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. 2026 Jun 3;22(39):e73943. doi: 10.1002/smll.73943

Modular Molecular Design and Self‐Assembled Nanostructures of Saccharide‑Appended Cyclic Dipeptides for Glycosidase‑Responsive Supramolecular Hydrogels

Shintaro Sugiura 1, Ryuta Tanaka 2, Sayuri L Higashi 1,3,4, Aya Shibata 2, Koichiro M Hirosawa 5, Kenichi G N Suzuki 5,6, Masato Ikeda 1,2,4,5,7,✉
PMCID: PMC13360816  PMID: 42233212

ABSTRACT

Glycosidase‐responsive supramolecular hydrogels represent a promising class of soft materials for biomedical applications, as glycosidases play pivotal roles in various physiological processes and viral infections. In this study, saccharide‐appended cyclic dipeptides are designed as modular building blocks for the construction of supramolecular hydrogel systems through aqueous self‐assembly. Notably, β‐galactosidase‐responsive gel‐to‐sol and neuraminidase (sialidase)‐responsive sol‐to‐gel transitions are achieved by employing cyclic dipeptides appended with galactose and N‐acetylneuraminic acid, respectively, under appropriate formulations.

Keywords: cyclic dipeptide, glycosidase, nanostructures, self‐assembly, soft materials, supramolecular hydrogel

Saccharide‐appended cyclic dipeptides are designed and developed as building blocks for glycosidase‐responsive supramolecular hydrogels. Their aqueous self‐assembly enables β‐galactosidase‐triggered gel‐to‐sol and neuraminidase‐triggered sol‐to‐gel transition systems, highlighting their potential as glycosidase‐responsive soft materials for biomedical applications.

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1. Introduction

Glycosidases belong to the glycoside hydrolase family and catalyze the hydrolysis of glycosidic bonds in complex carbohydrates [1, 2, 3]. These enzymes play pivotal roles in energy metabolism and a wide range of physiological processes, and their dysregulation is associated with severe disease states [4], including Gaucher's [5] and Pompe disease [6]. Moreover, the overexpression of β‐galactosidase (β‐Gal) in senescent cells (SA‐β‐Gal) has been reported and is widely recognized as a hallmark of cellular senescence [7, 8]. This discovery has stimulated increasing interest in SA‐β‐Gal as a biomarker for the detection of senescent cells both in vitro and in vivo [9]. Viral infections can also be potentiated by glycosidases. A representative example is the influenza virus, which encodes its own glycosidase, sialidase or neuraminidase (NA) [10, 11], to remove terminal sialic acid residues from the host cell surface. This enzymatic activity is essential not only for facilitating viral entry by overcoming mucosal barriers but also for promoting the efficient release of newly generated virions from infected cells, thereby enhancing viral dissemination [12]. Therefore, self‐assembled soft materials capable of glycosidase responsiveness may hold potential biomedical and diagnostic applications [13].

Cyclic dipeptides (CDPs) are versatile molecular scaffolds that have been explored for a wide range of applications, from therapeutics [14] to bulk materials [15], owing to their compact, rigid, yet chemically diversifiable structures. Supramolecular hydrogels [16, 17, 18, 19, 20, 21, 22] composed of low‐molecular‐weight compounds, including CDPs [23, 24, 25, 26], have attracted increasing attention for (bio)applications because of their biocompatibility and flexible molecular designability. In this context, our ongoing projects have revealed that cyclo‐l‐isoleucyl‐l‐tyrosyl ( c‐IY) [27] is one of the simplest hydrogelators based on CDPs. In this study, saccharide‐bearing CDPs ( c‐IYS ), including galactose (Gal) with β‐linkage and N‐acetylneuraminic acid (Neu5Ac) with α‐linkage, namely c‐IYGal and c‐IYNeu5Ac , respectively, are newly designed, as shown in Figure 1. The corresponding glycosidases, which are capable of removing the saccharide moieties (i.e., β‐Gal and NA for c‐IYGal and c‐IYNeu5Ac , respectively), are expected to induce phase transitions through the modulation of self‐assembly.

FIGURE 1.

