Enhancing the ROS Sensitivity of a Responsive Supramolecular Hydrogel Using Peroxizyme Catalysis

Hydrogels that can disintegrate upon exposure to reactive oxygen species (ROS) have the potential for targeted drug delivery to tumor cells. In this study, we developed a diphenylalanine (FF) derivative with a thioether phenyl moiety attached to the N-terminus that can form supramolecular hydrogels at neutral and mildly acidic pH. The thioether can be oxidized by ROS to the corresponding sulfoxide, which makes the gelator hydrolytically labile. The resulting oxidation and hydrolysis products alter the polarity of the gelator, leading to disassembly of the gel fibers. To enhance ROS sensitivity, we incorporated peroxizymes in the gels, namely, chloroperoxidase CiVCPO and the unspecific peroxygenase rAaeUPO. Both enzymes accelerated the oxidation process, enabling the hydrogels to collapse with 10 times lower H2O2 concentrations than those required for enzyme-free hydrogel collapse. These ROS-responsive hydrogels could pave the way toward optimized platforms for targeted drug delivery in the tumor microenvironment.


■ INTRODUCTION
Hydrogels made by self-assembly of small molecules have been drawing attention for over two decades due to the perspective of using these soft materials for applications ranging from drug delivery to tissue engineering. 1,2 The initial fascination over serendipitously discovered gels was soon replaced by the need to understand the forces that drive gel formation in a way to design a priori molecules that are able to gelate. 3,4 Considering the pool of self-assembled structures in biological systems, 5 nature was once again where researchers found inspiration. The first example of peptide-based low-molecular-weight gelator (LMWG), reported in 1995, was used to realize a thermoreversible gel as a carrier for antigen delivery and presentation. 6 Then, the recognition of diphenylalanine (FF) as a basic structure able to form ordered nanostructures 7 led to the derivatization of this sequence to obtain a large variety of novel hydrogelators. 8−11 The biocompatibility, the possibility of bottom-up fabrication, and easy chemical modification made diphenylalanine the natural choice as a short peptide sequence for biomedical materials. 12−14 Subsequently, this versatile building block was functionalized with groups sensitive to various stimuli such as pH, 15,16 enzymes, 17,18 ultraviolet (UV) light, 19,20 and redox change 21 to realize responsive hydrogels. Reactive oxygen species (ROS) overproduction is typical in many tumor and diseased cells, causing a redox imbalance in the microenvironment. Ikeda and co-workers prepared FF-type peptides with a boronoaryl group, which fragments in the presence of hydrogen peroxide (H 2 O 2 ), one of the most used ROS. 22 Besides the boronates, sulfides are commonly used oxidation-sensitive groups for stimuli-triggered nanomedicine. 23,24 In the aqueous medium, the oxidation of a sulfide into sulfoxide or sulfone produces a switch in solubility that can be used to destabilize self-assembled structures bearing these moieties. 25 The solubility switch from thioether to sulfoxide to trigger the gel−sol transition of hydrogels is not unprecedent, 26−28 but to the best of our knowledge, it has not been used for short peptide-based gelators. 29,30 Recently, we developed a thioether phenyl ester-based cascade mechanism in which the oxidation to sulfoxide induces the hydrolysis of the ester. 31 We functionalized diphenylalanine with the thioether phenyl moiety, aiming to realize a hydrogelator able to respond to H 2 O 2 through a logic gate mechanism that enables the gel to disrupt. Thus, we synthesized MTpcFF (Figure 1), an aromatic amphiphile able to form a selfassembled network in an aqueous environment at both pH values 6 Among the chloroperoxidases used for oxidation of thioanisole-type moieties, we chose CiVCPO for its robustness and high catalytic activity even in the presence of an organic solvent. 35−38 The production of HOCl is optimal at pH below 7; 39 therefore, this system is suitable for mildly acidic conditions, which are often present in tumor tissues. 40 In the MTpcFF gel, we also incorporated rAaeUPO, which can catalyze the oxidation of aromatic sulfides with H 2 O 2 at neutral pH 41,42 and it is known to have an enzymatic activity in water with a cosolvent and even in a pure organic solvent. 43,44 Despite the different mechanisms of the two peroxizymes, we found that their influence over the gel−sol transition of MTpcFF hydrogels upon the addition of H 2 O 2 is comparable. This establishes a versatile strategy that allows us to obtain ROS-responsive hydrogels in both mildly acidic and neutral conditions. ■ EXPERIMENTAL SECTION Materials. Diphenylalanine (FF) and triphenyl alanine (FFF) were purchased from Sigma-Aldrich. 4-(Methylthio)phenol and 4-(methylsulfonyl)phenol were purchased from Sigma-Aldrich and TCI, respectively.
