Frequency sweep tests revealed distinct viscoelastic differences among FG gel, κ-CG gel, and FG/κ-CG composite gels. At 10 °C, FG gel (9% w/v) displayed elastic-dominated behavior (G′ > G″) at frequencies below 70 Hz (Figure 5A), but shifted to viscous dominance (G″ > G′) at higher frequencies, indicating a weaker structural network which is sensitive to dynamic perturbation. By contrast, κ-CG gel (0.8% w/v) maintained solid-like behavior (G′ consistently greater than G″) across the entire frequency range (0.1–100 Hz; Figure 5B), confirming demonstrating confirming the stability of their rigid double-helix network. The weakening of the FG gel network was more pronounced at elevated temperature (60 °C; Figure 5C), whereas the κ-CG gel network retained its solid-like behavior even at 70 °C (Figure 5D) (28).

Composite 6.8% FG + 1.2% κ-CG gels exhibited improved viscoelasticity compared with FG gel alone. At 10 °C, G′ remained greater than G″ throughout the tested frequency range (Figure 5E), and no crossover was observed. This demonstrated that the dual-network structure effectively reinforced the elastic framework, suppressing the frequency-induced transition to viscous dominance seen in FG gel. At 70 °C (Figure 5F), the composite still maintained G′ > G″, confirming good thermal stability (29). Thus, composites integrated the elasticity of FG with the rigidity of κ-CG, achieving more stable solid-like viscoelasticity across the tested temperatures and frequencies.

The enhanced storage and loss moduli indicate that κ-CG addition reinforces the gel network, improving elasticity and resistance to deformation. Biologically, this mechanical reinforcement provides stability under physiological stresses and maintains gel integrity during digestion or implantation, which is essential for consistent peptide release and tissue interaction.

Temperature ramp tests further demonstrated the thermal stability of different gels. FG gel exhibited a sharp melting transition near 40 °C, where G′ rapidly decreased below G″ (Figure 6A), confirming their limited thermal resistance (30). In contrast, κ-CG gel underwent a sol–gel transition upon cooling around 30 °C (Figure 6B) and retained its elastic dominance until melting occurred at 70 °C during heating (Figure 6C), demonstrating superior thermal stability.

Composite 6.8% FG + 1.2% κ-CG gels displayed intermediate but significantly improved thermal behavior. Cooling scans revealed a gelation onset at 35 °C (Figure 6D), slightly higher than FG gel, while heating scans showed a melting point at 65 °C (Figure 6E), approximately 25 °C higher than FG gel and close to κ-CG gel. These results highlighted the synergistic contribution of the κ-CG network in stabilizing the gelatin framework, ensuring that the composite gels not only resist thermal degradation but while also maintain mechanical integrity under physiological and processing conditions.

The improved thermal stability of the FG/κ-CG system arises from dual-network crosslinking between gelatin polypeptide chains and carrageenan sulfate groups. Such stability is crucial for oral or nutritional peptide delivery, as the gel must tolerate temperature and pH fluctuations without premature disintegration. This property also suggests better peptide protection during processing and storage.

The improved mechanical strength and thermal stability observed in the FG/κ-CG gels highlight the advantages of combining protein- and polysaccharide-based networks. To better understand the relevance of these properties, it is helpful to considering how this dual-network structure compares with commonly used peptide carriers is significant. PLGA microspheres, for example, are well known for their high structural rigidity and durability; however, their stiffness and hydrophobic nature often limit their compatibility with soft or highly hydrated biological environments. In contrast, alginate hydrogels provide excellent water retention and biocompatibility but are generally susceptible to mechanical deformation and reduced stability under stress.

Positioned between these two extremes, the FG/κ-CG gel offers a balanced profile. The interpenetrating network created by gelatin’s flexible chains and κ-carrageenan’s ionic interactions grants the material sufficient elasticity while maintaining structural integrity. This combination allows the gel to better accommodate physiological movement and environmental fluctuations without compromising its cohesiveness. Such material behavior is especially relevant for oral or nutritional peptide formulations, where the matrix must remain intact long enough to protect the active component while withstanding temperature variations and hydration changes.

Taken together, these comparisons indicate that the FG/κ-CG system provides a complementary alternative to both rigid synthetic carriers and mechanically weak natural hydrogels. Its balanced mechanical and thermal characteristics support its suitability as a protective matrix for bioactive peptides, laying a strong foundation for the subsequent biological evaluation.

FG gel was prepared by dissolving FG powder in deionized water at concentrations ranging from 6 to 10% (w/v). The mixtures were stirred continuously at 70 °C until complete dissolution, degassed, and subsequently cooled at 4 °C for 8 h to facilitate gelation.