FIGURE 1

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(A) Chemical structures of the cyclic dipeptide c‐IY (hydrogelator), composed of isoleucine (I) and tyrosine (Y), and two saccharide‐appended cyclic dipeptides c‐IYS bearing galactose (Gal, β‐linkage) and N‐acetylneuraminic acid (Neu5Ac, α‐linkage). (B) Plausible mechanism of the glycosidase‐induced conversion of c‐IYS to c‐IYOH as an intermediate, yielding c‐IY via β‐1,6‐elimination. (C) Schematic illustration of the aqueous self‐assembly of c‐IY leading to hydrogel formation in a concentration‐dependent manner (β‐Gal: β‐galactosidase; NA: neuraminidase; CAC: critical aggregation concentration; MGC: minimal gelation concentration).

The modulation of the self‐assembly behavior of galactose‐appended molecules by β‐Gal has been extensively investigated [28, 29, 30, 31, 32, 33, 34], including applications in intracellular environments [35]. To the best of current knowledge, however, this study presents the first example in which NA induces the self‐assembly of a small molecule under biologically relevant conditions, leading to the formation of supramolecular hydrogels and associated nanofiber networks. Although nanoparticles bearing polysialic acid with NA responsiveness have only recently been explored [36, 37], their underlying mechanisms and self‐assembly behaviors differ from those described in this study, and hydrogel formation is not involved. Hydrogel formation induced by NA may offer a promising approach for the on‐demand entrapment of influenza viruses [10, 11, 12] and other pathogens [38, 39] that express NA, reminiscent of the functional properties of native mucus hydrogels [40, 41, 42, 43]. To this end, the hydrogel formed in response to NA is shown to entrap nanobeads with a diameter of approximately 100 nm within the nanofiber network. This size is comparable to that of spherical influenza virions (∼100 nm) and falls within the range of filamentous forms, which can exceed 300 nm in length [44, 45]. Furthermore, although the expression and translocation of NAs play significant roles in physiological and pathological processes [46], they have been less extensively explored as target stimuli than other glycosidases [47, 48]. Therefore, the present molecular self‐assembly systems based on CDPs, including NA‐responsive aqueous soft materials, hold significant promise for biomedical applications, particularly in the inhibition of viral and bacterial infections.

2. Results and Discussion

2.1. Modular Molecular Design of Saccharide‐Appended CDPs (c‐IYS)

After screening a small library of CDPs containing one tyrosine (Y) residue, it was found that the simple cyclic dipeptide c‐IY is capable of self‐assembling into a fibrous network that forms hydrogels (details will be reported elsewhere). Unless otherwise stated, the aqueous conditions used in this study were phosphate‐buffered saline (pH 7.4):DMSO = 95:5 (v/v). The minimal gelation concentration (MGC) of c‐IY was determined to be 15 mm (0.41 wt.%), at which a stable hydrogel formed within 30 min after the cessation of heating. In contrast, only a partial gel was obtained at a lower concentration of 7.5 mm (0.21 wt.%), as evaluated by the conventional tube‐inversion method (Figure 2A).

FIGURE 2.

FIGURE 2

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(A) Summary of gelation tests {states: Sol = solution, Gel = transparent or translucent gel, pGel = partial gel (part of the solution was gelled), Ppt = precipitation} 30 min after heating was stopped for cyclic dipeptides c‐IY and c‐IYS (S = Gal or Neu5Ac). (B) Representative photographs showing hydrogels of (i) c‐IY and (ii) c‐IYGal , and an aqueous solution of (iii) c‐IYNeu5Ac . Representative (C) atomic force microscopy height (tapping mode, mica) and (D) confocal laser scanning microscopy (CLSM) images (Nile red staining, [Nile red] = 0.30 µm) of (i) c‐IY and (ii) c‐IYGal (15 mm) gels. Scale bars: 1 µm for panel C and 10 µm for panel D. Conditions: phosphate‐buffered saline [PBS (–), pH 7.4]:DMSO = 95:5 (v/v), ambient temperature.

