# NIR-responsive dextran / poly(lactide) hydrogels: Characterization of cleavable hydrogels and photoactivated release of proteins

Mickael Guerreiro, Anissa Benkhedim, Doriane Voisin, Clémence Farine, Audrey Bethry, Cédric Paniagua, Hélène van den Berghe, Benjamin Nottelet

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## ► To cite this version:

Mickael Guerreiro, Anissa Benkhedim, Doriane Voisin, Clémence Farine, Audrey Bethry, et al., NIR-responsive dextran / poly(lactide) hydrogels: Characterization of cleavable hydrogels and photoactivated release of proteins. Carbohydrate Polymers, 2025, 368, 10.1016/j.carbpol.2025.124068. hal-05320265

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https://hal.science/hal-05320265v1

Submitted on 17 Oct 2025

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# NIR-responsive dextran / poly(lactide) hydrogels: Characterization of cleavable hydrogels and photoactivated release of proteins

Mickael Guerreiro<sup>a</sup>, Anissa Benkhedim<sup>a</sup>, Doriane Voisin<sup>a</sup>, Clémence Farine<sup>a</sup>, Audrey Bethry<sup>a</sup>, Cédric Paniagua<sup>a</sup>, Hélène Van Den Berghe<sup>a,*</sup>, Benjamin Nottelet<sup>a,b,*</sup>

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## ARTICLE INFO

**Keywords:**

Polysaccharide polyester hydrogel

Reactive oxygen species ROS

Near-infrared (NIR)

Photodegradation

BSA release

## ABSTRACT

Polysaccharide-based hydrogels often lack mechanical strength and, when used for protein delivery, are generally limited to diffusion-based release. In this work, we developed robust polysaccharide- and polyester-based near-infrared (NIR)-responsive hydrogels. Hydrogels are made from photo-crosslinked methacrylated dextran (DEX-MA), methacrylated polylactide containing oxygen reactive species (ROS) sensitive thioketal groups (PLA-TK-MA), and covalently bound protoporphyrin IX (PPIX) that generates ROS under NIR irradiation. We investigated the effects of DEX methacrylation degree (3, 12 and 45 %), PLA molecular weight (2500 vs. 5000 g.mol⁻¹) and DEX/PLA ratios (95/5 to 50/50) on the gels' rheological properties. We then examined the NIR-sensitivity of selected hydrogels by comparing their water uptakes and storage/loss moduli. Finally, we studied the release of a model protein, bovine serum albumin (BSA), from the hydrogels with and without NIR-irradiation. Results confirmed that protein release kinetics can be controlled by NIR-irradiation, with faster and complete release of BSA compared to non-irradiated samples.

## 1. Introduction

Hydrogels are highly hydrated polymer networks held together by physical or chemical crosslinks. With their high water content, loose network and biocompatibility, they offer physicochemical properties close to the extracellular matrix of soft tissues, making them ideal for biomedical applications like protein delivery, tissue engineering, and regenerative medicine (Buwalda, Vermonden, & Hennink, 2017). Hydrogels made from degradable or bioeliminable polymers are especially attractive, as they break down completely or are fully excreted, leaving no long-term residue. Among them polyether-polyester based hydrogels, such as those using polyethylene glycol (PEG) and poly(lactide) (PLA) or poly(ε-caprolactone) (PCL), have been extensively studied due to their tunable properties, which depend on factors like the nature and crystallinity of the hydrophobic blocks, the type of bonds between polyether and polyester blocks, the copolymers topology (e.g. linear di- or triblocks copolymers, graft or star copolymers), or their thermo-responsive behavior (Basu et al., 2016; Buwalda, Nottelet, & Coudane, 2017; Dethe et al., 2022). Polysaccharide-polyester copolymer hydrogels offer similar benefits but with more chemical versatility,

thanks to the functional groups on polysaccharide chains, which allow further modifications (Basu et al., 2016). Despite these advantages, polysaccharide-polyester hydrogels have been less explored due to inherent incompatibilities, such as solubility issues, making synthesis more complex. This is the case for dextran (DEX) and PLA which common solvents are scarce making the preparation of DEX/PLA hydrogels more difficult. DEX has a backbone of d-glucopyranose units linked with α-(1 → 6) bonds and branched by α-(1 → 2), α-(1 → 3), α-(1 → 4) linkages depending on the bacterial strain used for production. It is biocompatible, biodegradable, acts as a protein-repellent hydrophilic polymer, and has numerous hydroxy groups along its backbone, allowing for the coupling of targeting or labelling molecules (Varshosaz, 2012).

A limited number of physical and chemical DEX/PLA hydrogels have been reported so far for drug delivery applications. Physical DEX/oligoPLA hydrogels were prepared by stereocomplexation between DEX-g-oligoPLA-LA and DEX-g-oligoPD-LA to deliver IgG and lysozyme (de Jong et al., 2001), whereas a reverse structure PLA-g-DEX was investigated to yield hydrogel nanofibers used as scaffolds with tunable hydrophilicity for tissue engineering (J.-F. Zhang et al., 2017). In other approaches,

<footnote><sup>a</sup> Department of Polymers for Health and Biomaterials, IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote><sup>b</sup> Department of Pharmacy, Nîmes University Hospital, 30900 Nimes, France</footnote>

<footnote>* Corresponding authors at: Department of Polymers for Health and Biomaterials, IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France. E-mail addresses: helene.van-den-berghe@umontpellier.fr (H. Van Den Berghe), benjamin.nottelet@umontpellier.fr (B. Nottelet).</footnote>
hydrolytically degradable chemical hydrogels have been prepared by
radical polymerization of DEX-g-(2-(methacryloyloxy) ethyl-L-lactate)
(Cadée et al., 1999), by UV crosslinking of mixtures of diacrylate oli-
goPLA and acrylated-DEX to deliver indomethacin, albumin or insulin
(Y. Zhang & Chu, 2001, 2002a, 2002b), or via radical crosslinking of
dextrin-g-PLA or dextrin/PLA mixtures in the presence of methylene
bisacrylamide (Das et al., 2014, 2016).