Preparation of CiVCPO. The vanadium-dependent chloroperoxidase from C. inaequalis CiVCPO was produced in recombinant Escherichia coli TOP10 pBADgIIIB VCPO following previously described procedures. 45 CiVCPO was purified via heat treatment according to a recently reported protocol. 46 Preparation of rAaeUPO. The expression-engineered variant of the recombinant unspecific peroxygenase from A. aegerita (rAaeUPO, PaDaI mutant) was used as a concentrated supernatant derived from a 2500 L pilot-scale cultivation of recombinant Pichia pastoris X-33. 47,48 Synthesis of 4-(Methylthio)phenyl 4-Nitrophenyl Carbonate. 4-Nitrophenyl chloroformate (0.97 g, 4.80 mmol) and 4-(methylthio)phenol (0.56 g, 4.00 mmol) were dissolved in 30 mL of dichloromethane, and the solution was cooled to 0°C in an ice bath. Triethylamine (0.67 mL, 4.80 mmol) was added dropwise; the mixture was allowed to warm to room temperature and stirred for 4 h. TLC analysis (silica; eluant: dichloromethane) indicated that no 4-(methylthio)phenol remained. The reaction mixture was washed with water (2 × 25 mL) and brine (25 mL). The organic layer was dried over Na 2 SO 4 , filtered, and the solvent was removed by rotatory evaporation. Recrystallization from toluene (160 mL) gave the pure product (0.92 g, yield 77%) as a yellow solid. 1 49 Synthesis of 2-(((4-(Methylthio)phenoxy)carbonyl)amino)ethane-1-sulfonic Acid (1). To a solution of 4-(methylthio)phenyl 4-nitrophenyl carbonate (0.15 g, 0.50 mmol) in THF (2.0 mL) was added dropwise taurine (0.09 g, 0.75 mmol) and DIPEA (0.13 mL, 0.75 mmol) in distilled water (2.0 mL) at 0°C. The reaction mixture was allowed to warm at room temperature and stirred overnight. 50,51 After the removal of THF by rotatory evaporation, the mixture was extracted with ethyl acetate to remove the excess of p-nitrophenol. The aqueous layer was freeze-dried overnight. The crude was dissolved in acetonitrile, allowing the precipitation of the free taurine as a white solid. After filtration through syringe filters (45 μm), the acetonitrile was evaporated in vacuo. The mixture was dissolved in BuOH and further purified by flash chromatography over silica gel (CH 3 COOH/H 2 O/BuOH 5:5:90) to remove the excess of DIPEA. Compound 1 was obtained (0.07 g, yield 48%) as a slightly yellow solid. 1 H NMR (400 MHz, CD 3 OD) δ = 7.29 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 2H), 3.62 (t, J = 6.9 Hz, 2H), 3.06 (t, J = 6.9 Hz, 2H), 2.48 (s, 3H). 13     Rheology of MTpcFF Hydrogels. Oscillatory experiments were performed using a rheometer AR G2 from TA Instruments in a straincontrolled mode. The rheometer was equipped with a steel plate and plate geometry of diameter 25 mm and a water trap. The temperature Biomacromolecules pubs.acs.org/Biomac Article of the plates was controlled at 25 ± 0.2°C. The gel mixtures were prepared as reported above, obtaining a total volume for each gelation experiment of 0.1 mL. After stirring the vial by vortexing for 3 s, the gel was pipetted on the bottom plate of the rheometer and the upper plate was slowly rotated to equally spread the gel. The storage and loss moduli G′ and G″ were followed over time with the rheometer during the formation of the gel, setting