stability in the bloodstream but undergoes rapid reduction in the cell interior owing to the reductive potential stemming from the presence of reduced glutathione [21]. Interestingly, cancer cells demonstrate more than 10 times the normal levels of glutathione found in healthy cells.

The present study sought to create a redox-responsive material combining dextran and thiol/disulfide chemistry, accomplishing this by adding thiol groups to the dextran backbone. The resulting products were initially analyzed using characterization methods, i.e. ¹H NMR and

<footnote><sup>a</sup> Université Paris Est Creteil, CNRS, Institut Chimie et Matériaux Paris Est, UMR 7182, 2 Rue Henri Dunant, Thiais 94320, France</footnote>

<footnote><sup>b</sup> LEESU, Univ Paris-Est Creteil, École des Ponts, Creteil, France</footnote>

<footnote><sup>c</sup> Sciences et Ingénierie de la Matière Molle, ESPCI Paris, CNRS, Université PSL, Sorbonne Université, Paris 75005, France</footnote>

<footnote><sup>d</sup> Université Paris Cité, CNRS, INSERM, Institut Necker Enfants Malades-INEM, Paris F-75015, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: sabrina.belbekhouche@cnrs.fr (S. Belbekhouche).</footnote>
the Ellman test. We proceeded to examine the physical-chemical char-acteristics of the dextran derivative in water and conducted an analysis of its redox-responsive properties. Additionally, we developed redox-responsive dextran derivative particles.

The dextran-based particles were designed to encapsulate Nile red as a proof of concept, and their colloidal stability in a physiological environment, as well as the drug release upon treatment with glutathione, were also investigated.

## 2. Materials and methods

### 2.1. Materials

1,3-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino) pyridine (DMAP), 3-mercaptopropionic acid ($C_3SH$), 11-mercaptoundecanoic acid ($C_{11}SH$), 6-mercaptohexanoic acid ($C_6SH$), dimethyl sulfoxide (DMSO), aminoethanethiol, cystamine, chloramine T, Ellman's reagent, and glutathione and dextran (Dex, theoretical molar masses, 6 000 g/mol) were purchased from Sigma-Aldrich. Nile red was acquired from TCI.

### 2.2. Chemical modification of dextran: thiolated dextran

Dextran (1.23 mM of anhydroglucose unit), thiol derivative ($C_3SH$ or $C_6SH$ or $C_{11}SH$ 1.23 mM), 1,3-dicyclohexylcarbodiimide (1.23 mM), and 4-(dimethylamino)pyridine (0.246 mM) were simultaneously dissolved in dimethyl sulfoxide (procedure adapted from Choi et al. [22]). The resulting reaction mixture was stirred for a duration of 48 h at a temperature of 80°C. Subsequently, the solution was cooled and precipitated into 500 ml of ethanol. The resulting solid was filtered and thoroughly washed multiple times with ethanol. It was then dissolved in water, and the obtained solution underwent dialysis against water using a spectra/por dialysis membrane with a cut-off of 3.5 kDa. Following lyophilization, a powder was produced. The samples were designated as "DexMn-SH," where "Mn" indicates the theoretical number average molar mass of dextran as provided by the supplier, and "-SH" denotes the modified dextran. "Dex6" is the label of a pristine dextran sample with a theoretical molar mass of 6000 g/mol. "Dex6-$C_3SH$", "Dex6-$C_6SH$" and "Dex6-$C_{11}SH$" corresponds to dextran modified with $C_3SH$, $C_6SH$ and $C_{11}SH$ respectively. "Dex6-SH" refers to thiolated dextran without any distinction.

### 2.3. Characterization of dextran derivative

#### 2.3.1. Chemical characterization

The FTIR spectra were obtained using a Bruker Tensor 27 spectrometer (Bruker Optik GmbH, Germany), with the samples positioned on the ATR accessory crystal and 32 co-added scans at 4 cm⁻¹ resolution taken for each sample over the range of 4000–400 cm⁻¹. ¹H NMR analyses were performed using a Bruker UltrashieldTM plus 400 Hz spectrometer at a temperature of 343 K. A total of 10 mg of the dextran derivative was dissolved in 500 µL of DMSO-d6.