All these DEX/PLA hydrogels release their payload through classic
mechanisms like diffusion, swelling, and erosion, although many
stimuli-responsive hydrogels stimuli have been reported with specific
release triggers. These triggers can be physiological (e.g. glucose con-
centration, enzymes, pH or redox) or external (e.g. temperature, ultra-
sounds, magnetic fields, or light) to control spatiotemporal cargo release
(Thambi et al., 2016). NIR-sensitive hydrogels are particularly inter-
esting due to the NIR optical window in tissues, which enables NIR light
to penetrate several millimeters into tissues depending on the intensity

and wavelength, without causing phototoxicity to cells when suitable
irradiation time and power of NIR irradiation are used (Khan et al.,
2015; Rapp & DeForest, 2020; Weinstain et al., 2020). NIR-cleavable
bonds, which degrade under local NIR-irradiation, can be used to
trigger drug release from hydrogels. Such NIR-cleavable bonds are rare
(Peng et al., 2011). For this reason, most NIR-sensitive systems rely in
fact on concepts inspired by photodynamic therapy (PDT) (J. Chen et al.,
2020), where NIR-sensitive photosensitizers are used to generate reac-
tive oxygen species (ROS) under NIR-irradiation to break down ROS-
degradable bonds. By incorporating ROS-cleavable bonds into hydro-
gel networks, this approach enables the dual benefit of leveraging ROS
for therapeutic purposes while simultaneously achieving NIR-
degradable hydrogels. Various ROS-cleavable groups, such as thio-
ethers, diselenides, thioketals, aminoacrylates, boronic acid esters,
peroxalate esters and polyprolines, have been developed for ROS-
responsive materials (Saravanakumar et al., 2017). While recent

<figure><img src="image_5.png" /><figcaption>**Scheme 1.** Schematic illustration of the preparation of drug loaded ROS-sensitive DEX/PLA hydrogels, of their partial degradation following NIR-irradiation and of the subsequent protein release (PpIX = protoporphyrin IX; PLA-TK-MA = thioketal dimethacrylated polylactide; DEX-MA = methacrylated dextran)</figcaption></figure>
studies report using such ROS-responsive bonds to degrade particles embedded in hydrogels following NIR-irradiation of photosensitizers (Ding et al., 2022; Wu et al., 2021), only few focus on directly degrading the hydrogel network. Boronic esters have been used to release 5'-deoxy-5-fluorocytidine from core-shell poly(hydroxyethyl methacrylate)-chitosan microgels using Pheophorbide A as photosensitizer and NIR at $\lambda$ = 671 nm (Lee et al., 2019). In another study, alginate hydrogels with diselenide-based crosslinkers were developed to release doxorubicin using indocyanine green and NIR at $\lambda$ = 808 nm (Anugrah et al., 2019). It was also shown that hydrogels composed of a mixture of dithiolated poly(ethylene glycol) and dextran functionalized with acrylate-modified o-nitrobenzyl moieties could be cleaved under NIR-irradiation. Using a focused pulsed near infrared (NIR) laser beam (740 nm) the simultaneous absorption of two photons was triggered leading to the cleavage of the o-nitrobenzyl moieties. Although interesting, the photo-cleavage is limited spatially to the focal point of the laser (Peng et al., 2011). In another example, thermo-reversible C≡C double bonds formed between aldehyde groups of oxidized hyaluronic acid and cyanoacetate groups of cyanoacetate-functionalized dextran were cleaved following NIR-irradiation by exploiting the photothermal conversion ability of black phosphorus present in the gel (Ding et al., 2024). Finally, Xu et al. developed hyaluronic acid hydrogels crosslinked with thioketal linkers and bearing protoporphyrin IX (PpIX) to release doxorubicin under NIR-irradiation at $\lambda$ = 633 nm (Xu et al., 2020).

The originality of our approach lies in the design of a DEX/PLA NIR-sensitive hydrogel by crosslinking under UV a mix of methacrylated DEX (DEX-MA), methacrylated PLA bearing ROS-cleavable thioketal groups (PLA-TK-MA), and PpIX as a NIR-photosensitizer (Scheme 1). The covalent linkage of PpIX in the network allows under NIR-irradiation to generate ROS that induce the cleavage of thioketal groups and the subsequent partial degradation of the hydrogel network. We hypothesize that this approach can yield NIR-sensitive hydrogels with high, tunable stiffness controlled by DEX/PLA ratios, PLA molecular weight, and methacrylation degree. We also propose that these mechanically robust hydrogels could modulate protein release through NIR-induced ROS production from PpIX, leading to thioketal crosslink cleavage. To test our hypotheses, we discuss the properties of the resulting hydrogels based on the DEX-MA/PLA-TK-MA ratio and compare them before and after NIR-irradiation at $\lambda$ = 650 nm. Finally, we demonstrate the ability to load and control the in vitro release of the fluorescein-labeled model protein BSA-FITC from the hydrogels.

## 2. Material and methods

### 2.1. Materials

Dextran (DEX) from Leuconostoc spp., ($M_r$ = 15,000–25,000 g·mol⁻¹; experimental molecular weight $M_{n,SEC}$ = 10,000 g·mol⁻¹ and $D$ = 1.8 as determined by SEC in acetate buffer pH 4.5), glycidyl methacrylate (GMA, 97.0 %), 4-dimethylaminopyridin (DMAP, 99.0 %), hydroquinone bis(2-hydroxyethyl)ether (DHQ, 98.0 %), tin(II) 2-ethylhexanoate (92.5–100.0 %), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, 98.0 %), protoporphyrin IX (PpIX, ≥95 %), methacryloyl chloride (97.0 %), triethylamine (TEA, >99.0 %), acetone (>99.0 %), 3-mercaptopropionic acid (>99.0 %), lithium aluminum hydride solution (2.0 M in THF), sodium hydroxide, albumin-fluorescein isothiocyanate conjugate (BSA-FITC), toluene (>99.7 %), dichloromethane (DCM, >99.9 %), anhydrous N,N-Dimethylformamide (DMF, >99.8 %), Dulbecco's phosphate buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Diethyl ether (>99.8 %) was purchased from Honeywell (Riedel-de Haën). Dimethylsulfoxide (DMSO), anhydrous tetrahydrofuran (THF) were purchased from Carlo Erba reagents. Ethanol absolute was purchased from VWR chemicals. $D_{51}$-lactide was purchased from Corbion. All chemicals were used without further purification except for DMSO,

DMF, toluene, $D_{51}$-lactide, DHQ, TEA and DCM. DMSO and DMF were dried on molecular sieve at least 48 h before use. Toluene and DCM were freshly distilled before use. $D_{51}$-lactide and DHQ were dried overnight in a desiccator before use. TEA was dried on molecular sieve and stored under argon atmosphere.