up the instrument with a frequency of 1.0 Hz and under 1.0% strain. The measurements were stopped when no further increase of G′ was observed. A frequency sweep was measured in the range of 0.01−100 rad/s, confirming that the moduli are constant in the frequency range chosen and the strain sweep revealed that the applied strain percentage is in the linear strain regime. G′ was greater than G″, and both G′ and G″ were frequencyindependent, which indicated the typical viscoelastic behavior of a hydrogel consisting of fiber networks. The rheological properties of MTpcFF gels were not significantly influenced by the presence of the enzymes CiVCPO and rAaeUPO. In the previous work, we demonstrated the hydrolytic lability of the 4-(methylthio)phenyl ester when the thioether moiety is oxidized to the corresponding sulfoxide. 31 Here, we synthesized the taurine derivative 1 to study the oxidation process of the thioanisole moiety and whether sulfoxidation triggers carbamate hydrolysis. This model compound was also used to determine the ideal concentration of enzymes to accelerate the oxidation of the thioether unit. To avoid the inactivation of CiVCPO in the presence of inorganic phosphate, we performed all experiments with this chloroperoxidase in citrate buffer (CB, 50 mM, pH = 6.2, 140 mM NaCl). First, we followed product formation from 20 mM 1 in CB upon the addition of 1.5 equiv of H 2 O 2 in the absence of enzyme (Figure 2A) at 37°C.
After the first hour, the appearance of low field peaks in 1 H NMR revealed the formation of sulfoxide 2 and the 4-(methylsulfinyl)phenol 3 ( Figure S1). At 3 h, the conversion to 2 and 3 was 55 and 12%, respectively, and 33% of 1 was still present. Compound 1 was completely consumed after 8 h, but the amount of 2 dropped to 45%, while 3 increased to 52%. Sulfoxide 2 is an intermediate that hydrolyzes over time to form 3 and free taurine, demonstrating the hydrolysis of the carbamate moiety in these conditions. It took 16 h to reach more than 80% conversion to phenol 3, indicating that the hydrolysis rate from 2 to 3 is lower than the rate of oxidation of 1 to 2. Considering the difficulties in enhancing the rate of hydrolysis, we focused on the catalysis of the oxidation step. Therefore, we tested CiVCPO in concentrations of 0.1 and 1.0 μM to seek for the optimal oxidation conditions of 1. In the presence of 0.1 μM of CiVCPO, compound 1 converted into 80% of 2 in 2 h after the addition of H 2 O 2 ( Figure 2B, yellow line). With 1.0 μM of CiVCPO, 85% of 2 was produced in 10 min ( Figure 2B, purple line). This result demonstrates that the chloroperoxidase accelerates the oxidation of 1 more than two times for 0.1 μM and about 30 times for 1.0 μM enzyme. In addition, at 3 h, the conversion of 3 was 19% for 0.1 μM of CiVCPO and 26% for 1.0 μM of CiVCPO against 12% for the enzyme-free sample ( Figure 2C). However, for the samples with the chloroperoxidases, the formation of 3 was about 80% after 12 h. This indicates that the hydrolysis rate is influenced by the concentration of 2, but this effect fades with the consumption of the sulfoxide and the oxidant, translating into a minor difference in the kinetic profile toward the end of the reaction.