#### 2.3.2. Assessment of thiol concentration in a dextran derivative

The quantification of thiol groups was performed utilizing Ellman's reagent [23]. A solution of a dextran derivative at a concentration of 10 g/L, measuring 250 µL, was combined with 2.5 ml of PBS buffer (0.1 M, pH 8) and 50 µL of Ellman's reagent (4 g/L). Following 15-min incubation at room temperature, shielded from light, the absorbance was recorded at 412 nm using quartz cuvettes. The thiol content was calculated based on a standard curve established ($ε_{exp} = 13795 L.mol⁻¹.cm⁻¹$).

### 2.4. Demixing experiments for octanol/water

The water/octanol emulsion was prepared by mixing a 1 g.L⁻¹

aqueous solution of thiolated dextran and octanol at a volume ratio of 50/50 (50/50 Oil/Water Ratio) or 80/20 (Oil-in-Water (O/W) emulsion). The two phases were vortexed during 1 minute and was followed by 10 minutes mixing with a magnetic stirrer and the time for demixing was measured.

### 2.5. Fabrication of dextran-based nanoparticles

Thiolated dextrans (Dex6-SH) at 1 g/L were mixed in water with subsequent stirring in air as oxidizing media to favor particles formation. To highlight the time required to oxidize thiols in air. We set up the required conditions using the Ellman test described below (in Section 3.2) and commercial model compounds, namely aminoethanethiol and its disulfide-oxidized derivative (cystamine). The idea was to solubilize aminoethanethiol at the same thiol concentration as that contained in thiolated dextran, leave the solution to stir in air and then, at different times, withdraw solution and place it in the presence of Ellman's reagent. The oxidation time required for the thiolated dextran will be taken to be the time needed to achieve identical absorption between the aminoethanethiol exposed to air and the cystamine.

### 2.6. Hydrophobic probe loading in thiolated dextran particles and subsequent release in redox media

As a general procedure, Nile red (1 mg.mL⁻¹ in ethanol) was drop-wise added to thiolated dextran particles prepared in Section 2.5. After overnight stirring, the amount of hydrophobic encapsulated in the dextran-based particles was estimated using the molar extinction coefficient determined experimentally of Nile red as 34 226 L.mol⁻¹.cm⁻¹

The degradation of as-obtained particles in response to redox stimulus was examined via the size variation (measured by dynamic light scattering) in 10 mM glutathione.

### 2.7. Assessment of the toxicity of modified dextran (Dex-SH)

HEK293 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM, Invitrogen), which was supplemented with 1 % Penicillin/Streptomycin and 10 % Fetal Bovine Serum (FBS). The cells were maintained in a humidified incubator set at 37 °C with 5 % CO₂. For cell viability assays, the cells were seeded in a 96-well plate to achieve 80 % confluence. Following the seeding process, the cells were incubated for 24 h in DMEM supplemented with 10 % FBS and various concentrations of a dextran derivative that had been dissolved in water. Cytotoxicity was evaluated through photocolorimetric analysis using the Cell Counting Kit 8 (WST-8) according to the manufacturer's guidelines (Abcam, Ab228554). This involved adding CCK8 to DMEM with SVF at a 1:10 dilution, followed by the addition of 100 µl of the dye to each well. The cells were then incubated for one hour in a 37 °C, 5 % CO₂ environment. Absorbance readings were taken using a ClarioStar plate reader (BMG, Ger) at an optical density of 460 nm. Each measurement was conducted in duplicate across five independent experiments, with results expressed as a percentage of the untreated control. A 1 % SDS solution served as a positive control. Cells treated with 100 µg/ml of each dextran derivative solution for 24 h were visualized using an EVOS M5000 (Invitrogen).