κ-CG gel was obtained by dispersing κ-CG powder in deionized water at concentrations between 0.8 and 1.6% (w/v) under stirring at 70 °C. The solutions were then cooled at 4 °C for 8 h to form gels.

Composite FG/κ-CG gels were fabricated using a solute displacement approach, maintaining a total polymer concentration of 8% (w/v). Specifically, κ-CG was incorporated at concentrations of 0.8, 1.0, 1.2, 1.4, and 1.6% (w/v) by displacing an equivalent mass of FG, resulting in corresponding FG concentrations of 7.2, 7.0, 6.8, 6.6, and 6.4% (w/v). The mixtures were homogenized at 70 °C for 10 min, degassed in a water bath at the same temperature for 30 min, and finally cooled at 4 °C for 8 h to form stable composite gels.

Based on previous studies (13), SOP exhibits optimal osteogenic activity at micromolar concentrations. Therefore, SOP was incorporated into the 6.8% FG + 1.2% κ-CG composite solution at a final concentration of 70 μM prior to the gelation process. The mixture was homogenized at 70 °C for 10 min, degassed for 30 min, and cooled at 4 °C for 8 h to obtain the FG/κ-CG-SOP composite gel.

The morphology, transparency, and color of gels were assessed visually under standardized light conditions. Images were recorded using a digital camera. Colorimetric evaluation was performed using the CIE-Lab* system to quantify changes in brightness and chromaticity. To minimize variability, evaluations were conducted under controlled environmental conditions using the standardized scoring scales outlined in Table 1.

Water content ( W c ) :

Water content ( W c ) was determined by drying the centrifuged samples at 60 °C until a constant weight was achieved. The water content was calculated by:

where m d is the dry weight after drying.

Dried gelatin samples were immersed in distilled water or physiological saline [0.9% (w/v) NaCl] for 24 h at room temperature to assess their rehydration behavior, reflecting the gel’s ability to absorb and retain moisture. After soaking, surface moisture was carefully removed using filter paper. The rehydration percentage was calculated using:

where m r is the mass after rehydration, and m d is the initial dry mass. This parameter is critical for applications where gels undergo dehydration and subsequent moisture recovery.

The CIE-Lab* color space was employed for quantitative color evaluation. Measurements of lightness ( L ∗ ) , redness-greenness ( a ∗ ) , and yellowness-blueness ( b ∗ ) values were recorded using a precision colorimeter calibrated before each measurement session. Total color difference (ΔE) relative to reference samples was calculated as:

where L 0 ∗ , a 0 ∗ , b 0 ∗ represent reference sample values. The parameter Δ E provides a single metric for visual color deviation, essential for product quality control.

Dynamic rheological properties of gel samples were characterized using a HAAKE MARS III rheometer equipped with a 35 mm parallel plate geometry. We conducted measurements under controlled strain within the linear viscoelastic region to avoid structural damage. Frequency sweep tests ranged from 0.1 Hz to 100 Hz at a constant temperature of 10 °C to probe viscoelastic behavior.

Temperature sweeps were performed by cooling from 70 °C to 10 °C and heating back from 10 °C to 70 °C at a rate of 2 °C/min to evaluate gelation and melting transitions. The storage modulus G ′ (elastic response) and loss modulus G ″ (viscous response) were recorded. Gelation point ( T g ) and melting point ( T m ) were identified as the temperatures at which:

Indicating the transition between liquid-like and solid-like behavior. Apparent viscosity ( η ′ ) was also measured as a function of temperature and frequency to characterize the flow resistance of the gels.

Wild-type AB-strain zebrafish (Danio rerio) were obtained from the China Zebrafish Resource Center (CZRC, Beijing, China). Adults were maintained under standard laboratory conditions (28.5 ± 0.5 °C; 14 h light/10 h dark cycle) in a recirculating aquaculture system. Embryos and larvae were raised in fish water (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄) at 28.5 °C. All procedures were approved in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

Developing zebrafish embryos at 72 h post-fertilization were collected and randomly divided into six groups (n = 11 per group):

All treatments lasted 4 days with daily replacement of fresh medium at corresponding concentrations. At day 7 post-treatment, zebrafish larvae underwent indicator assays.

Seven-day-old zebrafish larvae were anesthetized with 0.015% tricaine methanesulfonate (MS-222) and placed in a 2% calcein solution for 6 h under dark conditions. After staining, larvae were rinsed three times with aquarium water to remove surface dye. Larvae were mounted on 3% methylcellulose, and fluorescence images of the head and spinal regions were acquired using a fluorescence microscope under identical exposure parameters.