These results motivated the design and synthesis of saccharide‐appended CDPs ( c‐IYS ; Figure 1A). To enable saccharide incorporation, a spacer was introduced between the phenolic moiety of tyrosine and saccharide units, primarily for practical and strategic considerations in synthesis (Scheme S1). This spacer can be removed via β‐1,6‐elimination following the glycosidase‐mediated removal of the saccharide units (Figure 1B). The cleavage mechanism, as well as the synthetic protocols for saccharide units and their incorporation, have been previously reported by other research groups in the context of molecular sensors [48, 49, 50] and prodrug development [49, 51], with a focus on glycosidase selectivity. Herein, the c‐IYS compounds were characterized by 1H and 13C NMR spectroscopy, as well as high‐resolution mass spectrometry (Figures S1–S4).

2.2. Aqueous Self‐Assembly of c‐IYS

c‐IYGal exhibited enhanced hydrogelation ability compared with c‐IY, as evidenced by a lower MGC of 10 mm for c‐IYGal vs. 15 mm for c‐IY (Figure 2A). (This trend is reversed when expressed in wt.%: 0.54 wt.% for c‐IYGal and 0.41 wt.% for c‐IY.) In contrast, the self‐assembly capability of c‐IYNeu5Ac was markedly reduced (Figure 2A), presumably due to electrostatic repulsion arising from the conditionally negative charge of the carboxylic group at the C2 position of N‐acetylneuraminic acid, which is pH‐dependent (pK a = 2.6 for free N‐acetylneuraminic acid [52]), as well as its intrinsic hydrophilicity resulting from the presence of multiple hydroxyl groups. A transparent aqueous solution of c‐IYNeu5Ac could be easily prepared at concentrations of up to 15 mm (1.0 wt.%, Figure 2B) without the need for heat treatment or sonication.

Critical aggregation concentrations (CACs) were estimated using Thioflavin T (ThT) as a fluorescent probe (Figure S5). Consistent with its inability to form a hydrogel, c‐IYNeu5Ac exhibited the highest CAC at 7.8 ± 2.6 mm, and the CAC of c‐IY (0.98 ± 0.01 mm) was higher than that of c‐IYGal (0.79 ± 0.05 mm). These values reflect the relative self‐assembly abilities of the compounds in the order c‐IYGal > c‐IY > c‐IYNeu5Ac (from strongest to weakest), consistent with their gelation abilities in terms of molar concentration.

Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) analysis further supported these observations. The retention times (t R) of c‐IYGal (15.0 min) and c‐IY (11.0 min) correlated with their self‐assembling abilities (Figure S6A). In contrast, c‐IYNeu5Ac exhibited the longest t R (15.6 min), which can be attributed, at least in part, to its larger molecular weight (M w: 673.3 vs. 544.2 for c‐IYGal and 276.1 for c‐IY) [53] and the acidic conditions of the eluent, containing trifluoroacetic acid to protonate the carboxylic group at the C2 position. To gain further insight into this result, RP‐HPLC analysis was performed on a model compound ( c‐IYOMe ) bearing a 4‐methoxy group (Figure S6C) in place of the hydrophilic glycosyl groups. The t R of c‐IYOMe (23.4 min, Figure S6B) was the longest among the tested compounds, compared with the glycosylated derivatives ( c‐IYGal : 11.6 min; c‐IYNeu5Ac : 12.4 min) under higher elution strength HPLC conditions (Figure S6B). Collectively, these results indicate that the introduction of a p‐phenyl‐based spacer between the saccharide units and tyrosine moiety enhances the hydrophobicity of the resulting compounds, whereas the incorporation of saccharide units increases their hydrophilicity.

Microscopic observation of the c‐IYGal and c‐IY gels revealed entangled nanofibers, as shown in atomic force microscopy images (Figure 2C). Clear morphological differences were observed: the nanofibers of the c‐IY gel (Figure 2Ci) appeared straighter than those of the c‐IYGal gel (Figure 2Cii). Similar fibrous morphological differences were observed in the gels using confocal laser scanning microscopy (CLSM) under aqueous conditions without sample drying, following the addition of Nile red, a conventional hydrophobic fluorescent probe (Figure 2D). These findings suggest that the cross‐linking density of the entangled nanofibers was higher in the less straight structures of the c‐IYGal gel, which may account for its relatively superior hydrogelation ability in terms of the MGC.