### 2.8. Methods

#### 2.8.1. Absorbance measurement

We used a Varian Cary 100 UV-Visible spectrophotometer to record the absorbance spectra

#### 2.8.2. Fluorescence spectroscopy measurements

First, a 10⁻³ mol.L⁻¹ pyrene stock solution was prepared in acetone, followed by introducing a 400 µL aliquot of this solution into empty vials and allowing the solvent to evaporate under vacuum. Following the
evaporation process, the vials were loaded with 1 L of distilled water and softly agitated for 24 h, resulting in a final pyrene concentration of $4.10^{-7}$ mol.L$^{-1}$. At this minimal concentration, we did not detect an excimer band resulting from the interaction between an excited state pyrene and a ground state pyrene. [24,25]. The method involved preparing the sample by mixing various concentrations of dextran derivative in an aqueous solution and stirring it for 24 h to ensure that the pyrene was incorporated into any existing polymer hydrophobic domains. Fluorescence measurements were conducted using a Spectrofluorometer (Jasco, FP8300, Japan) with a 150 W xenon lamp for excitation, and it was computer-controlled by the Spectra Manager II software package. All samples were excited at 332 nm and the emission spectra (350-450 nm, each 0.2 nm) of pyrene showed 5 vibronic peaks notably at 372 nm (fluorescence intensity $I_1$) and at 382 nm (fluorescence intensity $I_3$). The intensity ratios $I_1/I_3$, calculated with an accuracy of less than 5 %, were used to estimate the pyrene micro-environment polarity based on the emission spectra obtained from all samples excited at 332 nm, showing 5 vibronic peaks, particularly at 372 nm (intensity $I_1$) and 382 nm (intensity $I_3$) [26].

### 2.8.3. Surface tension measurements

The equilibrium surface tension of the aqueous solutions of dextran derivative was measured according to the pendent drop technique (Teclis tensiometer, France). The surface tension, $\gamma$ (mN/m), was measured at ambient temperature as a function of concentration to determine the critical micellar concentration (CMC). Several measurements were performed per drop and on several drops of the same solution to reduce uncertainties. The measuring time per drop was 15 min, which was sufficient to reach the equilibrium state.

## 3. Results

### 3.1. Modification of dextran through the introduction of thiol groups presenting different alkane chain length

Thiol-modified dextrans were produced through a single-step reaction utilizing DMSO as a solvent along with thiol derivative containing carboxylic acid in the presence of DCC and DMAP (refer to Fig. 1). The as-obtained polymers were subsequently purified via repetitive precipitation and dialysis using a semi-permeable membrane.

Following the reaction illustrated in Fig. 1 and subsequent purification, the FTIR analysis of native dextran, thiol-modified derivative, and Dex6-SH were performed to provide a more comprehensive understanding of the chemical groups within the polymer structure (Fig. 2A). The flexibility of dextran's chain is indicated by the presence of a peak at around $1010 \text{ cm}^{-1}$, which corresponds to the $\alpha$-(1,6) linkages in the glycosidic bond [27]. The $\alpha$-glycosidic bond is indicated by a peak at $914 \text{ cm}^{-1}$, while the covalent vibrations of the C-O-C bond and glycosidic bridge are represented by the band at $1150 \text{ cm}^{-1}$. Furthermore, the band at $3370 \text{ cm}^{-1}$ is associated with the hydroxyl stretching vibration of the polysaccharide, and the one at $2918 \text{ cm}^{-1}$ corresponds

to the C-H stretching vibration [28]. The characteristic bands of pristine dextran and thiol derivative can be found in Dex-SH, confirming the dextran modification through the appearance of a new peak at $1727 \text{ cm}^{-1}$, associated with the -C=O stretching of the ester bond.

The resulting dextrans (Dex-SH) were also analyzed using 1 H NMR spectroscopy. Fig. 2B depicts the $^1$H NMR spectrum in DMSO-d$_6$ of Dex6-C$_{11}$SH and indicates the presence of signals associated with both thiol derivative and dextran. For the dextran, the distributions of $^1$H NMR resonances are detailed within the range of 3-5.4 ppm. Specifically, the resonances corresponding to H-2, H-3, H-4, H-5, and H-6 are found in the 3-4.2 ppm range, while the proton associated with the anomeric carbon of the $\alpha$-1,6 linkage (H1) is observed at 4.9 ppm [29], while the peaks located in the 5.1-5.4 ppm range are attributed to branching in dextran [30]. For the thiol derivative, the protons associated with the -CH$_2$ group are detected in the 1.1-2.6 ppm range. We did not conduct a thorough analysis of the specific location where the substitution occurred within the dextran chain.

