Design and Characterization of Bovine Serum Albumin Hydrogels: A Promising Platform for Controlled Drug Delivery

Abstract

Protein-based hydrogels have attracted increasing interest as biocompatible matrices for drug delivery; however, simple systems based exclusively on albumin remain relatively underexplored. Here, we report the formation of bovine serum albumin (BSA) hydrogels generated through mild thermal aggregation (65 °C) without chemical cross-linkers, employing low protein concentrations (1–1.5%). The influence of the buffering environment (PBS vs Tris-HCl) on hydrogel architecture was identified as a key structural determinant. The resulting hydrogels were systematically characterized in terms of their structure, swelling behavior, water content, biodegradability, and morphology. Microscopic analyses revealed that Tris-HCl promoted the formation of a homogeneous and dense amyloid-like fibrillar network, whereas PBS led to more heterogeneous and irregular architectures. These structural differences affected the capacity to absorb and release model compounds with distinct physicochemical properties, producing sustained and matrix-dependent release profiles. In vitro assays demonstrated progressive degradation under physiological conditions, while in vivo murine models showed minimal immunological response.

1. Introduction

Hydrogels are three-dimensional polymeric networks capable of sequestering substantial volumes of water or biological fluids, a property that renders them highly attractive for diverse biotechnological, biomedical, and pharmaceutical applications. − In recent years, increasing attention has been shifted toward matrices derived from natural polymers, owing to their inherent biocompatibility, biodegradability, and minimal immunogenic profile. , Among these systems, protein-based hydrogels have emerged as particularly versatile materials.

Biomolecules, such as collagen, gelatin, and albumin, integrate structural plasticity with intrinsic biological functionality. , The abundance of reactive functional groups, including amino, hydroxyl, and carboxyl moieties, facilitates the assembly of complex networks stabilized by noncovalent interactions, such as hydrogen bonding, electrostatic forces, and hydrophobic interactions. , Consequently, protein-based scaffolds exhibit inherent biocompatibility and biodegradability, making them suitable candidates for biomedical and pharmaceutical applications that require mild processing conditions and biological compatibility.

Recent advances in protein-based materials have demonstrated that thermally induced protein aggregation can be effectively harnessed to generate structured hydrogel systems with biomedical relevance, circumventing the need for potentially toxic chemical cross-linkers. At the molecular level, the transition from a soluble state (monomer) to a cohesive hydrogel is driven by a sophisticated orchestration of structural reorganization processes involving partial unfolding, secondary structure rearrangement, and subsequent self-assembly.

Under finely tuned conditions of temperature, pH, ionic strength, and protein concentration, globular proteins undergo conformational transitions that expose naturally buried functional groups. This promotes the establishment of robust intermolecular interactions, mainly hydrophobic forces, hydrogen bonding, and electrostatic interactions , that collectively stabilize the three-dimensional hydrogel networks. Such physical restructuring provides a versatile and tunable strategy for hydrogel fabrication, relying solely on inherent molecular interactions to dictate the final material properties. ,

Among globular proteins, bovine serum albumin (BSA) represents a particularly interesting building block due to its abundance, remarkable structural stability, and well-documented biocompatibility. Recent investigations have reported the use of BSA-based hydrogels and albumin-derived supramolecular assemblies for drug delivery and other biomedical applications, primarily exploiting their biocompatibility and capacity to interact with a diverse range of ligands, including small therapeutic interactions with a diverse range of ligands, including small therapeutic molecules and proteins. , These multifaceted functionalities and safety profiles position albumin as a paradigmatic candidate for the development of smart, biobased platforms. However, a simple hydrogel system based exclusively on thermally aggregated albumin and formed under mild conditions remains underexplored.

Because of its biocompatibility and structural versatility, BSA is a highly attractive building block for protein-based hydrogels. In this work, we aimed to systematically investigate the formation of BSA hydrogels induced by thermal treatment and mechanical agitation, characterizing their structural, physicochemical, and immunological properties under different preparation conditions. By elucidating the interplay between buffer composition and network stability, this study provides a comprehensive framework for designing bioinert and enzymatically stable protein scaffolds suitable for advanced drug-delivery applications.

2. Methodology

2.1. Reagents

BSA, pepsin from porcine gastric mucosa (EC 3.4.23.1), trypsin from bovine pancreas (EC 3.4.21.4), α-chymotrypsin TLCK-treated bovine pancreas (EC 3.4.21.1), thioflavin-T (ThT), trycine, imidazole, acrylamide, and N-N-methylene-bis-acrylamide were purchased from Sigma-Aldrich (St. Louis, MO). Commercial buffers HEPES and Tris-HCl, Coomassie Brilliant Blue G-250 (MW 854), and Phenol Red (MW 354.4) were obtained from Genbiotech (Argentina). Analytical grade HCl, NaCl, KCl, Na₂HPO₄, KH₂PO₄, NaOH, and Bromophenol Blue sodium salt (MW 691.9) were obtained from Cicarelli (Argentina). Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor was purchased from Roche (Argentina). Solutions were prepared using Milli-Q water and filtered using a 0.22 μm filter. To perform in vivo assays, all solutions were filtered or sterilized in an autoclave; final BSA solutions were filtered to maintain sterile conditions.

2.2. In Vitro BSA Aggregation

To obtain BSA hydrogels, several experimental conditions were evaluated by varying the buffer compositions and pH. Aggregation was carried out at a fixed temperature slightly above the denaturing point (65 °C) under constant orbital stirring (600 rpm) using a tabletop thermomixer C (Eppendorf, Hamburg, Germany). BSA solutions were prepared in the following buffer solutions: HEPES 40 mM/NaCl 60 mM, pH 7.4; Tris-HCl 20 mM, pH 7.4 and 5.0. The pH was adjusted using HCl 3 M, and PBS 10 mM (137 mM NaCl), pH 7.0. The protein concentrations used were 5, 10, and 15 mg/mL. The aggregation reaction was monitored for 120 h; 50 μL aliquots were withdrawn at different time points for further analysis. Final products (from 24 to 120 h incubation) were preserved at −20 °C. The concentration of the free BSA monomer (BSA_m) in the reaction supernatants was directly measured using a DS-11 spectrophotometer (deNovix, Delaware) and used to calculate the aggregation rate according to the following equation:

$$\mathrm{Aggregation}\mathrm{rate}=\frac{(C_{\mathrm{i}}-C_{\mathrm{f}})}{C_{\mathrm{i}}}\times 100$$ 1

where C _i is the initial BSA_m concentration, and C _f is the final BSA_m concentration. All experiments were performed at least in triplicate, unless otherwise stated. Data are presented as mean ± standard deviation.