### 2.2. Synthesis of hydrogels

#### 2.2.1. Synthesis of methacrylated dextran (DEX-MA)

DEX was methacrylated to yield a photo-crosslinkable DEX using a method reported in literature (Van Dijk-Wolthuis et al., 1995). Briefly, 4 g of dextran ($M_n$ (glucopyranosyl unit) = 166 g·mol⁻¹, 0.024 mol, 1 eq. of glucopyranosyl units) were dissolved in 40 mL of dried DMSO at 40 °C for 2 h under argon atmosphere to prevent DEX oxidation. After dissolution, defined amounts of DMAP (147 mg, 0.05 eq.; 293 mg, 0.1 eq.; or 804 mg, 0.27 eq. per glucopyranosyl unit) and GMA (0.32 mL, 0.1 eq.; 0.64 mL, 0.2 eq.; or 1.8 mL, 0.54 eq.), calculated according to the targeted degree of substitution, were added dropwise. The solution was stirred at 40 °C for 3 h, then at RT for 48 h under argon atmosphere. The reaction mixture was concentrated by evaporation of DMSO and then added dropwise in 400 mL of cold EtOH. The white precipitate was filtered off, dissolved in 200 mL of water and purified by ultrafiltration using a Sartorius, Vivaflow 50 R (5000 MWCO HY), ultrafiltration system with a peristaltic MasterFlex Cole-Parmer pump (flow at 2 mL·min⁻¹ rate, $P$ = 1.5 bar) until the volume of concentrate and the volume of filtrate = 100 mL. DEX-MA concentrate was lyophilized to obtain white, fluffy product. Nature, purity and degree of substitution (DS = number of methacrylate groups per 100 glucopyranosyl groups) of the modified dextran were determined by SEC and ¹H NMR in $D_2O$. DEX-MA with DS of 3 %, 12 % and 45 % were obtained with yields around 75 %.

¹H NMR (600 MHz, $D_2O$): $\delta$ (ppm) = 6.25 (m, 1H, Ha), 5.80 (m, 1H, Ha), 5.00 (s, 1H, $H_1$ anomeric), 3.55–4.02 (m, $H_{2-6}$ glucosidic), 2.00 (s, 3H, Hb) (Fig. S1).

¹³C NMR (600 MHz, $D_2O$): $\delta$ (ppm) = 169.34 (Cd = 0), 135.49 (Cc = CH₂), 127.26 ($H_2$Ca = C), 97.58 (C₁ anomeric), 69.37–73.27 (C₂–5 glucosidic), 65.35 (C₆ glucosidic), 17.40 (C₆H₃) (Fig. S2).

#### 2.2.2. Synthesis of 3,3'-[(1-methylethylidene)bis(thio)]bis-propan-1-ol (TK)

TK was prepared according to a two steps procedure of the literature (McEnery et al., 2016). In the first step, 3 mL of acetone and 8,8 mL mercaptopropionic acid were stirred for 96 h at 50 °C in a round bottom flask. The mixture was poured dropwise into a beaker containing iced water. The solution was then filtered, washed with water and dried with a desiccator for 24 h. 3,3'-[(1-methylethylidene)bis(thio)]bis-propanoic acid was obtained with a 20 % yield.

¹H NMR (400 MHz, DMSO-d₆): $\delta$ (ppm) = 12.28 (m, 2H, Ha), 2.74 (t, 4H, $^3J_{Hc}$, $Hb$ = 7.2 Hz, Hc), 2.51 (t, 6H, $^3J_{Hb}$, $Hc$ = 7.2 Hz, Hb + DMSO), 1.53 (s, 6H, Hd) (Fig. S5).

In a second step the carboxyl groups of the intermediate were reduced to produce the hydroxy-terminated TK. Under positive nitrogen pressure, 1.7 g of intermediate was introduced in a dried round bottom flask fitted with a condenser and a constant pressure dropping funnel capped with a rubber stopper. Using anhydrous techniques, 170 mL of anhydrous THF were added in the dropping funnel and were partly dropped in the flask to dissolve the intermediate. After solubilization the reactor was placed in an ice bath and 18 mL of a 2 M THF solution of $LiAlH_4$ was added in the dropping funnel and added dropwise to the intermediate solution at 0 °C. After addition of the $LiAlH_4$ solution, the ice bath was removed, and the reaction medium was heated for 2 h at reflux under nitrogen at 55 °C. The reaction mixture was then slowly added into 80 mL deionized water to quench the excessive $LiAlH_4$ and few drops of 15 % NaOH aqueous solution was added to adjust the pH value around 7–8. The resulting suspension was filtered with a Buchner funnel to remove the by-products. The filtrate was introduced in a flask and concentrated under reduced pressure. The solution was then placed
in a separation funnel and washed with diethyl ether (3 × 70 mL). The organic phases were dried on MgSO₄, the solvent was removed by rotary evaporation and the product dried under vacuum overnight to yield dry TK with a 20 % yield.

¹H NMR (400 MHz, DMSO-d₆): δ(ppm) = 4.49 (m, 2H, Ha), 3.44 (t, 4H, ³J Hd,Hc = 8.0 Hz, Hd), 2.60 (t, 4H, ³J Hb,Hc = 4.0 Hz, Hb), 1.64 (dd, 4H, ³J Hc,Hb = 8.0 Hz, ³J Hc,Hd = 4.0 Hz, Hc), 1.53 (s, 6H, He) (Fig. S6).

### 2.2.3. Synthesis of thioketal dimethacrylated PLA (PLA-TK-MA)

PLA and ROS-cleavable PLA-TK were obtained by ring opening polymerization using DHQ or TK as initiator, respectively. Procedures were similar with exception of the initiator and the following illustrates the synthesis of PLA-TK. Typically for a targeted M_n = 5000 g.mol⁻¹, 4 g of D,L-lactide (LA, 28 × 10⁻³ mol, 34 eq.) and 184 mg of TK (8.2 × 10⁻⁴ mol, 1 eq.) were introduced into a dried Schlenk. Three argon-vacuum cycles were applied and the vacuum was maintained during 1 h. Then, 0.065 g of Sn(Oct)₂ (1.6 × 10⁻⁴ mol, 0.2 eq.) and 50 mL of freshly distilled toluene were added to the Schlenk. The medium was stirred for 30 min to ensure complete dissolution of LA and the Schlenk was placed in a hot oil bath heated at 100 °C for 48 h. After polymerization, toluene was eliminated under reduced pressure to yield an oil that was as precipitated in cold Et₂O. The final copolymer was filtered off and dried under reduced pressure to constant mass to obtain PLA-TK as a white powder (75 % yield).

¹H NMR (400 MHz, CDCl₃) δ(ppm) = 5.15 (m, 2H, Hc), 4.35 (m, 2H, Hb), 4.20 (m, 4H, He), 2.62 (t, 4H, Hg), 1.91 (m, 4H, Hf), 1.54 (m, 6H, Ha; 6H, Hd; 6H, Hh) (Fig. S7).

Methacrylation was then performed following the same procedure with PLA and PLA-TK. Briefly, 1 g of PLA-TK (M_n = 5000 g.mol⁻¹, 1 eq.) was introduced in a two-necked flask fitted with a condenser and a dropping funnel and dissolved in 40 mL of freshly distillated DCM. 0.21 mL of TEA (10 eq.) were added to the reactor which was subsequently placed in an ice bath for 1 h. After that, 0.15 mL of methacryloyl chloride (10 eq.) were diluted in 5 mL of freshly distillated DCM and added dropwise using the dropping funnel. The ice bath was maintained 1 h and the reactor was covered with aluminum foil. After another hour at room temperature, the reaction was performed at 45 °C for 72 h. The mixture was then washed with water (3 × 30 mL), dried over MgSO₄, and evaporated under reduced pressure. The orange solution was precipitated in cold Et₂O and the solid was dried overnight. PLA-TK-MA was obtained as a yellow powder (70 % yield).