Ellman's reagent was subsequently employed to ascertain the presence of a thiol group within the dextran chain of Dex-SH and to quantify the thiol content if it was indeed present. The optical absorption measurement of thiolated dextran using Ellman's reagent at 412 nm serves as an indicator of the presence of a thiol group in the modified dextran, thereby confirming the successful conjugation of the hydroxyl groups of dextran with the carboxyl group of the examined thiol derivative. Following this, we determined the concentration of thiol groups in each Dex6-SH by utilizing molar absorptivity i.e. 1.07 mM, 7.31 mM and 2.93 mM for Dex6-C$_3$SH, Dex6-C$_6$SH and Dex6-C$_{11}$SH, respectively.

It is important to highlight that the formation of disulfide bonds was observed during the reaction by introducing a reducing agent (NaBH$_4$) to the DexSH solution prior to and following Ellman's test for Dex6-C$_6$SH and Dex6-C$_{11}$SH. Indeed, thiol amount can only be estimated after NaBH$_4$ addition.

In contrast to the thiolated dextran previously documented in the literature, we modified the molar mass of dextran. Additionally, to our knowledge, there exists only one publication that employs a similar methodology for thiolated dextran, specifically the incorporation of a thiol into a dextran chain utilizing DCC/DMPA coupling. The research conducted by Choi et al. involved the thiolation of a 10 kDa dextran utilizing DCC/DMPA for the coupling process only with C3-SH [22]. The value of the DS and the physico-chemical characterization of the thiolated dextran were not reported, which prevents us from making a comparison between our findings and theirs. Choi et al. developed a thiolated dextran that was conjugated to gold nanorods for the purpose of photothermal ablation of inflammatory macrophages. In the majority of the studies published, thiolated dextrans are synthesized utilizing EDC as a coupling agent (e.g. Gupta et al. [31] thiolated a dextran of 40 000 g/mol) or even two steps, e.g., carboxymethylation of dextran by sodium chloroacetate and the addition of cysteamine hydrochloride (Mytnyk et al. [32] found a DS 10 % for modifying a dextran 20 kDa) or the dextran hydroxyl groups were activated with 4-nitrophenyl chloroformate (4-NC) and 4-NC-activated dextran react with a thiol

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** One-step modification of dextran for incorporating thiol function presenting different length. An example of the substitution at position C-2 is provided.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>B)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 2. A) FTIR spectra of mercaptopropionic acid (—), dextran (—) and dex-SH (—), B) ¹H NMR spectra of dextran (dex6) and modified dextran (dex6-C₁₁SH) in DMSO-D₆. An example of the substitution at position C-2 is provided.</figcaption></figure>

derivative, e.g. 2-aminoethanethiol (Chen et al. [33] found a DS 20 % for modifying dextran).

Thus, the successful thiolation of dextran was confirmed through
FTIR NMR, and Ellman's test.

## 3.2. Demonstration of redox-responsive properties and physicochemical characterization of the synthesized thiolated dextran in aqueous environments

3.2.1. Association behaviour of thiolated dextran in aqueous environment

The ratio of pyrene emission intensities I₁/I₃ serves as a distinctive
indicator of the polarity surrounding pyrene [34]. The reduction of this
ratio indicates the development of hydrophobic clusters resulting from
the aggregation of amphiphilic polymers.

It is important to observe that the fluorescence spectra of pristine dextran, within the concentration range of 10⁻⁴ g/L to 10 g/L, consistently yield an I₁/I₃ ratio exceeding 1.8. In contrast, for thiolated dextran (Fig. 3), there is a noticeable decline in the I₁/I₃ ratio as the concentration of Dex-SH increases. This indicates a potential associative behavior of thiolated dextran or the presence of a hydrophobic region that emerges with rising concentration. The fluorescence spectra of pyrene in thiolated dextran, at the highest concentration, yielded an I₁/I₃ ratio exceeding 0.90 for Dex6-C₃SH and Dex6-C₆SH and down to 0 for Dex6-C₁₁SH. Thus, it is clear that the Dex6-C₁₁SH is more hydrophobic than Dex6-C₆SH, and the Dex6-C₃SH. This result is consistent with the variation in alkyl chain length in each system. The critical micelle concentration (CMC) values are determined at the inflection point of the decline in the I₁/I₃ ratio in relation to log C in Fig. 3 [35] and which
<figure><img src="image_7.png" /><figcaption>Fig. 3. Variation of fluorescence characteristics of pyrene as function of thiolated dextran concentration at room temperature.</figcaption></figure>

depends on the final hydrophobicity of the system. In our study, the objects formed are of the micelle type as in the work of Francis et al. [36].