Aiming to investigate the reaction in neutral conditions, we decided to follow the oxidation and hydrolysis of 1 with 1.5 equiv of H 2 O 2 in phosphate buffer (PB, 50 mM) at pH = 7.0. In Figure 2D, we show the conversion of 1 and its products in PB in the absence of enzyme. At 3 h, the production of 2 and 3 was, respectively, 39 and 28%, with 33% of 1 remaining. At the same time point, 1 was consumed equally whether in phosphate or in citrate buffer, but the higher concentration of 3 in PB indicates that the hydrolysis is indeed faster than in CB. Due to the incompatibility of CiVCPO and phosphate buffer, we employed rAaeUPO as a peroxizyme to catalyze the oxidation of 1 for these conditions. Similar to CiVCPO, we used 0.1 and 1.0 μM of rAaeUPO to accelerate the oxidation of 1 with H 2 O 2 . Compound 2 reached 90% in only 10 min with 1.0 μM of rAaeUPO and 48% in 2 h with 0.1 μM of rAaeUPO ( Figure 2E). The hydrolysis profile of 2 to 3 ( Figure 2F) was similar in all cases, reaching about 90% in 12 h. The use of 0.1 μM of rAaeUPO barely accelerated the oxidation of 1 compared to the uncatalyzed case. In the presence of 1.0 μM of rAaeUPO, we have an almost immediate full conversion to 2, similar to the use of the same concentration of CiVCPO. Finally, at 12 h, the hydrolysis rate reached 90% in PB against 80% in CB. Additionally, after 12 h in the absence of oxidant, 14% of 4-(methylthio)phenol had formed in PB (orange line, Figure 2F) against about 5% in CB (orange line, Figure 2C), confirming the former condition as more advantageous for hydrolytic degradation. Considering the comparable efficiency of both CiVCPO and rAaeUPO at a concentration of 1.0 μM in the H 2 O 2 -driven oxidation of the thioether moiety and the balance between responsiveness and stability of the nonoxidized substrate, we chose to explore these conditions at the material level in both PB and CB with the corresponding 1.0 μM enzyme.
Properties of MTpcFF Hydrogels. Short peptide-based amphiphiles have great potential to form gels encapsulating large amounts of water. 52,53 Considering its promising gelation properties, 54 we chose FF as a peptide building block and functionalized its N-terminus with the 4-(methylthio)phenyl moiety to obtain MTpcFF. Using the tube-inversion method, we found that this dipeptide derivative has a critical gel concentration (CGC) of 0.70 wt % in CB ( Figure S5) and 0.95 wt % in PB ( Figure S6).
Knowing that tripeptide-based hydrogelators have generally lower CGC, we also synthesized MTpcFFF. However, in the tube-inversion tests, the gelation of this derived tripeptide was often difficult to achieve and replicate. We indeed noticed poor solubility in aqueous solution even at a concentration of 0.1 wt %, and without the possibility of heating to avoid the This result is not surprising if we consider the influence that pH and Hofmeister effect have over the gelation of other phenylalanine-based LMWG. 55−60 Lower pH leads to a higher degree of protonation of the terminal carboxylic acid of the dipeptide amphiphile, making the functionalized FF more hydrophobic and therefore favoring the gelation in aqueous solution. Nevertheless, obtaining G′ values always higher than the G″ means that both conditions produced gels with viscoelastic properties typical of fiber networks. 61 Observation of the hydrogels with optical microscopy ( Figure S3) and cryo-EM images (Figures 3C and S4) confirmed the presence of nanofibers that can aggregate in bundles with microscopic diameters. Analysis of cryo-EM images showed average fiber diameters of 7.9 ± 2.0 (citrate buffer, Figure S4C) and 4.0 ± 0.7 nm (phosphate buffer, Figure S4F) for the MTpcFF hydrogels.
Additives and impurities can affect the gelation of LMWG. Thus, to exclude that the encapsulation of CiVCPO and rAaeUPO influences the mechanical properties of MTpcFF gels, we performed additional rheological analysis on the enzyme-loaded hydrogels. The resulting data confirmed that adding 1.0 μM of CiVCPO to 0.75 wt % of MTpcFF in CB and 1.0 μM of rAaeUPO to 1.0 wt % of MTpcFF in PB produced G′ and G″ values that are only slightly lower than the corresponding gels without peroxizymes ( Figure 3F). We also performed strain amplitude measurements within the linear viscoelastic region for all four different conditions. MTpcFF gels in CB both with and without CiVCPO showed a slight drop in G′ and G″ values at 3% strain. In contrast, despite their lower storage and loss moduli, MTpcFF gels in PB demonstrated good stability even at 5% strain. Knowing these minor differences in the mechanical properties of MTpcFF hydrogel in different conditions, we pursued to investigate their response to H 2 O 2 .