2.3. Thioflavin-T assay and Second Degree Light Scattering

A stock solution of ThT 2.5 mM was prepared in Milli-Q water, filtered, and stored, as previously described. Briefly, a mixture of 110 μL of ThT and 10 μL of the hydrogels in a previous dilution was prepared to reach a final concentration of 25 μM ThT and 0.045 mg mL^–1 of protein. Each sample was analyzed using a 3 mm Micro Quartz fluorometer cell (HORIBA Instruments) for fluorescence and second-degree light scattering using a Fluoromax-4 spectrofluorometer (HORIBA Scientific). For fluorescence analysis, the excitation wavelength (λ_ex) was set at 450 nm, and the emission (λ_em) was read at 482 nm. For second-degree light scattering, the values were λ_ex = 300 nm and λ_em = 600 nm. Slit widths of 2 were set for excitation and emission of light paths. Background spectra from blank buffer and ThT 25 μM solutions were extracted from sample readings. End point data were analyzed for 0, 24, and 120 h incubation time. Values represent the means of at least three independent experiments with their respective standard deviation.

2.4. Swelling Ratio Determination

Swelling capacity was determined using BSA aggregates previously dehydrated using a centrifugal evaporator Savant (ThermoFisher) at 36.5 °C and 3000 rpm overnight (16 h) and maintained sealed at −20 °C. To determine the swelling ratio (SR), all products were weighed at a standard weight (1 mg each), rehydrated with 200 μL of bidistilled H₂O or DMEM culture, and left for 2 h to stabilize at room temperature (RT). Excess fluid was eliminated with absorbent paper, and the products were weighed again. SR and water content (WC %) were calculated using the following formulas ,

$$\mathrm{SR}=\frac{W_{\mathrm{w}}-W_{\mathrm{d}}}{W_{\mathrm{d}}}$$ 2

$$\mathrm{WC}\%=100\times \left[\frac{W_{\mathrm{w}}-W_{\mathrm{d}}}{W_{\mathrm{w}}}\right]$$ 3

where W _w is the wet weight, and W _d is the dry weight of the BSA hydrogels. At this point, BSA aggregates were named as follows: (a) 10-BSAg-PBS, (b) 15-BSAg-PBS, (c) 10-BSAg-Tris-HCl, and (d) 15-BSAg-Tris-HCl, where the number indicates the initial BSA_m concentration (10 or 15 mg mL^–1); letter “g” indicates gel formation, and the buffer used (PBS 10 mM or Tris-HCl 20 mM); all products were obtained from 120 h of incubation. Values represent the means of at least three independent experiments with their respective SEM.

2.5. Dye Release Experiment

A releasing molecule assay was performed using dehydrated BSA aggregates prepared as described above. Coomassie Brilliant Blue G-250, Bromophenol Blue sodium salt, and Phenol Red were dissolved in Milli-Q water to obtain a 0.8 mM solution. One milligram of dried hydrogel was weighed and rehydrated with 200 μL of dye solution at 65 °C for 1 h, and then left to stabilize at room temperature overnight. The remaining dye solution was removed, and the volume was adjusted to 1 mL with isotonic saline solution (NaCl 0.9% w/v). The vessels were incubated for 7 days at 37 °C under static conditions. Absorbance was measured by taking 1.5 μL from the vessels at different times during the first 24 h and then once a day using a DeNovix CS-11 spectrophotometer. The concentration of released dyes was calculated using calibration curves prepared from pure dye solutions at their respective maximum absorbance wavelengths (phenol red at 429 nm, bromophenol blue at 592 nm, and CBB-G250 at 583 nm), as previously reported, and the results are expressed as the concentration coefficient (C _f/C _i), where C _f is the final concentration, and C _i is the initial concentration. Dye release data were expressed as normalized concentration ratios (C _f/C _i) to enable comparative analysis of release behavior among hydrogels with different swelling capacities, rather than quantifying absolute dye loading efficiency. In all cases, experiments were performed at least in triplicate, unless otherwise stated.

2.6. In Vivo Studies

To evaluate the potential of BSA hydrogels to induce inflammatory and/or adaptive-immune responses, in vivo assays were performed using a murine model. Young adult male BALB/c mice (5–6 weeks old) were obtained from the CERELA animal facilities (Centro de Referencia para Lactobacilos, San Miguel de Tucumán 4000, Argentina). Mice were derived from a closed breeding colony, maintained under specific pathogen-free conditions and housed under controlled environmental conditions (22 ± 2 °C, 55 ± 2% relative humidity) with a 12 h light/dark cycle. Mice were fed a standard diet and had ad libitum access to water. All experimental procedures were preapproved by the CERELA Animal Protection Committee (CRL-BIOT-LI-2012/B) and conducted in accordance with the guidelines established by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Animals were randomly divided into experimental groups according to the BSAg used and the administration route. Each group was identified by the hydrogel code, followed by the letter “o” for oral or “s” for systemic administration (e.g., 10-BSAg-PBSs). The control group was sham-inoculated with 10 mM PBS to determine the baseline immune parameters. The animals received three inoculations on alternate days consisting of 100 μL of hydrogel (500 μg mL^–1) diluted in sterile 0.9% NaCl. For systemic stimulation, hydrogels were administered by subcutaneous injections, whereas for mucosal stimulation, oral gavage was used. Twenty-one days after the last inoculation, the mice were anesthetized with ketamine-xylazine for blood collection and subsequently euthanized by cervical dislocation for small intestine removal. Serum was obtained by centrifuging at 1500 rpm for 15 min at 4 °C and stored at −20 °C. The small intestine was flushed with 1 mL of cold sterile PBS 10 mM. Intestinal fluids were centrifuged at 3000 rpm for 15 min at 4 °C to remove fecal traces, and stored with PMSF 1 mM at −20 °C. For the in vivo studies, three animals were used per experimental group.