### 3.2.2. Emulsifying properties of thiolated derivative Dex6-SH

The thiolated derivative contains hydrophobic chain. In contrast, dextran is water soluble and only dissolves to some extent in some aprotic polar solvents as DMSO or DMF. Therefore, it is expected that thiolated dextrans show surfactant behaviour - this is confirmed by surface tension measurements, which is around 45 mN/m. Thus, it is anticipated that these thiolated derivate in a water/organic 2-phase system, may be positioned at the interface and be able to behave as emulsifier. The emulsifying ability of Dex6-SH was evaluated for a water/octanol biphasic mixture. Note that these two phases separate completely in less than a minute after stirring. For all the thiolated dextran, good emulsions have been obtained (an example is given Fig. 4) and remains stable for over 24 h.

This data provides additional support for the previously proposed hypothesis that these thiolated derivatives may have the ability to form colloids and thus that alkyl chain lead to self-assembling.

### 3.2.3. Influence of thiolated dextran on cellular viability

The evaluation of the biocompatibility of the synthesized thiolated dextran was conducted by analyzing its impact on the viability of HEK293 cells. As depicted in Fig. 5, the cell viability was measured following a 24-h incubation with different concentrations of Dex-SH and pristine dextran, which served as a reference, diluted in culture media (DMEM supplemented with SVF). The assessment of cell viability was performed using the colorimetric CCK-8 assay. The findings demonstrated that thiolated dextran had no adverse effects on cell viability.

### 3.2.4. Preparation and characterisation of thiol dextran particles under air as oxidizing media

In this section, we first seek to highlight the conditions required to obtain thiol oxidation in air as an oxidizing medium. The Ellman's test [37] was thus used to determine the conditions for switching from a thiol derivative (aminoethanethiol) to its counterpart in oxidized form (disulfide bridge, cystamine) by utilizing Ellman's reagent (5,5'-dithiod bis-(2-nitrobenzoic acid), DTNB) (Fig. 6A). Thiol can indeed react with Ellman's reagent, cleaving the disulfide bond to give 3-thio-6-nitrobenzoate (TNB-), which ionizes to the TNB²⁻ dianion in water at neutral pH. This TNB²⁻ ion has a yellow color and shows an absorbance peak at

<figure><img src="image_8.png" /><figcaption>Fig. 5. The impact of dextran and its derivatives on HEK293 cells was investigated. (A) HEK293 cells were treated for 24 h with varying concentrations (100 µg/ml, 250 µg/ml, and 500 µg/ml) of each polysaccharide derivative, and cell viability was assessed using the CCK-8 assay.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 4. Emulsifying ability of Dex6-SH for a water/octanol mixture ((1) without Dex6-SH and (2) with Dex6-SH a 1 g.L⁻¹).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** A) UV-visible spectra of aminoethanethiol and cystamine solutions in both case in presence of Ellman reagent, B) UV-visible spectra of an aminoethanethiol solution in the presence of Ellman's reagent, measured in air at different times.</figcaption></figure>

412 nm which can be easily measured by UV. As expected, at the same concentration, the cystamine solution in the presence of Ellman's reagent shows no peak at 412 nm, whereas this is the case for aminoethanethiol. Interestingly, when the aminoethanethiol solution is stirred in air, the peak at 412 nm gradually decreases a clear sign that the thiol is oxidizing (Fig. 6B). A time of 24 h seems to be required, and this is the time we'll be using next.

We then investigated the possibility of preparing thiolated dextran-
based particles under air as oxidizing agent (having stirred the
dextran derivative solution in air for 24 h). It deserves noting that ex-
pected no thiol group was evidenced by the Ellman test at the end of this
time. All the nanoparticulate formulations without any hydrophobic
cargo were first analysed via dynamic light scattering to evidence the
possibility of forming particles and to characterize their size and size
distribution. Based on the experimental results, it is clear that the
examined thiolated dextrans form nanoparticles and have the following
sizes and dispersities: 175 nm (PDI = 0.2), 165 nm (PDI = 0.5) and 130
nm (PDI = 0.5) for Dex6-C₃SH, Dex6-C₆SH and Dex6-C₁₁SH,
respectively.