¹H NMR (400 MHz, CDCl₃) δ(ppm) = 6.20 (m, 2H, Ha), 5.63 (m, 2H, Ha), 5.14 (m, 2H, Hc), 4.20 (m, 4H, He), 2.63 (t, 4H, Hg), 1.96 (m, 4H, Hf), 1.57 (m, 6H, Hb; 6H, Hd; 6H, Hh) (Fig. 1A).

### 2.2.4. Preparation of ROS-cleavable hydrogels

Depending on the targeted DEX-MA/PLA-TK-MA weight ratio, a total of 150 mg of DEX-MA and PLA-TK-MA were dissolved in DMF. In a

<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Characterization of PLA-TK. (A) ¹H NMR (400 MHz, CDCl₃) of PLA-TK-MA. (B) Kinetics of ROS-cleavage of PLA-TK under NIR-irradiation in the presence of PpIX (λ = 650 nm, THF) by SEC analysis. (C) Illustration of the cleavage of the TK intermediate 3,3'-[(1-methylethylidene)bis(thio)]bis-propanoic acid in the presence of PpIX after 1 h irradiation at λ = 650 nm with top row reaction scheme and bottom row ¹H NMR peaks of interest to follow the cleavage before and after 1 h of irradiation in deuterated DMSO. (D) Proof of quantitative ROS-cleavage of PLA-TK upon oxidation by H₂O₂ (DMF, 37 °C, 72 h) followed by SEC.</figcaption></figure>
typical procedure, 85/15 DEX-MA/PLA-TK-MA gels were prepared as follows. In a first vial, DEX-MA (127 mg) was dissolved in 0.9 mL DMF and stirred for 30 min protected from light. PLA-TK-MA (23 mg) was then added and the solution was further stirred for another 30 min. Then, 0.1 mL of a PpIX solution (1 mg.mL⁻¹ in DMF) and 0.5 mL of Irgacure 2959 solution (4 mg.mL⁻¹ in DMF) were added. The resulting solution was poured in a mold and irradiated for 10 min with a UV Dmax Bluewave® device equipped with a mercury bulb (280–400 nm). DMF was then removed via solvent exchange by putting the gel in a large excess of water (100 mL) for 2 days with frequent water changes.

### 2.2.5. PLA-TK thioetal bonds cleavage

For model kinetic studies, the TK intermediate 3,3'-[(1-methyl-ethylidene)bis(thio)]bis-propanoic acid was cleaved under NIR-irradiation in the presence of PpIX. In a typical experiment, 10 mg TK intermediate (1 eq.) and PpIX (0.04 eq.) were dissolved in 0.6 mL DMSO-d₆ or deuterated DMF. The solution was then irradiated for different times at 650 nm using a Flexpoint FP/65 laser (900 mW). For each time, the solution was analyzed by ¹H NMR.

PLA-TK degradation was studied under oxidative conditions using H₂O₂ as oxidant and CuCl₂ as catalyst (B. Chen et al., 2019). In brief, PLA-TK (110 mg, 10 µmol TK) was dissolved in a solution of DMF (0.8 mL) and H₂O₂ aqueous solution at 800 mM (0.18 mL), before addition of CuCl₂ solution (74 µM, 17 µL). The reaction medium was heated at 37 °C for 72 h before analyses by SEC.

PLA-TK degradation was also studied under NIR-irradiation. In a typical experiment, 100 mg of PLA-TK (Mₙ = 5000 g.mol⁻¹, 2 × 10⁻⁵ mol TK) was dissolved in 3 mL of THF at room temperature. Then 1.1 mg of PpIX (2 × 10⁻⁶ mol, 10 eq.) was added to the solution. The latter was divided into Eppendorf vials, one for each irradiation time of the study (0 min, 5 min, 10 min, 15 min, 20 min, 30 min). The mixture was then irradiated under NIR-irradiation at 650 nm (900 mW). This wavelength was selected as PpIX strongly absorbs over a domain of wavelengths ranging from 380 to 650 nm (Croizat et al., 2022) and that PDT that requires NIR tissue penetration uses wavelength between 600 and 800 nm (Desgranges et al., 2022). Each solution was then analyzed by SEC.

## 2.3. Characterization

¹H, ¹³C, ¹H¹H COSY, ¹H¹³C HSQC and ¹H¹³C HMBC nuclear magnetic resonance (NMR) spectra were recorded using a Bruker Avance III HD spectrometer operating at 400 or 600 MHz at room temperature. The solvents used were deuterium oxide (D₂O), deuterated chloroform (CDCl₃), deuterated dimethyl formamide (DMF-d₇) or deuterated dimethyl sulfoxide (DMSO-d₆). The chemical shifts were expressed in ppm.

Average molecular weights (Mₙ) and dispersities (D) of polysaccharides were determined by SEC in acetate buffer (pH 4.5) using a Waters 515 HPLC pump equipment fitted with TSKgel PW5000 and TSKgel PW3000 columns (Tosoh Bioscience) coupled with a Waters 410 refractometric detector at 35 °C. Calibration was performed with pullulan standards (pullulan calibration kit PSS-PULKITR1, Agilent). The samples were analyzed with acetate buffer (pH 4.5) as mobile phase at 1 mL.min⁻¹ flow rate. Results were analyzed using OmniSec software. Polymer samples were dissolved in acetate buffer (10 mg.mL⁻¹) and then filtered through Chromafil Xtra filter (regenerated cellulose) before injection (20 µL).

Average molecular weights (Mₙ) and dispersities (D) of polyesters were determined by SEC in THF with a Shimadzu equipment using PLGel MIXED-C 2 × 300 mm columns and a Shimadzu RI detector 20-A (40 °C thermostatic analysis cells). THF was the mobile phase with 1 mL. min⁻¹ flow at 30 °C (column temperature). Polymer was dissolved in THF to reach 10 mg.mL⁻¹ concentration and filtered through a 0.45-mm PTFE Millipore filter before injection. Mₙ and D were expressed according to calibration using polystyrene standards with LabSolutions Lite Main software.

The mechanical properties (elasticity modulus G' and viscosity modulus G") of hydrogels were evaluated using an Elastosens Bio² (Rheolution, Quebec, Canada, now named Elastosens™ Bio) in stiff mode (temporal step = 15 s) at 25 °C and 37 °C. Hydrogels (concentration of 100 g/L, volume of 1.5 mL) were prepared in a well plate (diameter of 2.2 cm), removed from the mold after irradiation, washed with water and transferred to the Elastosens Bio² sample holders for analysis. Data were analyzed with Elastoview software version 18.12.8.