The viscoelastic properties of the c‐IYGal and c‐IY gels were assessed using conventional oscillatory rheological measurements. As shown in Figure S7, a linear viscoelastic region was observed at low strain, where the storage modulus (G′) exceeded the loss modulus (G″), a characteristic feature of hydrogels composed of entangled nanofibers [54]. In addition, the G′ value of the c‐IYGal gel was higher than that of the c‐IY gel (4.3 kPa vs. 0.23 kPa, at 0.2% strain and 1.0 rad/s), which can be attributed to the differences in their fibrous morphologies.

In contrast, atomic force microscopy observations (Figure S8A) revealed the formation of nanoparticles in the aqueous solution of c‐IYNeu5Ac , with an average hydrodynamic diameter of 228 ± 44 nm as determined by dynamic light scattering measurements (Figure S8B). The non‐networked nanoparticles formed by c‐IYNeu5Ac are associated with its inability to form a hydrogel.

2.3. Glycosidase‐Responsiveness of c‐IYS

2.3.1. β‐Gal‐responsive Gel‐to‐Sol Transition of c‐IYGal

Escherichia coli β‐Gal (EC 3.2.1.23) [55, 56] was employed to investigate the β‐Gal‐responsiveness of c‐IYGal gel. After the addition of β‐Gal (final concentration: 4.7 U/mL), the c‐IYGal gel underwent a gel‐to‐sol (ppt) transition, with its concentration decreasing from 11 to 10 mm (still above the MGC of c‐IYGal ), as shown in Figure 3Ai. This macroscopic phase transition occurred within 3 h; however, the resulting sol phase was not fully clear and contained precipitates. No such phase transition was observed without the addition of β‐Gal (Figure 3Aii). A lower concentration of β‐Gal (final concentration: 2.3 U/mL) was sufficient to induce a similar phase transition (Figure 3Aii), although the process required a longer time (6 h vs. 3 h).

FIGURE 3.

FIGURE 3

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(A) (i, ii) Photographs illustrating the β‐Gal‐responsiveness of c‐IYGal gel and (iii) summary of the β‐Gal‐induced gel‐to‐sol transition of c‐IYGal . (B) (i) High‐performance liquid chromatography (HPLC) traces [detection wavelength: 275 nm, *p‐methoxyphenol was used as an internal standard] monitoring the molecular transformation of c‐IYGal following β‐Gal addition (final concentration: 4.7 U/mL). (ii) Time‐dependent consumption (%) of c‐IYGal estimated from HPLC trances. The line connecting data points is provided for visual guidance. A proposed transformation scheme of c‐IYGal is shown. (C) Representative CLSM images (Nile red staining, [Nile red] = 0.30 µm) during the β‐Gal‐induced gel‐to‐sol (ppt) transition of c‐IYGal . Scale bar: 10 µm. (D) Circular dichroism spectra (l = 0.1 mm, demountable quartz cell) of c‐IYGal before and 3 h after β‐Gal addition (final concentration: 4.7 U/mL) and of c‐IY, including the corresponding HT data. Conditions: [ c‐IYGal ] = 10 mm (11 mm before β‐Gal addition), [ c‐IY] = 10 mm (for panel D), PBS (–) (pH 7.4):DMSO = 95:5 (v/v), room temperature.

RP‐HPLC analysis was conducted to monitor the molecular transformation of c‐IYGal after β‐Gal addition. As shown in Figure 3B, the peak corresponding to c‐IYGal (t R = 15.1 min) nearly disappeared within 3 h (Figure 3Bi), accompanied by the appearance of a new peak at t R = 20.5 min. The t R of this new peak differed significantly from that of c‐IY (t R = 10.8 min), suggesting the formation of a distinct product catalyzed by β‐Gal. To clarify this discrepancy, the β‐Gal‐treated sample was exposed to basic conditions by adding aqueous NaOH and reanalyzed. A peak with a t R of 10.3 min, nearly identical to that of c‐IY, was then observed (Figure 3Bi), indicating that the product formed after β‐Gal treatment is most likely c‐IYOH (Figure 1B), generated following galactose removal but prior to the β‐1,6‐elimination of the spacer unit. The longer t R assigned for c‐IYOH compared with c‐IY would be related to the long t R of c‐IYOMe , whose structural difference is relatively small (i.e., hydroxyl group against methoxy group) (vide supra).