In the development of a drug delivery carrier, a key consideration is
the adequate degradability of the particle in reaction to a specific
stimulus. This is essential to avoid the bioaccumulation of the resulting
degradation products and to promote the release of the cargo at the
intended site. To examine the degradation behavior, dynamic light
scattering (DLS) measurements were conducted to monitor the changes
in hydrodynamic size of dextran-based particles following treatment
with 10 mM glutathione (GSH).

The treatment of Dex-SH particles suspended in PBS remains stable (Fig. 7). In contrast, when suspended in 10 mM GSH results in a measurable increase in size, as indicated by dynamic light scattering (DLS), demonstrating a swelling effect. This phenomenon can be linked to the degradation of pre-existing disulfide bonds. Over time, the cleavage of these disulfide bonds becomes more pronounced, leading to the degradation of the particles. Glutathione activity produces free thiol groups, which exchange disulfide bonds and promote particle swelling. The particles disintegrate rapidly following thiol-disulfide reshuffling, which yields unlinked DexSH chains.

3.2.5. *Proof of concept: loading of the hydrophobic probe, namely Nile Red*

A solution of Nile red, which is a hydrophobic and colored dye, was
used to investigate the loading. A reference solution was made using
unmodified dextran in air. When the colored Nile red solution was
added, colored aggregates formed, highlighting the non-encapsulation

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Hydrodynamic size of Dex6-C11SH particles a) in PBS at pH 7.4 at 37 °C and regular time intervals for 120 h and b) in the presence of 10 mM GSH at regular time intervals showing the nanoparticle degradation.</figcaption></figure>

of this compound as expected. However, as soon as Nile red is added
to the solution of particles formed by thiolated dextrans, the solution
becomes colored, indicating incorporation of Nile into the hydrophobic
core formed by the alkyl chains in the modified dextran. The fluorescent
microscopic image of Nile red loaded in Dex6-SH clear evidenced Nile
red loading and the spherical shape of the particles (see Fig. 8). As
anticipated, the introduction of 10 mM GSH triggers the release of Nile
red for all the particles examined (see Figure SI 1).

**4. Conclusion**

In conclusion, we successfully developed thiolated dextrans (Dex-SH) using a one-step method, confirmed by NMR and Ellman's test and still be water solubility. These Dex-SH polymers exhibit amphiphilic behavior, as demonstrated by pyrene probe and tensiometric measurement, and display redox-responsive behavior. Furthermore, cytotoxicity studies indicate that all Dex-SH derivatives are non-toxic. Our findings demonstrate the successful production of an emulsifier and redox-responsive dextran derivative particles with a size under 200 nm
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Photographs of beaker with A) a solution of dextran (Dex6) subsequent addition of Nile red lead to the formation of colored aggregated and B) a solution of left Dex6-SH subsequent addition of Nile red lead to a pink colored solution indicating the Nile red loading inside particles. Fluorescent microscopic image of Nile red-loaded Dex6-SH based particles in PBS, C) Fluorescent microscopic image of Nile red-loaded Dex6-SH based particles.</figcaption></figure>

through dynamic light scattering analysis.

The developed polysaccharide has potential for loading hydrophobic dye (Nile red), and we have demonstrated the ability to regulate the loading quantity based on the length of dextran and initiate delivery using a redox stimulus (glutathione).

### CRediT authorship contribution statement

**Chesneau Cléa:** Formal analysis, Data curation, Conceptualization.
**Goffin Angélique:** Formal analysis.
**Pelletier Andréa:** Formal analysis.
**Wickramanayaka Madara:** Investigation, Formal analysis, Data curation, Conceptualization.
**Jørgensen Loren:** Formal analysis.
**Belbe-khouche Sabrina:** Writing - review & editing, Writing - original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation.
**Hinzpeter Alexandre:** Investigation, Formal analysis, Data curation, Conceptualization.

### Declaration of Competing Interest

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

### Acknowledgement

We gratefully acknowledge the Agence Nationale de la Recherche (grants ANR-22-CE92-0086) for the financial support during this research project.

### Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.colSURF.2025.114603.

### Data availability

No data was used for the research described in the article.

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