## 2.4. Characterization of hydrogels and evaluation of the impact of NIR-irradiation

For the swelling kinetics, the water uptake of the gels immersed in PBS at pH 7.4 and 37 °C as a function of the time of immersion was followed. The initial weight (W₀) and the weight at defined time points (W) were measured. The water uptake was calculated from Eq. 1.

$$ Water\ uptake\ (%) = \frac{W - W_0}{W_0} \times 100 \qquad (1) $$

For the comparison of the water uptakes before and after NIR irradiation, the gels containing PpIX were dried in a freeze-dryer overnight and weighed to obtain their dry weight (W₀). They were then immersed in 20 mL of PBS solution for 30 min at 37 °C, recovered and weighed again to obtain the hydrated mass (W). Excess of PBS was removed with absorbent paper and gels were then placed in a glass vial and NIR-irradiated at 650 nm for 2 h. At the end of the irradiation, they were immersed once more in a 20 mL PBS solution for 30 min and weighed at the end (W'). The water uptakes were determined before and after irradiation according to Eq. 1.

To evaluate the impact of NIR-irradiation on the moduli of the gels, swollen gels were placed in the sample holder of Elastosens Bio² rheometer and were then continuously NIR-irradiated at 650 nm with measurements of the storage modulus every 40 min.

## 2.5. Loading and release of model protein

### 2.5.1. Loading of hydrogel with BSA-FITC

Based on water uptake measurements, the average volume of PBS solution absorbed by a predefined weight of hydrogel was determined. According to this average volume, 3 mL of a BSA-FITC solution was prepared in PBS with a predefined concentration allowing to incorporate 2.5%wt of protein. Dry hydrogels (20 mg) were immersed into BSA-FITC solution at 37 °C for 30 min under constant orbital shaking (Heidolph Unimax 1010) and then weighed to extrapolate the amount of incorporated BSA-FITC based on the initial protein concentration in buffer. BSA-FITC was selected over BSA for an easy detection by UV/ visible spectrophotometry in the visible domain at 495 nm.

### 2.5.2. In vitro BSA-FITC release study

In vitro BSA-FITC release experiments were performed in PBS (pH 7.4) at 37 °C under constant orbital shaking for 24 days. Non irradiated hydrogels or hydrogels photo-irradiated at 650 nm for 2 h, were immersed into 2 mL of PBS. At specific time points, 1 mL of medium was removed and replaced by 1 mL of fresh PBS. Of notice this volume was sufficient to guarantee sink conditions for the readily soluble BSA-FITC in PBS. Collected samples were analyzed by UV-vis spectrophotometry at 495 nm (PerkinElmer Lambda 35 UV/vis spectrophotometer) with reference to a calibration curve of BSA-FITC in PBS. The amount of released BSA-FITC (RBSA-FITC) was calculated based on eq. 2 with mR BSA-FITC the mass of released BSA-FITC and mBSA-FITC the mass of BSA-FITC initially loaded in the hydrogel.

$$ R_{BSA-FITC} (\%) = 100 \times m_{R BSA-FITC} / m_{BSA-FITC} \qquad 2 $$
## 2.6. Statistical analyses

Reported values are presented as the average of three samples per condition and expressed as means ± standard deviation. The statistical comparisons of the results were evaluated using R software (V 3.6.1). Nonparametric data results were analyzed using Wilcoxon rank test. A p value < 0.05 was considered statistically significant. In addition, for Fig. 4A and B, nonparametric data results were analyzed using Wilcoxon rank test with Benjamini-Hochberg procedure applied to control False Discovery Rate (FDR) at a 5 % level for data.

# 3. Results and discussion

## 3.1. Synthesis of polymer precursors

To prepare NIR-cleavable PLA/DEX hydrogels, two photo-crosslinkable polymeric precursors were synthesized. At first, methacrylated-dextran (DEX-MA) was obtained by reacting DEX ($M_n = 10,000 \text{ g.mol}^{-1}$) and glycidyl methacrylate. The methacrylation ratio was modulated to evaluate the influence of this parameter on the rheological properties of the hydrogels. Three ratios were therefore targeted based on the DEX-MA of the literature (see Table S1): a low ratio (5 % target), an intermediate ratio (15 % target) and a high ratio (50 % target). By changing the GMA equivalents, DEX-MA were obtained with 3 % (DEX-MA$_3$), 12 % (DEX-MA$_{12}$) and 45 % (DEX-MA$_{45}$) of methacrylate groups. $^1$H NMR of DEX-MA derivatives showed the peaks characteristics of DEX ($H_1$ anomeric at 5.01 ppm; $H_{2-6}$ glucosidic at 3.55–4.02 ppm) as well as the peaks of methacrylate groups, i.e. the Ha alkene protons at 6.25 and 5.80 ppm and the Hb methyl protons at 2.00 ppm. The extent of methacrylation was calculated by comparison of the intensities of the peak at 5.00 ppm, corresponding to the H1 anomeric proton, and of the peaks of Ha alkene protons (6.25 and 5.80 ppm) (Fig. S1, for clarity typical NMR spectra used for illustration purposes correspond to DEX-MA$_{45}$). $^1$H-$^1$H COSY NMR confirmed the assignments of methacrylate chemical shifts with a correlation between the Ha alkene protons and the Hb methyl protons (Fig. S3). $^{13}$C NMR were also performed on DEX-MA derivatives and typically showed the C1 anomeric signal at 97.58 ppm while C2–5 appeared in the region 69.37–73.27 ppm and C6 at 65.35 ppm. The methacrylate groups were identified at 169.34 ppm (Cd = O), 135.49 (Cc = CH$_2$), 127.26 ($H_2Ca = C$) and 17.40 ppm ($CbH_3$) (Fig. S2). $^1$H-$^{13}$C HSQC and HMBC experiments were performed on DEX-MA derivatives to confirm the assignments of the $^1$H and $^{13}$C NMR analyses. Based on comparison of reported literature, the α-configuration of sugar residues was confirmed with a typically single $^{13}$C anomeric signal observed at 97.58 ppm which correlated to the $H_1$ anomeric proton at 5.00 ppm in the HSQC spectrum (Fig. S4A). These chemical shifts are characteristics of α-(1 → 6) glucosyl residues linkages (Yang et al., 2015). The assignments of methacrylate chemical shifts were also confirmed in the HSQC spectrum. These different correlations were further corroborated from HMBC experiment (Fig. S4B) and the literature (Nonsuwan & Matsumura, 2019; Ridella et al., 2024; Yang et al., 2015).