H 2 O 2 -Responsive Gel−Sol Transition of MTpcFF Hydrogels.
To test the sensitivity of MTpcFF gels toward oxidation, we followed the gel−sol transition in time after the addition of H 2 O 2 via the tube-inversion method. MTpcFF hydrogels (0.75 wt %, 16 mM) were prepared in CB with and without CiVCPO (1.0 μM) and visually followed after the addition of varying equivalents of H 2 O 2 ( Figure 4A).
The peroxizyme-free gels turned into solution 5 h after the addition of 1.0 equiv of H 2 O 2 , while CiVCPO-loaded MTpcFF hydrogels became solutions within 30 min in the presence of an equimolar amount of hydrogen peroxide ( Figure 4A). The hydrogels with CiVCPO fully disrupted in 1 and 3 h with 0.5 and 0.25 equiv of H 2 O 2 , respectively. Meanwhile, the CiVCPO-loaded control gels, where the oxidant was not added, remained intact for over 8 h. To investigate the molecular mechanism behind the material change, we analyzed MTpcFF hydrogels as soon as they had disintegrated after the addition of 1.0 equiv. of H 2 O 2 with the high-performance liquid chromatography (HPLC). In the chromatogram, we found that after this time the peak of MTpcFF has decreased, while an unidentified peak appeared at t R = 18.7 min. LC-MS analysis revealed that we obtained the corresponding sulfoxide of the gelator ( Figure S9). Despite the presence in the HPLC analysis of about 15% FF 5 h after the addition of H 2 O 2 , we concluded that the main cause of the disassembly of the hydrogel is the oxidation of MTpcFF into the more hydrophilic sulfoxide. This result comes unexpected since the sulfoxidation-related change in hydrophilicity was previously considered to be too small to lead to the disassembly of block copolymers based on this motif. 31 On the other hand, the observed gel disruption at the oxidation   Figure 4A, bottom left). The HPLC analysis was performed on the solutions obtained after 5 h for the hydrogels exposed to high oxidant concentration, while the partially disrupted gels exposed to 0.1 equiv of H 2 O 2 and the control experiments were analyzed after 8 h. The HPLC data in Figure 4B show that the presence of 0.1 equiv of H 2 O 2 caused the consumption of about 10% gelator. Considering the corresponding gel picture, we can conclude that this variation in the MTpcFF concentration, and therefore the addition of 0.1 equiv of H 2 O 2 , is not sufficient to completely degrade the gel. In contrast, upon the addition of 0.25 equiv of H 2 O 2 we obtained full collapse of the gels in 3 h, with about 75% of the remaining gelator according to HPLC analysis. This suggests that the threshold for full solubilization of an MTpcFF gel (0.75 wt %) in CB is in the range of 0.1−0.2 equiv of H 2 O 2 . All of these experiments were carried out either with hydrogen peroxide and either NaCl or KBr, needed for the production of hypohalites by CiVCPO. The chloroperoxidase is able to convert H 2 O 2 and NaCl into HOCl, while in the presence of KBr, HOBr is formed. In principle, HOCl has a higher oxidation potential than HOBr, while the latter is more electrophilic and can react faster with the thioether. 62 In the tube-inversion tests, the use of NaCl together with H 2 O 2 led to solutions that appeared slightly less viscous than when KBr was used. In the correlating HPLC analysis, for additions of 1.0 and 0.5 equiv of H 2 O 2 , about 6% less MTpcFF was measured when NaCl was added instead of KBr. However, the difference is not significant, making it difficult to assess what species has more impact on the system. On a second note, considering the abundant presence of chlorine in the cellular environment and the tendency of HOBr to react with a broad range of substrates other than the thioether, 63,64 the conditions with NaCl should be favorable for biological applications.