2.7. Immunological Parameter Determination

Serum levels of specific IgG antibodies and cytokines interleukin 4, 6, and interferon γ (IL-4, IL-6, and IFN-γ) were determined by ELISA. The mucosal immune response was evaluated by measuring the total secretory IgA (S-IgA) in small intestinal fluids. Cytokines were measured using commercial kits (BD OptEIA mouse cytokine ELISA sets) according to the manufacturer’s instructions. Total S-IgA and specific IgG antibodies were measured, as previously described by Velez et al. IgA determination, goat-antimouse IgA affinity-purified (BETHYL Laboratories Inc., Montgomery, TX) was used as the capture antibody. Purified mouse IgA Kappa immunoglobulin (SIGMA, Saint Louis) was used as a standard, and goat-antimouse IgA (α-chain specific) peroxidase conjugated (SIGMA, Saint Louis) was used as a detection antibody. For specific IgG determination, ELISA plates were coated with BSA hydrogels, and detection was performed using biotin SP-conjugated goat-antimouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA). All reactions were revealed with enzyme concentrate streptavidin-HRP (BD OptEIA, San Diego) and TMB reagent (3,3′,5,5′-tetramethylbenzidine; BD Biosciences, San Diego), and then stopped with H₂SO₄ 2 N. The absorbance was measured at 450 nm. Results were expressed as pg mL^–1 for cytokine quantification, absorbance (optical density at 450 nm) for specific IgG, and μg mL^–1 for S-IgA. Values represent the means of at least three independent experiments, with their respective SEM.

2.8. Long-Term In Vitro Stability/Degradation Assay in PBS

Hydrogels were first washed three times by centrifugation at 12,000 rpm in Mili-Q water to remove residual salts and unaggregated BSA monomers. Pure hydrogels were then mixed with fresh sterile 10 mM PBS at a final ratio of 1:2 hydrogel/PBS (v/v) and incubated at 37 °C for 3 weeks under mild agitation (300 rpm) in a Thermomixer. 50 μL aliquots were withdrawn from the mixture at times t₀: 1 min, t₁: 6 h, t₃: 24 h, and subsequently every 24 h for up to 21 days, while maintaining sterile conditions. The samples were stored at −80 °C. The free BSA released into the medium was quantified by UV–Visible spectroscopy and expressed as a concentration (mg mL^–1). All assays were carried out in triplicate.

2.9. In Vitro Enzymatic Digestion Assay

The digestion process was considered in two consecutive stages: gastric and intestinal degradation, following the instructions from Minekus et al., adapted to small volumes. The simulated gastric fluid (SGF) stock solution was prepared containing KCl 6.9 mmol, KH₂PO₄ 0.9 mmol, NaHCO₃ 25 mmol, NaCl 47.2 mmol, MgCl₂(H₂O)₆ 0.1 mmol, (NH₄)₂CO₃ 0.5 mmol, and CaCl₂(H₂O)₂* 0.75 mmol, adjusted to pH 3 with HCl 1M. The simulated intestinal fluid (SIF) stock solution contained KCl 6.8 mmol, KH₂PO₄ 0.8 mmol, NaHCO₃ 85 mmol, NaCl 38.4 mmol, MgCl₂(H2O)₆ 0.33 mmol, and CaCl₂(H₂O)₂* 0.6 mmol, which was adjusted to pH 7 with NaOH 1 M. Stock solutions were prepared at 1.25× and stored at −20 °C. For the gastric phase, hydrogels and gastric fluid were mixed to a final ratio of 50:50 v/v (five parts of hydrogel with four parts of GF). To complete the GF content, pepsin was added to a final concentration of 2000 U mL^–1 with CaCl₂(H₂O)₂* and the required volume of water to reach 1× concentration. Reaction was carried out for 2 h at 37 °C in a Thermomixer. The intestinal phase was prepared after the gastric phase with a 50:50 ratio, with five parts of the remaining gastric mix + four parts of IF. The reaction mix was completed by adding trypsin (100 U mL^–1) and chymotrypsin (25 U mL^–1) with CaCl₂(H₂O)₂* and the necessary amount of water to achieve 1× IF. The intestinal phase was followed for 2 h at 37 °C in a Thermomixer. Samples from each phase were withdrawn at different times (t₀: 1 min, t₁: 10 min, t₂: 30 min, t₃: 60 min, t₄: 90 min, t₅: 120 min); the reaction was stopped using a protease inhibitor (PMSF 1 mM) and snap-frozen at −80 °C. All assays were carried out in triplicate.

2.10. Blue Native Gel Electrophoresis

Degradation products from the PBS stability/degradation assay and enzymatic digestion assay were analyzed using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE). 15 μL samples were prestained with Coomassie Blue G-250 (2.5% stock solution prepared in a 1:1 ratio of water/glycerol solution). BN-PAGE was performed using stain-free gels at fixed concentrations (4% and 8%). Colored cathodes and anode buffers were prepared and used as specified by Wittig in a Bio-Rad Mini PROTEAN system.

2.11. Transmission Electron Microscopy and Scanning Electron Microscopy

Transmission and scanning electron microscopy (TEM and SEM) were employed to analyze the micro- and nanostructural organization of the BSA hydrogels, providing complementary insights into their internal morphology, pore structure, and surface characteristics. For TEM analysis, 15-BSAg-PBS and 15-BSAg-Tris-HCl hydrogels were diluted to a final protein concentration of 1 mg mL^–1 to ensure appropriate contrast and resolution of the nanostructures. Samples were prepared following the procedure described by González-Lizárraga et al. TEM images were acquired using a Zeiss Libra 120 transmission electron microscope at the Centro Integral de Microscopía Electrónica (CIME, CONICET-UNT, Tucumán, Argentina). For SEM analysis, 100 μL of the hydrogel samples were fixed with Karnovsky’s fixative (2.5% glutaraldehyde, 1.5% paraformaldehyde in phosphate buffer, pH 7.2) for 30 min at room temperature. After fixation, the samples were dehydrated in a graded ethanol series (30%, 50%, 70%, 90%) followed by 100% acetone, dried using liquid CO₂, and coated with a 5 nm-thick gold layer by sputtering. SEM analysis was performed using a ZEISS SUPRA 55 VP scanning electron microscope at CIME.