In a second step, 3,3'-[(1-methylethylidene)bis(thio)]bis-propan-1-ol (TK) was prepared in two steps according to the literature (McEnery et al., 2016) (Figs. S5 and S6) and used as ROS-sensitive thioketal-based initiator for the ring opening polymerization (ROP) of PLA. To favor the hydrophilicity and degradation of the targeted PLA/DEX hydrogels, the amorphous P(D,L-LA) was selected over its semi-crystalline counterparts. ROP was conducted under standard conditions and PLA with a central thioketal group (PLA-TK) was obtained in good yield (75 %). An average number molecular weight of 5200 g.mol$^{-1}$ was calculated based on the $^1$H NMR spectrum using the average integration of the peak at 2.62 ppm of TK and of the peak at 4.35 ppm of the methine group of PLA chain end, compared to the peak at 5.15 ppm of the methine group of PLA chain (Fig. S7). Size exclusion chromatography showed a monomodal distribution corresponding to an average number molecular

weight of $M_{nSEC} = 7500 \text{ g.mol}^{-1}$ with a dispersity of 1.25. The quantitative methacrylation of the hydroxy chain-ends to yield PLA-TK-MA was confirmed by $^1$H NMR by comparison of the intensities of the peaks at 6.20 and 5.63 ppm of the alkene protons with the one of the peak at 5.14 ppm corresponding to the methine of the PLA chain (Fig. 1A). Table 1 summarizes the characteristics of the polymers obtained.

## 3.2. Evaluation of thioketal cleavage

The cleavage of PLA-TK was tested in THF, a good solvent for the polymer, and tracked using SEC analysis. Chromatograms in Fig. 1B show the extent of cleavage by comparing peak areas. ROS-induced PLA chain scission reached 5 % after 5 min and about 12 % after 30 min. Next, the cleavage of the thioacetal group alone, in the presence of NIR-sensitive PpIX, was evaluated based on irradiation time and solvent. Solutions of 3,3'-[(1-methylethylidene)bis(thio)]bis-propanoic acid in deuterated DMSO or DMF were irradiated at 650 nm (900 mW) in closed NMR tubes. These solvents were chosen because they dissolve both PLA-TK-MA and PpIX, allowing ROS-sensitivity testing for this hydrophobic polymer. Fig. 1C shows the cleavage reaction and $^1$H NMR spectra before and after irradiation. Shifts of the methylene peaks from 2.74 and 2.48 ppm in the thioketal compound to 2.88 and 2.62 ppm in the resulting mercaptopropionic acid, and the new peak at 2.09 ppm corresponding to released acetone, were used to measure the extent of cleavage. The kinetics spectra are in the supporting information (Figs. S8 and S9). Results confirmed ROS-induced cleavage of the thioketal group after NIR-irradiation of PpIX, with faster and more complete cleavage in DMF (46 % after 1 h, 100 % after 2 h) compared to DMSO (11 % after 1 h, 81 % after 2 h).

Finally, we studied the cleavage of PLA-TK solubilized in DMF under classical oxidative conditions ($H_2O_2$ as oxidant and $CuCl_2$ as catalyst) without NIR-irradiation (Chen et al., 2019). SEC analyses of the polymer before and after oxidative stress confirmed that PLA-TK could be quantitatively cleaved under these conditions (Fig. 1D).

This set of data confirmed the possibility to generate the oxidative degradation of the PLA-TK pre-polymer, which led us to further develop the hybrid DEX/PLA-TK hydrogels.

## 3.3. Preparation of hydrogels

### 3.3.1. Selection of optimal hydrogels compositions with non-cleavable systems

A first generation of hydrogels was prepared by mixing the non-cleavable PLA-MA and DEX-MA. The objective of this study was to investigate the influence of various parameters on the swelling and rheological properties of the resulting gels. The studied parameters were the ratio of DEX-MA/PLA-MA, the molecular weight of PLA-MA and the methacrylation degree of DEX-MA.

To perform these tests, non-cleavable PLA-MA were first synthesized under the same conditions as PLA-TK-MA by replacing TK by hydroquinone bis(2-hydroxyethyl) ether (DHQ) as initiator. Molar masses of 5000 g.mol$^{-1}$ (PLA-MA$_{5k}$) and 2500 g.mol$^{-1}$ (PLA-MA$_{2.5k}$) were targeted and $M_{n,NMR} = 4600 \text{ g.mol}^{-1}$ and 2570 g.mol$^{-1}$ were calculated from $^1$H NMR spectra (not shown).

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

Characteristics of the DEX-MA and PLA-TK-MA synthesized in this work.</caption><thead><tr><th>Polymer</th><th>Molecular weight ($M_n$, g.mol<sup>-1</sup>)</th><th>Methacrylation <sup>b</sup> (%)</th></tr></thead><tbody><tr><td>DEX-MA<sub>3</sub></td><td rowspan="3">10 000<sup>a</sup></td><td>3</td></tr><tr><td>DEX-MA<sub>12</sub></td><td>12</td></tr><tr><td>DEX-MA<sub>45</sub></td><td>45</td></tr><tr><td>PLA-TK-MA</td><td>5200<sup>b</sup></td><td>100</td></tr></tbody></table>

<footnote><sup>a</sup> determined by SEC analysis in acetate buffer (pH 4.5)</footnote>

<footnote><sup>b</sup> determined by $^1$H NMR analysis.</footnote>
Gels were then prepared as described in the experimental part by mixing pre-polymers at defined ratios in DMF before UV-crosslinking. Hydrogels were obtained by diffusional replacement of DMF by water. Four series of hydrogels were prepared to evaluate the impact of the DEX-MA/PLA-MA ratios (ranging from 95/5 to 50/50), of the PLA-MA molecular weights (PLA-MA<sub>5k</sub> or PLA-MA<sub>2.5k</sub>), and of the methacrylation degree of DEX-MA (3, 12 or 45%). For this screening, the visual aspect and the rheological properties of all samples were assessed using an ElastoSens Bio<sup>2</sup> Rheolution at 25 °C and 37 °C. Since no significant difference was found on the storage modulus (G') for these two temperatures (Fig. S10), all following experiences were run at the physiological temperature of 37 °C. Stiffer and more homogenous gels were obtained with increased ratios of DEX-MA<sub>45</sub> in the gels due to the higher content of photo-crosslinkable methacrylate groups. Stiffer gels were also obtained when PLA-MA<sub>5k</sub> was used over PLA-MA<sub>2.5k</sub> (Fig. S11). In a second run of experiments the impact of DEX-MA methacrylation was assessed. No gels formed with DEX-MA<sub>3</sub>, regardless of the DEX-MA/PLA-MA ratio or the PLA-MA molecular weight. For DEX-MA<sub>12</sub> no gels were obtained for ratios below 90/10. At this ratio, soft gels with similar G' values (between 1 and 2 kPa) were obtained for both DEX-MA<sub>12</sub> and DEX-MA<sub>45</sub>. G' remained unchanged for increased contents of DEX-MA<sub>12</sub>, while it increased with DEX-MA<sub>45</sub>, reaching 4 kPa (Fig. S12).