Subsequently, we proceeded to test the gel disruption upon the addition of H 2 O 2 between 0.1 and 1.0 equiv on 1.0 wt % of MTpcFF hydrogels (20 mM) in PB with and without rAaeUPO (1.0 μM). The photographs of these inverted vial tests are shown in Figure 4C. rAaeUPO-free hydrogels take 5 h to turn into a viscous solution in the presence of 1.0 equiv of the oxidant, while when rAaeUPO was encapsulated in the gel formulation, this time reduced to 30 min. The time to gel degradation was 1 and 3 h when, respectively, 0.5 and 0.25 equiv of H 2 O 2 were added. We found that 0.1 equiv of hydrogen peroxide is enough to achieve the gel−sol transition even if the time scale is extended to 8 h in this case. The corresponding HPLC results ( Figure 4D) showed that for 0.1 equiv of H 2 O 2 , the expected 90% of MTpcFF was detected, which appeared to be below the CGC of the gels when compared with the tube-inversion tests. Such a low threshold is in line with the need for increasing the gelator concentration when performing gelation in PB compared to that achieved in CB. Additionally, we tested the stability of previously formed MTpcFF hydrogels in both PB and CB solutions, showing that MTpcFF hydrogels are stable in 500 μL of citrate buffer for 20 h and in 200 μL of phosphate buffer for more than 8 h ( Figure   S7). These results demonstrate good stability of MTpcFF hydrogels compared to their responsiveness toward H 2 O 2 .
Despite the different concentrations and conditions of MTpcFF hydrogels with rAaeUPO and with CiVCPO, the response times in gel collapse upon the addition of H 2 O 2 were remarkably similar. The close catalytic effect of 1.0 μM of rAaeUPO and of 1.0 μM of CiVCPO (in the presence of halides) to oxidize the thioether with H 2 O 2 was already anticipated from the 1 H NMR study on 1 (Figure 2). The translation of this effect to the hydrogels with different formulations enables these materials to have comparable sensitivity toward H 2 O 2 (∼2.0 mM), in both mildly acidic and neutral settings.

■ CONCLUSIONS
This study presents a thioether carbamate-based dipeptide that can form stable hydrogels in the pH range of 6.0−7.0. The addition of H 2 O 2 leads to oxidation of the thioether to sulfoxide, triggering a solubility switch and disruption of the gel. We encapsulated two different peroxizymes in the hydrogel: the vanadium-dependent chloroperoxidase CiVCPO in citrate buffer at pH 6.2 and the heme-dependent peroxidase rAaeUPO in phosphate buffer at pH 7.0. Enzymatic hydrogen peroxide activation in both cases resulted in the gel collapsing 10 times faster than peroxizyme-free samples. The encapsulation of peroxizymes in the gel matrix proved essential to achieve a gel−sol transition at hydrogen peroxide concentrations below 2.0 mM. Moreover, we believe that the similar responsiveness of the hydrogel in two different conditions is a key feature for application of this system in neutral and mildly acidic environments with elevated ROS concentrations.
This simple dipeptide-based gelator could be used to create a library of thioanisole carbamate-based LMWGs with different mechanical properties and response thresholds, making it a promising material for devices sensitive to redox imbalance in biological settings. Additionally, although not essential in this study, the hydrolytic instability of the carbamate after oxidation makes the thioether phenyl group a potential ROSlabile protecting group for amines in pharmaceutically active compounds.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.3c00262. Methods description; additional synthetic and characterization procedures; 1 H NMR and 13 C NMR spectra of the synthesized molecular models and hydrogelators; hydrogel characterization procedures; photographs of the inverted tube tests; optical microscopy and cryo-EM images of the hydrogels, and frequency distribution of the gel fiber diameters and HPLC analysis (PDF)