To investigate the morphological changes associated with the β‐Gal‐induced phase transition, CLSM observations were conducted. As shown in Figure 3C, short fibrous structures were observed following β‐Gal addition. This transformation from entangled fibers (Figure 2D) to shorter fibrous assemblies suggests that lateral bundling may be facilitated, potentially accompanied by the weakening of the network structure and/or reduction of cross‐linking. Such a self‐assembled structural rearrangement appears to be closely linked to the destabilization of the gel state, resulting in a phase transition even without substantial dissolution of the fibrous structures, thereby yielding a dispersed (sol) state.

Circular dichroism (CD) spectroscopy was further employed to characterize the self‐assembled structures (Figure 3D). The CD spectrum of the c‐IYGal gel exhibited positive signals in the 250–285 nm region, which can be attributed to the two aromatic units. In contrast, c‐IY gel displayed negative signals at a relatively longer wavelength region than the c‐IYGal gel, arising from the tyrosine residue bearing a free hydroxyl group, and corresponding to the 1Lb band [57, 58]. Notably, the CD spectrum of c‐IYGal gel after β‐Gal addition differed from those of both the original c‐IYGal and c‐IY gels. This spectral change is consistent with the RP‐HPLC results, indicating transformation to c‐IYOH rather than c‐IY, which may result in distinct chiral arrangements of the aromatic units in the three molecules ( c‐IY, c‐IYGal , c‐IYOH ).

2.3.2. NA‐responsive Sol‐to‐Gel Transition of c‐IYNeu5Ac

Among the sialidases (NAs, EC 3.2.1.18), a commercially available Clostridium perfringens sialidase [59] was used to evaluate the NA‐responsiveness of c‐IYNeu5Ac . In contrast to c‐IYGal , which undergoes a β‐Gal‐responsive gel‐to‐sol transition, a sol‐to‐gel transition was observed by the addition of NA to an aqueous solution of c‐IYNeu5Ac (16 mm), as shown in Figure 4Ai. Gel formation was induced by NA at concentrations as low as 0.10 U/mL (final concentration) after 3 h in a 16 mm solution of c‐IYNeu5Ac (diluted to 15 mm after NA addition), whereas partial gelation occurred at 12.5 and 10 mm (data not shown). No such phase transition was observed without the addition of NA (Figure 4Aii). At NA concentrations exceeding 0.20 U/mL, a rapid sol‐to‐gel transition was observed within 30 min (Figure 4Aiii).

FIGURE 4.

FIGURE 4

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(A) (i, ii) Photographs demonstrating the NA‐responsiveness of c‐IYNeu5Ac and (iii) a summary of the NA‐induced sol‐to‐gel transition of c‐IYNeu5Ac . (B) (i) HPLC traces (detection wavelength: 275 nm; *p‐methoxyphenol used as an internal standard) monitoring the molecular transformation of c‐IYNeu5Ac after the addition of NA (final concentration: 0.15 U/mL). (ii) Time‐dependent consumption (%) of c‐IYNeu5Ac estimated from the HPLC trances. Lines connecting data points are provided as visual guides. A proposed transformation scheme of c‐IYNeu5Ac is shown. (C) Representative CLSM images (Nile red staining, [Nile red] = 0.30 µm) acquired during the NA‐induced sol‐to‐gel transition of c‐IYNeu5Ac . Scale bar: 10 µm. (D) Circular dichroism spectra (l = 0.1 mm, demountable quartz cell) of c‐IYNeu5Ac before and 180 min after the addition of NA (final concentration: 0.10 U/mL), together with the corresponding HT data. Conditions: [ c‐IYNeu5Ac ] = 15 mm {16 mm (except CLSM 18 mm) prior to NA addition}, [ c‐IY] = 15 mm (for panel D), PBS (–) (pH 7.4):DMSO = 95:5 (v/v), room temperature.