### 3.3.2. Cleavable hydrogels

Based on these results we prepared ROS-cleavable hydrogels with PLA-TK-MA<sub>5k</sub> and DEX-MA<sub>12</sub> or DEX-MA<sub>45</sub>. A DEX-MA/PLA-TK-MA ratio of 85/15 was chosen to create stiff hydrogels while ensuring enough thioketal groups for ROS-induced cleavage. To enable degradation under NIR-irradiation via ROS generation and thioketal cleavage, protoporphyrin IX (PpIX) was added to hydrogel structure. To prevent PpIX from leaching out, it was covalently bonded during photo-crosslinking using available alkene groups (Scheme 1). This method, previously reported in the literature (Yue et al., 2016) does not affect the photosensitizing function of PpIX. After thorough washing, the hydrogels showed a reddish color typical of bound PpIX. The water-uptake in phosphate buffer saline at pH 7.4 and 37 °C was assessed for hydrogels with and without PpIX (Fig. 2A). The incorporation of PpIX in the network showed some influence on the water-uptake with differences observed for different methacrylation degrees of DEX. For DEX-MA<sub>12</sub>/PLA-TK-MA<sub>5k</sub> this impact was limited as shown by the close values of ~1450% and ~1550% water absorption in hydrogels without and with PpIX, respectively.

This impact was higher in DEX-MA<sub>45</sub>/PLA-TK-MA<sub>5k</sub> hydrogels that

absorbed about half as much water ~750% without PpIX due to the higher crosslinking density from using DEX-MA<sub>45</sub>, but reached ~1050% when PpIX is introduced in the network (Fig. 2A). This difference is likely due to a more pronounced disturbance of the network formation in the more crosslinked hydrogel. For comparison, hydrogels made from DEX-MA (Mw 43,000 g/mol) with low substitution degree (5 photo-reactive groups per 100 anhydroglucose units) and low molecular weight PLA (Mw 740/mol) reached only 80% water uptake at a similar DEX:PLA weight ratio of 80:20 (Y. Zhang & Chu, 2002a). On the other hand, DEX-MA with similar properties and oligo-lactate (2 units) resulted in very high water uptake of 3000% (Cadée et al., 1999). Lastly, with shorter DEX-MA (Mw 10,000 g/mol) and PLA initiated directly from DEX hydroxy groups, intermediate water uptake of 300% was achieved at a DEX:LA ratio of 95:5 (Das et al., 2014, 2016).

Our objective being to evaluate NIR-responsive gels, the rheological properties of the PpIX hydrogels were then evaluated with an ElastoSens Bio<sup>2</sup> Rheolution equipment. Storage (G') and loss moduli (G") are shown in Fig. 2B. The covalent bonding of PpIX to the network did not affect the rheological properties, which were similar to those of the non-cleavable gels. Specifically, DEX-MA<sub>45</sub>/ PLA-TK-MA<sub>5k</sub> formed stiffer gels at 37 °C with a more elastic behavior (G' = 1.55 kPa, tan δ = 0.06) compared to the more viscous DEX-MA<sub>12</sub>/PLA-TK-MA<sub>5k</sub> (G' = 0.78 kPa tan δ = 0.25). The only example of DEX/PLA hydrogel reporting rheological data in the literature is the one developed by Das et al., which showed values of G' = 6 kPa and tan δ = 0.25 (Das et al., 2014, 2016). However, comparison is difficult due to the different gel formulations and synthetic approaches (PLA initiated directly from DEX backbone with no molecular weight characterization, DEX-MA Mw = 10,000 g/mol, DEX:LA wt % 95:5).

### 3.3.3. NIR-sensitivity of the hydrogels

The ability of the two hydrogels to respond to NIR-irradiation was assessed by comparing their water uptakes before and after 2 h of irradiation at 650 nm. NIR-irradiation affected water uptake depending on the methacrylation of DEX-MA. Water uptake increased by 11% for DEX-MA<sub>45</sub>/PLA-TK-MA<sub>5k</sub> (from ~1050% to 1200%) but decreased by 12.5% for DEX-MA<sub>12</sub>/PLA-TK-MA<sub>5k</sub> (from ~1550% to 1350%) (Fig. 3A). These differences were not found statistically significant using a Wilcoxon rank test. The results should therefore be considered as a trend for our materials based on mean ± SD values provided. This trend is explained by the initial crosslinking densities. In the stiffer, more crosslinked DEX-MA<sub>45</sub>-based hydrogel, NIR-irradiation loosens the network, restoring chain mobility and increasing water uptake. In contrast, the already loose DEX-MA<sub>12</sub>-based hydrogel experiences

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Characterization of the DEX-MA/PLA-TK-MA<sub>5k</sub> (85/15) hydrogels. (A) Maximal water uptake of DEX-MA<sub>45</sub>/PLA-TK-MA<sub>5k</sub> and DEX-MA<sub>12</sub>/PLA-TK-MA<sub>5k</sub> hydrogel without (dashed bars) and with (plain bars) covalently linked PpIX (values are expressed as mean ± SD, *n* = 3); and (B) storage (G') and loss (G") moduli of the DEX-MA/PLA-TK-MA<sub>5k</sub> (85/15) hydrogels containing PpIX as a function of the extent of methacrylation of DEX-MA (mean and standard deviation from *n* = 1 using 4 values of moduli measured once equilibrium on the swelling plateau was reached).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** NIR-sensitivity of the hydrogels. (A) Water uptake (values are expressed as mean ± SD, n = 3) and (B,C) typical rheological characterization (storage (G') and loss (G'') moduli) of DEX-MA₁₂/ PLA-TK-MA₅k (85/15) and DEX-MA₄₅/ PLA-TK-MA₅k (85/15) hydrogels before and after NIR-irradiation.</figcaption></figure>

further loosening, reducing its swelling capacity as the network struc-
tural integrity weakens. A swelling optimum, depending on crosslinking
density, has been described for chemically crosslinked hydrogels from
both natural and synthetic polymers (Dainiak et al., 2010; X. Yang et al.,
2023). In our case both hydrogels show different swelling behaviors
after NIR-irradiation, as their crosslinking densities are on opposite sides
of their respective optima.

Rheological evaluations further confirmed these behaviors. G'
remained unchanged for DEX-MA45/ PLA-TK-MA5k (G' ~ 1.6 kPa) due to
the high initial crosslinking density. However, cleavage of the thioketal
groups and the resulting breakage of PLA chains caused G'' to double
100 Pa to 200 Pa (Fig. 3B), explaining the more viscous behavior of the
viscoelastic gel and its increased swelling capacity after NIR-irradiation.
For DEX-MA12/ PLA-TK-MA5k the behavior was again the opposite. This
gel had a looser network and initially more viscous character, so G'' was
unaffected by the cleavage, staying around 200 Pa before and after NIR-
irradiation. However, G' dropped by 200 Pa, from 700 Pa to 500 Pa,
indicating a loss of crosslinking points, which was also reflected in its
lower swelling capacity after NIR-irradiation (Fig. 3C).