2.12. In Vitro Seeding Assay

A solution of BSA monomer (BSA_m) was prepared in NaCl 0.85%, pH 7.0, to mimic physiological conditions and maintained in cold (4 °C). Hydrogels obtained with 10 or 15 mg mL^–1 BSA in buffer Tris/HCl and PBS, pH 7.4–7.0, were diluted in NaCl 0.85%, pH 7.0, to the same concentration used in the in vivo assays (50 μg/100 μL). A mixture of 0.9 mL of BSA_m was prepared with 0.1 mL of each hydrogel and incubated at 37 °C with constant orbital stirring for 7 days. Samples were withdrawn at different times to study the aggregation of BSA_m induced by the already formed hydrogels. On days 3 and 5, 100 μL of hydrogel was added to each solution, and samples were collected. Aggregation was controlled by ThT fluorescence and second-order light scattering, as previously described. Results are presented as mean ± standard deviation (SD) of three independent assays.

3. Results and Discussion

3.1. Visible Aggregation Observed at 24 h with a Minimum BSA Concentration of 10 mg mL^–1

The development of protein-based supramolecular matrices has gained significant attention in the health and food industries due to their inherent biodegradability and biocompatibility. ,,, Among the various strategies for material fabrication, thermal induction is a versatile, solvent-free method that avoids potentially toxic chemical cross-linkers. ,− In this study, we employed mild thermal induction at 65 °C, a temperature strategically selected to be slightly above the BSA denaturation point (62 °C). According to the kinetic models proposed by Borzova et al., at this specific temperature, the protein unfolding stage and subsequent aggregation steps are markedly separated in time, allowing for the controlled formation of primary aggregates and minimizing the emergence of proteotoxic intermediate species. ,

To identify the optimal conditions for hydrogel assembly, an initial screening was performed by varying the protein concentration, buffer composition, and pH, as summarized in Table . The aggregation process was monitored at 24 and 120 h, revealing a marked dependence on these parameters.

1. Conditions for BSA Hydrogel Formation.

	BSA concentration
	5 mg mL–1		10 mg mL–1		15 mg mL–1
Buffer	24 h	120 h	24 h	120 h	24 h	120 h
Hepes/NaCl 40 mM, pH 7.4	++	++
Tris/HCl 20 mM, pH 7.4	+++	+++	+++	+	+	+
Tris/HCl 20 mM, pH 5.0	–	–	–	–	–	–
PBS 10 mM, pH 7.0	++	++	++	+++	+++	+++

Qualitative ThT fluorescence analysis at 24 h initially detected aggregates in 10 and 15 mg mL^–1 BSA samples prepared in Tris-HCl buffer (pH 7.4) and in 15 mg mL^–1 samples in PBS buffer (pH 7.0). However, due to the limited stability exhibited by these early stage species, the reaction was continued for up to 120 h to ensure full matrix consolidation.

Our results confirm that the formation of a self-supporting network is strictly governed by the critical gelling concentration (C ₀), a fundamental principle described by Yan and Pochan for various polypeptides and reported for BSA systems by Amdursky et al. , We observed that only BSA solutions with 10–15 mg mL^–1 yielded dense, stable aggregates, whereas the minimum concentration (5 mg mL^–1) failed to generate any visible structures regardless of the buffer used. Furthermore, environmental pH proved critical; under acidic conditions (pH 5.0), only disordered “white clots” were formed, which did not meet the structural criteria for functional hydrogels.

Table summarizes all the incubation conditions used to obtain BSA hydrogels. Aggregation was carried out at a constant temperature (65 °C) and stirring. The aggregation process was controlled by the qualitative ThT fluorescence intensity, shown with +.

In our system, the ionic microenvironment and temperature play a pivotal role in modulating the assembly kinetics of BSA hydrogels, aligning with previous reports that highlight the strong influence of physicochemical conditions on protein gelation. ,, Under Tris-HCl (pH 7.4), hydrogel formation proceeded efficiently, achieving aggregation rates exceeding 80% within the first 24 h, which correlated with the macroscopic gelation observed between 24 and 120 h. In contrast, gel formation occurred more slowly in PBS (pH 7.0), suggesting that the ionic environment in PBS stabilizes the native conformation of BSA, thereby kinetically delaying the structural rearrangements required for macroscopic hydrogel consolidation, despite the structural changes detected by fluorescence at 10 and 15 mg mL^–1.

The efficiency of the assembly process was quantified using the aggregation rate (Figure ). The observed differences between the buffers likely reflect the influence of ionic composition on protein conformational stability and intermolecular interactions during thermal assembly. Rather than promoting direct chemical cross-linking, the ions present in PBS modulate the process through electrostatic screening and specific ion–protein interactions. An environment containing Na⁺ can enhance the conformational stability of BSA by shielding the charged surface residues, thus reducing the long-range electrostatic repulsion. Under these conditions, thermal energy drives closer molecular proximity and promotes intermolecular hydrogen bonding and hydrophobic packing to govern the assembly. Interestingly, this stabilization of the native-like state in PBS paradoxically delays the abrupt unfolding events required for initial aggregation. This slow, more controlled assembly leads to a more ordered and denser fibrillar network during hydrogel formation compared to less saline environments like Tris-HCl. ,

1.

Aggregation rate of BSA under different buffer and time conditions. BSA was incubated at 65 °C with continuous stirring (600 rpm) at different concentrations (10 and 15 mg mL^–1). Bars with white and black squares correspond to aggregation performed in PBS (pH 7) for 24 h; bars with vertical stripes correspond to aggregation performed in PBS ( pH 7) for 120 h; bars with crossed black stripes correspond to aggregation performed in Tris-HCl (pH 7.4) for 24 h and bars with diagonal stripes correspond to aggregation performed in Tris-HCl (pH 7.4) for 120 h. Data are presented as mean ± standard deviation (n = 3).