RP‐HPLC analysis was performed to monitor the enzymatic reactions. As shown in Figure 4B, following the addition of NA (final concentration: 0.15 U/mL), the peak corresponding to c‐IYNeu5Ac (t R = 15.5 min) gradually decreased, accompanied by the simultaneous emergence of a new peak at t R = 20.9 min. This behavior closely resembled the changes observed for c‐IYGal after β‐Gal treatment (Figure 3B). A peak with a t R nearly identical to that of c‐IY was observed after NaOH treatment, indicating that c‐IYNeu5Ac was converted to c‐IYOH following NA addition.

CLSM observations revealed the emergence of entangled fibrous structures following the addition of NA to an aqueous solution of c‐IYNeu5Ac , which are responsible for the gel state, as shown in Figure 4C. Before the addition of NA, c‐IYNeu5Ac self‐assembles to form nanoparticles under these conditions (vide supra). Notably, microspherical structures were observed ∼10 min prior to gelation and subsequently underwent a morphological transition into entangled fibrous structures within 30 min, consistent with hydrogel formation.

RP‐HPLC analysis revealed that the consumption of c‐IYNeu5Ac was approximately 40% at the gelation point (30 min) and reached near saturation at approximately 50% after 24 h (Figure 4Bii). This modest conversion can be attributed, at least in part, to the immobilization of NA within the gel matrix, which may lead to the attenuation of its enzymatic activity. Because the concentration of residual c‐IYNeu5Ac (15 mm × 60% remaining after 30 min = 9.0 mm) remained above its CAC (∼7.8 mm) even in the gel state, the entangled fibrous structures observed by CLSM could be composed of co‐assemblies of c‐IYOH and c‐IYNeu5Ac . It is presumed that such co‐assemblies of c‐IYOH and c‐IYNeu5Ac influenced the modest conversion of c‐IYNeu5Ac to c‐IYOH , most likely due to the limited accessibility of NA to c‐IYNeu5Ac in the self‐assembled state compared with the molecularly dissolved and nanoparticle states.

The CD spectrum of the 16 mm aqueous solution of c‐IYNeu5Ac exhibited positive signals in the 250–285 nm region, similar to those observed for the c‐IYGal gel (Figure 4D). After the addition of NA, these positive CD signals were further enhanced, giving rise to a distinct spectral pattern compared with that of the c‐IY gel, yet resembling that of the c‐IYGal gel. These spectral features suggest differences in molecular arrangement within the self‐assembled fibrous structures, namely between the c‐IY and c‐IYGal gels, as well as the c‐IYNeu5Ac / c‐IYOH gel system.

The two glycosidase‐responsive phase transitions of c‐IYS are schematically depicted in Figure 5. In brief, the precise adjustment of the initial and working c‐IYS concentrations relative to their CAC and MGC values is critical for achieving the desired phase transitions as discussed in this section. For example, when the working concentration of c‐IYGal is set above its MGC (10 mm) but below that of c‐IY (15 mm), a β‐Gal‐induced gel‐to‐sol transition of the c‐IYGal gel can be expected. Conversely, when the working concentration of c‐IYNeu5Ac is set below its MGC (> 15 mm) but above that of c‐IY (15 mm), an NA‐induced sol‐to‐gel phase transition of the c‐IYNeu5Ac solution is anticipated.

FIGURE 5.

FIGURE 5

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Proposed schematics illustrating the (A) β‐Gal‐responsive gel‐to‐sol transition of c‐IYGal and (B) NA‐responsive sol‐to‐gel transition of c‐IYNeu5Ac .

This study demonstrates the predicted glycosidase‐responsive phase transitions. However, the product principally responsible for the transformations of the self‐assembled architectures triggered by glycosidases was not c‐IY itself, but rather an intermediate ( c‐IYOH ), which can be slowly converted to c‐IY under neutral aqueous conditions, with the conversion being accelerated by base addition, as indicated by HPLC analysis. Because c‐IYOH could decompose gradually, evaluation of its CAC and MGC using conventional analytical protocols after purification procedures would be difficult. Nonetheless, further investigations are considered necessary to regulate the rate of β‐1,6‐elimination, for example, through the introduction of electron‐withdrawing groups into the spacer [60] and/or modification of the spacer scaffold [61].