**3.3.4. NIR-triggered protein release**

The ability to control protein release from the gels using NIR-
irradiation was assessed to explore the potential of hydrogels for
photo-triggered release of biologics. The release of bovine serum
albumin-fluorescein isothiocyanate (BSA-FITC) from both irradiated
and non-irradiated hydrogels were tested under sink conditions. For
NIR-irradiated DEX-MA45/PLA-TK-MA5k hydrogels, the release of BSA-
FITC was not faster, with 60 % release in 3 days in both cases, and

full release in 24 days (Fig. 4A). The absence of statistical difference for
the release was confirmed using a Wilcoxon rank test. This was expected
due to the lower impact of thioketal cleavage in this highly crosslinked
hydrogel, where the extent of PLA-TK cleavage is likely too small to
significantly reduce the number of crosslinking nodes that are mainly
due to DEX-MA45.

This was different for DEX-MA₁₂/PLA-TK-MA₅k, where a faster and more complete release of BSA-FITC was achieved after NIR-irradiation (Fig. 4B). This difference was found statistically significant up to 192 h using a Wilcoxon rank test of release with a *p* value of 0.05. Of notice, this was not the case using the same test with the Benjamini-Hochberg procedure applied to control False Discovery Rate (FDR) at a 5 % level, which shows that our results should be considered as a trend of faster release following NIR-irradiation of DEX-MA₁₂/PLA-TK-MA₅k in the early time points. In more details, nearly complete release occurred in about 8 days for irradiated hydrogels, while a maximum of 88 % was reached without NIR-irradiation after 24 days. Additionally, under NIR-irradiation, 80 % of the BSA-FITC was released in just 2 days, compared to 63 % in non-irradiated hydrogels, showing the faster release rate induced by NIR-irradiation.

The release profiles were fitted to various models to better under-
stand the release mechanism. All profiles confirmed that the release
followed Fickian diffusion. Specifically, release from DEX-MA45/ PLA-
TK-MA5k before and after irradiation, as well as from DEX-MA12/ PLA-
TK-MA5k after irradiation, fit the simple Higuchi model (Fig. S13). For
DEX-MA12/PLA-TK-MA5k before irradiation, the more complex Weibull
model was used, with a β parameter value of 0.35 (Fig. S14).

Finally, we compare our results with the literature by presenting
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** NIR-triggered BSA-FITC release from hydrogels. Release kinetics of BSA-FITC before (red marks) and after (blue marks) NIR-irradiation with (A) DEX-MA<sub>45</sub>/PLA-TK-MA<sub>5k</sub> (85/15) and (B) DEX-MA<sub>12</sub>/PLA-TK-MA<sub>5k</sub> (85/15) hydrogels (values are expressed as mean ± SD, n = 4). Release curves on the right-hand side correspond to zooms of the full release curves over the first 35 h.</figcaption></figure>

relevant examples of chemically crosslinked DEX-based hydrogels used for BSA release, summarized in Table S1. Most are purely hydrophilic, made either of DEX alone or with PEG segments, and their release kinetics depend heavily on the molecular weight of DEX and DS (which affects crosslinking density), as seen in hydrogels 1 vs. 2 or 3 vs. 4 (Table S1). Due to their purely hydrophilic nature, comparison with our system is difficult, though some show similar release behavior (hydrogels 1 and 3). Notably, these examples lack the hydrolytic degradation provided by PLA blocks in our system, and, without rheological data, we expect lower moduli for these hydrogels. The most comparable system is hydrogel 5, which contains both DEX and short PLA segments (Mw 740 g/mol). Its BSA release is more limited than in our hydrogels, with only 50 % released after 60 days and no full release. These results confirm the potential of our hydrogels as protein reservoirs, where release profiles

can be adjusted using NIR-irradiation.

4. Conclusion

We successfully developed degradable ROS-sensitive hybrid hydro-
gels by combining methacrylated dextran (DEX-MA) with methacrylated
thioketal-functional poly(lactide) (PLA-TK-MA). Hydrogels were formed
through UV irradiation by mixing different ratios of PLA-TK-MA and
DEX-MA, with PpIX as a NIR-photosensitizer covalently bound to the
hydrogel. Initial studies on non-cleavable hydrogels showed that the
mechanical properties could be adjusted by changing the DEX meth-
acrylation degree, PLA molecular weight, and polymer ratios. Stiffer and
more homogeneous hydrogels were produced with higher DEX-MA<sub>45</sub>
content and PLA-TK-MA 5000 g/mol compared to PLA-TK-MA 2500 g/
mol. No gels formed with DEX-MA₃, and DEX-MA₁₂ had a negligible impact. Building on this, ROS-cleavable hydrogels were prepared using DEX-MA₄₅ or DEX-MA₁₂ and PLA-TK-MA (5000 g/mol) at an 85/15 weight ratio. Swelling and mechanical properties under NIR irradiation were studied. NIR exposure had opposite effects depending on the initial crosslinking density: DEX-MA₄₅/PLA-TK-MA hydrogels showed increased swelling and loss modulus, while DEX-MA₁₂/PLA-TK-MA had reduced swelling and storage modulus, confirming TK group cleavage. Protein release studies with BSA-FITC showed no accelerated release from DEX-MA₄₅/PLA-TK-MA due to high crosslinking density, whereas DEX-MA₁₂/PLA-TK-MA showed faster, quantitative release post-irradiation. Overall, these results demonstrate the system's ability to control protein release via NIR irradiation, highlighting the potential of our ROS-sensitive hydrogels for NIR-triggered delivery. Future work will focus on evaluating these hydrogels for therapeutic protein release and enhancing NIR-responsiveness.

## CRediT authorship contribution statement

Mickael Guerreiro: Writing – original draft, Investigation. Anissa Benkhedim: Writing – original draft, Investigation. Doriane Voisin: Investigation. Clémence Farine: Investigation. Audrey Bethy: Formal analysis. Cédric Paniagua: Investigation. Hélène Van Den Berghe: Writing – review & editing, Writing – original draft, Supervision, Conceptualization. Benjamin Nottelet: Writing – review & editing, Writing – original draft, Supervision, 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.

## Acknowledgements

Mickael Guerreiro's Master training course and Anissa Benkhedim's PhD were supported by LabUM Chimie at the University of Montpellier and received French Government funding managed by the "Agence Nationale pour la Recherche" under the "Investissements d'Avenir" program, reference ANR-16-IDEX-0006. The authors also wish to thank the support of the SynBio3 platform (IBMMontpellier) and Manuela Pastore et Christelle Reynes of the platform STATABIO Biocampus Montpellier.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.carbpol.2025.124068.

## Data availability

Data will be made available on request.

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