Finally, the transition from early, unstable aggregates observed at 24 h to the robust matrices shown in Figure at 120 h reflects a maturation process, where the network reaches a thermodynamic “sink,” as proposed by Jean et al. In this state, the fibrils become highly entangled and stabilized within a 3D supramolecular architecture. In contrast, using acidic conditions (pH 5.0) resulted in the formation of “white clots” (Table ). This phenomenon occurs because aggregation near the BSA isoelectric point (pI 4.7) is too rapid and disordered, failing to produce the functional properties and porous meshwork of a true hydrogel. Based on these considerations, 10 and 15 mg mL^–1 BSA products obtained either in PBS (pH 7.0) or Tris-HCl (pH 7.4) and 120 h incubation were selected for further characterization.

3.2. Structural Changes in BSA Aggregates

The formation of BSA hydrogels is driven by a molecular reorganization, initiated by protein unfolding and the subsequent transition of secondary structures into aggregated states. The formation of BSA hydrogels is driven by advanced molecular reorganization, initiated by protein unfolding and the subsequent transition of native secondary structures into aggregated states. Initially, the disruption of native stabilizing forces, primarily intramolecular hydrogen bonds and electrostatic interactions, exposes functional groups, which facilitate the establishment of new intermolecular associations. As gelation proceeds at 65 °C, hydrophobic interactions become the dominant force governing the final aggregation and cross-linking density of the protein network. ,

Given that the thermal treatment of BSA (e.g., at 64–65 °C) triggers a transition toward a molten-globule state prone to forming amyloid-like aggregates, , we employed the Thioflavin-T (ThT) probe to detect the presence of cross-β (cross-β) architectures. ThT is a benzothiazole dye that selectively intercalates into β-sheet-rich fibrils, resulting in characteristic fluorescence enhancement. Our results revealed relevant spectral shifts associated with cross-β structures, particularly in hydrogels prepared in PBS at 24 and 120 h (10 and 15 mg mL^–1 BSA; Figure A,B), which exhibited higher fluorescence intensities than those prepared in Tris-HCl buffer (15 mg mL^–1 BSA; Figure C,D). The positive ThT signal indicates a higher density of cross-β structures relative to the native state, consistent with recent findings by Panda et al., who demonstrated that heat-induced BSA (60 °C) undergoes a massive enhancement in ThT fluorescence linked to the appearance of β-sheet-rich aggregates. Although light scattering effects, common in dense protein matrices, contributed to intensities exceeding theoretical expectations, the signals remained diagnostic of supramolecular assembly. To confirm whether the increased ThT fluorescence correlated with the growth of larger aggregates, we monitored the second-order light scattering at 600 nm (λ_ex 300 nm). All four studied hydrogels exhibited significantly increased scattering signals at 24 and 120 h compared to the initial state (Figure A–D), confirming the successful consolidation of supramolecular BSA structures.

2.

Detection of amyloid-like structures in BSA hydrogels. BSA solutions (10 or 15 mg mL^–1) were incubated at 65 °C in PBS (pH 7.0) or Tris-HCl (pH 7.4) under constant stirring for 0h (bars with vertical stripes), 24 h (bars with black and white squares), or 120 h (bars with horizontal stripes). The presence of cross-β structures was evaluated by ThT fluorescence (λ_ex = 450 nm, λ_em = 482 nm). Increases in aggregate size were detected using second-order light scattering (λ_ex = 300 nm, λ_em = 600 nm). (A) and (B) show the results for hydrogels prepared in PBS (10 and 15 mg mL^–1, respectively), pH 7, while (C) and (D) correspond to samples prepared in Tris-HCl, pH 7.4. Data are shown as arbitrary units (a.u.); *p < 0.05 compared to that at time 0.

Our findings correlate with the macroscopic changes observed in the appearance of the BSA matrices and are further supported by the work of Panda et al., who reported a multifold increase in the hydrodynamic diameter of the protein following thermal treatment. This growth in the molecular size, accompanied by an increase in the polydispersity index, validates our observation of the formation of supramolecular structures and β-sheet-rich prefibrillar aggregates.

Complementary FTIR spectroscopy provides a detailed map of the structural transitions. The analysis confirmed that thermal denaturation triggers a shift from the predominantly α-helical native state to an aggregated β-architectural state. Specifically, a distinctive peak emerged at 1616 cm^–1, a critical structural marker for intermolecular cross-β-sheets. This band accounted for 7% of the hydrogel structure compared to 0.5% in native BSA. Recent literature identifies this 1616 cm^–1 band as a signature of stable intermolecular β-sheets and protein aggregates. This was accompanied by a decrease in the α-helical content (1653 cm^–1) and a concomitant increase in total β-sheets (1631 cm^–1), which represented 37% of the hydrogel vs 32% in the native protein (see Table S1 and Figure S1). Additionally, an increase in antiparallel β-sheets (1682 cm^–1) was identified as a distinctive feature, consistent with bands previously correlated to hydrogen-bonded antiparallel structures during protein aggregation. , While these amyloid-like motifs are not the sole components of the network, they act as critical structural anchors that provide stability and rigidity to the gel. This behavior supports a hierarchical assembly model, where stochastic nucleation leads to fibril elongation and, ultimately, the formation of a three-dimensional scaffold through fiber entanglement. In this framework, the density and entanglement of the fibers dictate the final mechanical properties and viscoelasticity of the BSA hydrogel.

3.3. BSA Aggregates Obtained in Tris-HCl (pH 7.4) Have a Good Swelling Response in H₂O and DMEM

The functional performance of hydrogels is largely dictated by their capacity to absorb and retain aqueous fluids, a property typically quantified through the swelling ratio (SR). , This parameter reflects the hydration capacity of the polymer network and is modulated by the cross-linking density and the balance between hydrophilic and hydrophobic interactions within the matrix. , Generally, highly hydrophilic or densely charged networks exhibit elevated SR values, whereas more hydrophobic structures show restricted swelling or even partial contraction upon dehydration. ,

To evaluate the suitability of BSA aggregates for biomedical applications, SR and Water Content (WC%) were measured in bidistilled water and DMEM, the latter serving as a physiological mimic for interstitial fluid. , Our results revealed that aggregates formed in PBS at 10 mg mL^–1 achieved the highest SR and WC (∼80%) in water (Figure A,B). However, this formulation lacked sufficient mechanical robustness and underwent structural disintegration during handling. Similar limitations have been documented for low-concentration BSA systems, where protein fractions below ∼3 wt % often fail to generate mechanically stable, free-standing hydrogels. ,

3.