2.4. Controlled Release From and Entrapment Within Gel Matrices Composed of Glycosidase‐Responsive c‐IYS

2.4.1. β‐Gal‐Responsive Gel‐to‐Sol Transition of c‐IYGal for the Controlled Release of Encapsulated Proteins as Models of Biomedicines

Using the newly developed β‐Gal‐responsive hydrogel ( c‐IYGal ), which undergoes a β‐Gal‐responsive gel‐to‐sol(ppt) phase transition, its capability for the controlled release of encapsulated substances within the gel matrix was evaluated. FITC‐labeled lysozyme [62] was selected as a model therapeutic protein and incorporated into the c‐IYGal gel (Figure 6A and Figure S9). Upon exposure of the c‐IYGal gel (10 mm, 100 µL) encapsulating FITC‐lysozyme (1.0 µm) to an aqueous solution of β‐Gal (26 U/mL, 1.0 mL; final concentration: 24 U/mL) applied to the gel surface, continuous release of FITC‐lysozyme was observed (Figure 6B). In contrast, only limited release occurred in the absence of β‐Gal, likely owing to the relatively large molecular size of lysozyme (14 kDa, pI = 11), which remained entrapped within the gel matrix. FITC‐lysozyme was found to adsorb onto the surface of the nanofibers within the c‐IYGal gel (Figure S10A), which may have further contributed to efficient encapsulation. In contrast, fluorescein (0.33 kDa) exhibited a significantly faster release profile, even in the absence of β‐Gal (Figure 6B). These encapsulation behaviors are comparable to those reported previously for supramolecular hydrogels [63, 64]. The relatively low release of FITC‐lysozyme, even in response to β‐Gal (30 ± 4% after 90 min), is attributed to sustained adsorption onto non‐networked yet bundled fibrous precipitates composed primarily of c‐IYOH , which persist in the sol(ppt) state (Figure 3D and Figure S10B). Indeed, the subsequent addition of NaOH induced further release of the adsorbed FITC‐lysozyme (∼71%).

FIGURE 6.

FIGURE 6

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(A) Schematic illustration of the β‐Gal‐responsive gel‐to‐sol transition of the c‐IYGal gel for the controlled release of encapsulated proteins as a model of biomedicines. (B) (i) Cumulative release (%) of encapsulated FITC‐lysozyme and fluorescein from the c‐IYGal gel in response to β‐Gal addition. (ii) Averaged release (%) of encapsulated FITC‐lysozyme and fluorescein from the c‐IYGal gel 90 min after β‐Gal addition. Corresponding photographs are provided in Figure S9. Conditions: Gel (100 µL): [ c‐IYGal ] = 11 mm, [FITC‐lysozyme or fluorescein] = 1.0 µm, PBS (–) (pH 7.4):DMSO = 95:5 (v/v); Supernatant (1.0 mL): [β‐Gal] = 26 U/mL, PBS (–) (pH 7.4); room temperature.

Because lysozyme is itself a glycosidase (1,4‐β‐d‐N‐acetyl muramidase) capable of cleaving the β‐(1,4)‐glycosidic bonds in peptidoglycan, a major structural component of bacterial cell walls, this result highlights that glycosidase selectivity can be incorporated into supramolecular hydrogel matrices through the introduction of appropriate saccharide units into self‐assembling CDP scaffolds via deliberate molecular design.

2.4.2. NA‐Responsive Sol‐to‐Gel Transition of c‐IYNeu5Ac for Entrapment of Nanobeads as a Virus Model

The potential of the NA‐triggered gel matrix to entrap nanoparticles with sizes comparable to those of spherical influenza virions [44, 45] was investigated (Figure 7A). Polystyrene nanobeads (micromer‐redF, micromod; size: 100 nm) labeled with fluorescent dyes (λex/λem = 552/580 nm) were employed for this purpose. To visualize the self‐assembled fibrous structures concurrently but separately from the fluorescence of the nanoparticles, ThT was used as a fluorescent probe instead of Nile red.