Swelling ratio (SR) and water content (WC) of BSA hydrogels formed in PBS or Tris-HCl. BSA hydrogels were prepared at concentrations of 10 or 15 mg mL^–1 in PBS (pH 7.0) or Tris-HCl (pH 7.4) and incubated for 120 h at 65 °C. The hydrogels were dehydrated by using a centrifuge evaporator. Swelling ratio (A) and water content (B) were evaluated in bidistilled water and DMEM. All values are shown as mean ± standard deviation (n = 3); *p < 0.05.

In contrast, the Tris-HCl-based formulations (10 and 15 mg mL^–1) maintained stable SR values of between 69–73% across both media while maintaining their macroscopic integrity (Figure A,B). This behavior suggests that the synergy between Tris-HCl (pH 7.4) and thermal incubation at 65 °C optimizes the intermolecular interactions sufficiently to stabilize the matrix even at relatively low protein concentrations (1–1.5% w/v).

Previous studies have reported that increasing the protein concentration generally reduces swelling by limiting the free volume of the hydrated network. However, in our system, Tris-HCl aggregates maintained a favorable balance between the structural stability and fluid uptake. Collectively, these data confirm that all four BSA aggregates exhibit characteristic hydrogel-like behavior, with Tris-HCl-based formulations providing the most mechanically stable matrices under aqueous conditions.

3.4. DYE Release Profile Depends on BSA Aggregate Type and Dye Nature Properties

The functional utility of hydrogels in drug delivery is primarily defined by their capacity to enable the controlled and sustained release of active compounds. This process is typically governed by passive diffusion, network swelling, or specific solute–matrix interactions. ,, In this study, we evaluated the intrinsic capacity of BSA-based hydrogels (10 and 15 mg mL^–1) prepared in Tris-HCl or PBS to absorb and release small molecules using three model dyes with well-characterized physicochemical properties: Coomassie Brilliant Blue G-250 (CBB-G250), Bromophenol Blue (BPB), and Phenol Red (PR). Dye uptake and release kinetics were monitored for 168 h at 37 °C using dried hydrogels rehydrated in 0.9% NaCl solution containing the respective probes.

As shown in Figure A, CBB-G250 was completely absorbed and retained by all four hydrogel formulations, resulting in clear supernatants after 168 h. No detectable release was observed even after an additional 7 days of incubation (data not shown). This behavior is consistent with the high affinity of CBB-G250 for proteins, driven by strong hydrophobic interactions and electrostatic binding to basic residues. , Such extensive protein–dye interactions likely immobilize the molecule within the fibrillar BSA network, effectively preventing its diffusion into the surrounding medium.

4.

Release profiles of model dyes from BSA-based hydrogels. Dry BSA hydrogels (10-BSAg-PBS, 15-BSAg-PBS, 10-BSAg-Tris-HCl, and 15-BSAg-Tris-HCl) were rehydrated in Coomassie Brilliant Blue G-250 (CBB-G250), Bromophenol Blue (BPB), or Phenol Red (PR), each dissolved in 0.9% NaCl solution (pH 7.0). Dye release was monitored over 168 h at 37 °C. (A) No release of CBB-G250 was detected. (B) BPB was gradually released from all hydrogels after an initial lag phase. (C) PR was continuously released without lag. Data are expressed as the ratio of dye concentration in the medium at a given time (C _f) to the initial concentration (C _i) plotted against the logarithmic time. Note: In some cases, the error bars are smaller than the symbols.

In contrast, BPB exhibited a markedly different kinetic profile characterized by a brief initial lag phase (∼1 h), followed by a progressive increase in release that reached a steady state after approximately 24 h (Figure B). Certain differences among the formulations were evident: Tris-HCl-based hydrogels generally displayed higher BPB release levels than their PBS counterparts. Notably, the 10-BSAg–Tris-HCl system achieved the highest C _f /C _i values during the steady-state phase, whereas the PBS-derived matrices exhibited lower release levels over the same period (superior retention). These variations correlate with the structural characteristics described in the previous section, where differences in the protein concentration and network organization are expected to influence the effective mesh size and, consequently, the diffusion of small solutes through the hydrated matrix. In addition, the partial physical entrapment of BPB during the loading step may have contributed to the sustained release observed over time.

Phenol red (PR) exhibited a slower but immediate release profile compared to that of BPB, with no evident lag phase, indicating rapid equilibration between the hydrogel matrix and the surrounding medium (Figure C). At pH 7.0, PR was initially sequestered by the 10-BSAg-PBS, 15-BSAg-PBS, and 15-BSAg-Tris-HCl hydrogels, while the 10-BSAg-Tris-HCl matrix showed minimal dye retention. This behavior aligns with previous reports, indicating that PR possesses relatively high diffusivity in protein or peptide matrices due to its limited capacity for electrostatic anchoring compared with more highly sulfonated dyes.

Collectively, these results demonstrate that the release behavior of small molecules from BSA hydrogels is a multifactorial process depending on both the physicochemical properties of the solute and the structural characteristics of the hydrogel matrix. Tris-HCl-derived hydrogels maintained sustained release profiles for BPB and PR for at least 24 h while preserving structural integrity, highlighting their potential as protein-based platforms for controlled molecular delivery in biological environments. Although the specific release mechanisms were not examined, assessing the hydrogel capacity for the uptake and release of an active compound is a critical first step. Further studies are required to clarify these mechanisms, which are beyond the scope of this work.

3.5. 15-BSAg-PBS and 15-BSAg-Tris-HCl Do Not Induce Immune Response In Vivo In Mice

An ideal drug delivery system should exhibit minimal cytotoxicity toward healthy tissues while maintaining selective activity toward its intended targets. In addition, such systems should possess “stealth” characteristics to allow them to evade immunosurveillance and avoid premature clearance by the mononuclear phagocyte system. Although albumin-based biomaterials are generally regarded as biocompatible, bovine serum albumin (BSA) is inherently xenogeneic in murine or human models and may elicit immune responses. Moreover, structural rearrangements occurring during thermal aggregation, specifically the transition from α-helices to β-sheet-rich, amyloid-like fibrils, may lead to the exposure of cryptic epitopes. These conformational changes could potentially alter the immunogenic profile of the protein, increasing its recognition by the host immune system. To address these concerns, we evaluated the systemic and mucosal immune responses that could possibly be induced by BSA-based hydrogels using an adult BALB/c mouse model. ,,

In this study, the administration of 10-BSAg-PBS (1.0% w/v) was identified as the only formulation that significantly increased specific IgG production (p ≤ 0.05) (Figure A). This humoral response was correlated with a significant elevation in IFN-γ levels in the same experimental group (Figure D), suggesting the induction of an active adaptive-immune response biased toward a Th1-type phenotype. − Th1 cells are well-characterized producers of IFN-γ, a cytokine that orchestrates macrophage activation and amplifies pro-inflammatory signaling during the early stages of the immune response. The observation that this immunological signature was unique to the 10-BSAg-PBS formulation implies that the hydrogel network formed at a lower protein concentration in PBS may exhibit reduced structural integrity. This reduced integrity potentially facilitates more rapid enzymatic degradation or mechanical breakdown in vivo, thereby promoting the premature release of immunogenic protein fragments or “danger signals” (DAMPs), triggering immune responses. In contrast, the more robust network (e.g., 15-BSAg-PBS or Tris-HCl-derived gels) likely sequestered the BSA monomer more effectively, minimizing its interaction with antigen-presenting cells, resulting in low production of specific IgG without significant biological relevance (Figure A). Consistent with these observations, IL-4 levels remained notably low in both the 10-BSAg-Tris-HCl and 15-BSAg-Tris-HCl groups (Figure E). Because IFN-γ is a well-known antagonist that downregulates IL-4 expression and suppresses Th2-mediated humoral responses, this cytokine cross-regulation likely explains the absence of a pronounced antibody response in these specific matrices. ,

5.

Systemic and mucosal immune responses to BSA hydrogels in BALB/c mice. Mice were immunized with BSA hydrogels prepared at 10 or 15 mg mL^–1 in PBS or Tris-HCl by intraperitoneal injection (systemic challenge “s”) or gavage (oral challenge “o”). (A) Serum levels of specific IgG (absorbance at 450 nm), (B) total secretory IgA in intestinal washes (μg mL^–1), and cytokines (pg mL^–1) (C) IL-6, (D) IFN-γ, and (E) IL-4 were quantified to assess systemic and mucosal immune responses. Data represents standard deviation (n = 3); *p < 0.05 compared to that of the normal control.

Notably, the 15-BSAg-Tris-HCl formulation exhibited no significant differences compared to the control group under systemic challenge (Figure A), indicating a favorable biocompatibility profile and a relatively bioinert behavior, suggesting that 15-BSAg-Tris-HCl evades significant immune recognition. These findings align with previous reports proposing that the hierarchical self-assembly of BSA into ordered fibrillar aggregates can yield structurally robust materials. In such cases, potentially immunogenic protein regions remain sterically shielded within the supramolecular network, minimizing their exposure to immunosurveillance.

Regarding IL-6, a pleiotropic cytokine typically secreted in response to infection, inflammation, or neoplastic pro, , our results revealed a significant increase only in mice treated with the 10-BSAg-TRIS-HCl group (Figure C). However, the absence of a concurrent specific IgG increase suggests that this formulation may trigger a transient inflammatory signal rather than a fully developed adaptive-immune response. In contrast, hydrogels prepared at higher protein concentrations (15 mg mL^–1) did not induce detectable changes in this cytokine, indicating enhanced immunological tolerance at higher network densities.

Finally, oral administration of 15 mg mL^–1 hydrogels did not elicit total secretory IgA (S-IgA) production in the intestinal lumen, and no detectable changes were observed in the serum cytokines analyzed (Figure B). These findings indicate that the hydrogels failed to activate the mucosal immune system under the experimental conditions tested. This behavior is consistent with previous observations, suggesting that protein-based hydrogels assembled through physical interactions may exhibit lower inflammatory potential than materials prepared using reactive chemical cross-linking.

Taken together, these results demonstrate that the 15-BSAg-Tris-HCl formulation offers the most favorable immunological profile, successfully combining structural stability with minimal systemic and mucosal activation. While these results are promising, further studies will be required to determine whether the immune responses observed in murine models are predictive of the performance of these protein-based hydrogels in human clinical settings. ,

3.6. 15-BSAg-Tris-HCl and 15-BSAg-PBS Hydrogel Morphology

For biomaterials intended for biomedical applications, elucidating the organization of the fibrillar network is essential to interpret the swelling behavior and diffusion properties. To evaluate how the buffer environment influences the supramolecular organization of BSA aggregates, hydrogels formed under mild thermal aggregation conditions (65 °C, 120 h) in PBS and Tris-HCl were characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

The TEM micrographs (Figure A–D) revealed distinct differences in the internal organization of the networks. Hydrogel formed in PBS displayed loosely packed, randomly oriented fibrillar networks with large, irregular voids (Figure A,B). In contrast, matrices prepared in Tris-HCl exhibited denser fibrillar bundles and a more organized internal architecture (Figure C,D). In both system, the fibrils extended several micrometers in length and were predominantly unbranched, consistent with previously reported thermally induced BSA fibrillar assemblies

6.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of BSA hydrogels formed in PBS and Tris-HCl after 120 h of incubation at 65 °C. Left panel (A–D): TEM images of BSA hydrogels prepared in PBS (A: 4000X, B: 10000X) and Tris-HCl (C: 4000X; D: 10000X). The hydrogels formed in PBS show loosely packed fibrillar networks with large, irregular pores, whereas Tris-HCl hydrogels exhibit denser and more compact fibrillar assemblies with smaller and uniformly distributed pores. Scale bars: 1 μm (A, C), 500 nm (B, D). Right panel (E–H): SEM images of BSA hydrogels prepared in PBS (E: 5000×; F: 10,000×) and Tris-HCl (G: 5000×; H: 10,000×). PBS hydrogels display a rough, highly porous surface with heterogeneous fiber distribution and randomly sized cavities, while Tris-HCl hydrogels exhibit a more homogeneous fibrous network with smaller, uniformly distributed pores. SEM images were acquired using an S2 detector to enhance the surface topography contrast. Scale bars are indicated. Scale bars: 1 μm (E, G) and 200 nm (F, H).