FIGURE 7.

FIGURE 7

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(A) Schematic illustration of the NA‐responsive sol‐to‐gel transition of c‐IYNeu5Ac for the entrapment of nanobeads as a virus model. (B) Representative time‐lapse CLSM images (15‐s intervals; magenta: nanobeads, green: Thioflavin T) showing the Brownian motion of nanobeads (micromer‐redF, 100 nm; 10 µg/mL) in a solution of c‐IYNeu5Ac (15 mm) (i) prior to, (ii) 30 min after, and (iii) 60 min after NA addition, corresponding to the Sol, pGel, and Gel states, respectively. NA was added at 0.90 U/mL (2.0 µL), resulting in a final concentration of 0.15 U/mL. Entrapped nanobeads are indicated by white arrowheads. Scale bar: 2 µm. Conditions: [Thioflavin T] = 2.8 µm, PBS (–) (pH 7.4):DMSO = 95:5 (v/v), room temperature.

As shown in Figure 7Bi, active Brownian motion was observed in an aqueous solution of c‐IYNeu5Ac . In contrast, Brownian motion was markedly suppressed 30 min after the addition of NA (partial gel state), as the gel state began to form (Figure 7Bii). Fiber networks, visualized by ThT fluorescence, gradually emerged (Figure 7Bii→iii). These results indicate that the entangled fibrous structures act as effective physical barriers to the movement of nanosubstances in the gel phase.

3. Conclusion

This paper presents a versatile modular molecular design for glycosidase‐responsive supramolecular hydrogels, employing saccharide‐appended CDPs as building blocks. These supramolecular hydrogel systems enable selective, glycosidase‐dependent control over the release and entrapment of bio‐relevant substances within the gel matrix. The inherent protease resistance and relatively low cytotoxicity of CDPs [14, 23], coupled with their structural compactness, underscore their potential for further bioactive applications involving elaborate molecular designs. Critically, the interplay between saccharide properties and the overall molecular balance dictates the self‐assembly pathway and outcome, yielding supramolecular architectures ranging from non‐networked nanoparticles in the sol state to networked fibers in the gel state [65, 66], as demonstrated in this study. These findings provide a foundation for the rational design of soft materials with tunable supramolecular architectures and enzymatic responsiveness, offering a promising platform for advanced biomedical and biotechnological applications.

Funding

This work was supported by Grant‐in‐Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (23H01815, M.I.), the joint research program of the J‐GlycoNet cooperative Network, accredited by the Minister of Education, Culture, Sports, Science and Technology (MEXT), Japan, As a Joint Usage/Research Center, Tokai Pathways to Global Excellence (T‐GEx), the MEXT Strategic Professional Development Program for Young Researchers (S.L.H.), and the THERS Interdisciplinary Frontier Next Generation Researcher Project and Make New Standards Program (JST SPRING, S.S.).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73943‐sup‐0001‐SuppMat.pdf.

SMLL-22-e73943-s001.pdf (3.7MB, pdf)

Acknowledgements

This work was supported in part by a Grant‐in‐Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (23H01815, M.I.), the joint research program of the J‐GlycoNet cooperative Network, accredited by the Minister of Education, Culture, Sports, Science and Technology (MEXT), Japan, as a Joint Usage/Research Center, Tokai Pathways to Global Excellence (T‐GEx), the MEXT Strategic Professional Development Program for Young Researchers (S.L.H.), and the THERS Interdisciplinary Frontier Next Generation Researcher Project and Make New Standards Program (JST SPRING, S.S.). The authors are grateful to Associate Prof. Naoko Komura and Prof. Hiromune Ando (Gifu University) for their valuable discussions regarding the synthesis of compounds containing N‐acetylneuraminic acid. The authors also acknowledge the Life Science Research Center, Gifu University, for instrument maintenance and support.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File: smll73943‐sup‐0001‐SuppMat.pdf.

SMLL-22-e73943-s001.pdf (3.7MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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