These structural differences were also evident at the surface level. SEM analysis showed that the PBS hydrogels presented a rough and heterogeneous morphology characterized by irregular cavities of variable dimensions (Figure E,F). Conversely, Tris-HCl hydrogels displayed a more homogeneous fibrous network with smaller and more uniformly distributed pores (Figure G,H). These observations were supported by a quantitative assessment of the pore size dispersion using Feret diameter analysis (see Supporting Information). PBS hydrogels showed a broad pore size distribution (30–120 nm) with high dispersion (maximum frequency at 40–60 nm). In contrast, Tris-HCl hydrogels displayed a narrower distribution (30–100 nm), reflecting a more regular network organization.

Although larger cavities with apparent diameters of several hundred nanometers were occasionally observed in the SEM images, these features are likely associated with pore coalescence or partial surface collapse during sample preparation and are therefore not representative of the intrinsic mesh size. Accordingly, the Feret diameter analysis provides a more reliable estimate of the effective pore dimensions governing mass transport within the material. The more compact and homogeneous architecture observed in Tris-HCl hydrogels aligns with reports indicating that buffer composition can influence protein–protein interactions and fibril organization through electrostatic effects. Similar fibrillar networks have been proposed for thermally induced BSA hydrogels, and organized fibrillar networks have been proposed as advantageous structural features in scaffolds designed to guide extracellular matrix formation.

Collectively, these results suggest that the Tris-HCl environment promotes the formation of a more ordered and topologically consistent fibrillar network in low-concentration BSA hydrogels.

3.7. In Vitro and Enzymatic Degradation of BSA Hydrogels

Evaluating the stability of hydrogels under physiologically relevant conditions is essential for understanding their degradation kinetics and predicting their performance as controlled drug delivery systems. To assess the intrinsic stability of the matrices, 15-BSAg-PBS and 15-BSAg-Tris-HCl hydrogels were incubated in sterile PBS (10 mM, pH 7.0) at 37 °C with constant agitation for 21 days. The release of soluble free BSA into the medium was quantified, and the remaining hydrogel fractions were analyzed by BN-PAGE.

As shown in Figure A, the degradation profiles varied according to the buffer used during assembly. The 15-BSAg-PBS matrices maintained relative structural integrity during the first week; however, a marked increase in protein release was observed starting on day 8 and continuing progressively until day 21. In contrast, the 15-BSAg-TRIS-HCl hydrogels exhibited a more gradual and sustained release profile at the onset of the assay. These differences suggest that the specific internal architecture of the hydrogel network modulates the rate of albumin erosion, consistent with previous observations of albumin-based scaffolds. Macroscopic observations corroborated these findings: freshly prepared hydrogels were handled as cohesive structures, whereas aged samples gradually lost stiffness, appeared softer, and were more prone to fragmentation after prolonged incubation. Such changes are consistent with the progressive disruption of the fibrillar network over time. BN-PAGE analysis of the supernatants (Figure B) confirmed the gradual release of protein fractions throughout the 21-day period (t₁ = 6 h, t₂ = 24 h, t₉ = 8 days, t₁₉ = 18 days), reflecting a slow but steady degradation of the albumin scaffold under physiological conditions relative to molecular weight markers (MWM) and monomeric BSA (BSA_m).

7.

In vitro degradation and protein release profiles of BSA hydrogels under physiological and enzymatic conditions. (A) Concentration of free BSA released into the supernatant (mg mL^–1) from 15-BSAg-PBS and 15-BSAg-Tris-HCl in sterile PBS (10 mM, pH 7.0) at 37 °C and 300 rpm for 21 days. Data are shown as mean ± SEM (*p ≤ 0.05 vs time 0). (B) BN-PAGE analysis from the degradation assay (A) showing protein fractions released at selected time points for 15-BSAg-PBS and 15-BSAg-Tris-HCl hydrogels (t₁: 6 h, t₆: 120 h, t₉:216 h, t₁₉ :432 h, equivalent to 5, 9, and 18 days of incubation). (C) Gastric degradation of 15-BSAg-Tris-HCl in simulated gastric fluid (SGF) with or without pepsin. The intact hydrogel is retained in the stacking gel (dotted arrows). (D) Intestinal degradation of 15-BSAg-Tris-HCl in simulated intestinal fluid (SIF) with or without trypsin/chymotrypsin. Residual aggregates are retained in the stacking gel (solid arrow), and the band intensity decreases progressively from t₀ to t₅ (2 h). MWM: molecular weight marker; BSA_m: monomeric BSA. PH: untreated hydrogel.

Given the potential for oral administration, the enzymatic resistance of the 15-BSAg-Tris-HCl hydrogel was evaluated using the standardized INFOGEST static digestion protocol described by Minekus et al. The gastric and intestinal phases were prioritized, assuming that orally administered materials undergo minimal residence time in the oral cavity (unless otherwise indicated). The hydrogel samples were sequentially exposed to simulated gastric fluid (SGF) and intestinal fluid (SIF). The electrophoretic profiles, therefore, correspond to specific sampling times (t₀–t₅), while PH denotes the untreated pure hydrogel, and BSA_m corresponds to the monomeric BSA used as a reference control. In addition, controls with and without enzymes in the gastric and intestinal fluids were used.

The gastric digestion profile (Figure C) showed that during the early stages of incubation (t₀ = 1 min, t₂ = 30 min, dotted arrows), substantial fractions of the hydrogel remained trapped in the stacking gel, indicating limited proteolysis. Partial degradation products only began migrating into the separation gel after 120 min (t₅), and did partial degradation products migrate into the separation gel (black rectangle), suggesting a degree of gastric resistance.

A different pattern emerged during the intestinal phase (Figure D). Although an initial fraction remained in the stacking gel (black arrow), the band intensity decreased progressively from t₀ (1 min) to t₅ (120 min), reflecting active enzymatic cleavage. This behavior is consistent with the action of trypsin, a serine protease present in intestinal fluid that targets lysine and arginine residues, both amino acids abundant in the BSA sequence. ,

Overall, these results indicate that BSA hydrogels undergo slow self-degradation under physiological conditions while retaining partial resistance to gastric digestion, ultimately remaining susceptible to intestinal proteases. Similar behavior has been reported for enzyme-responsive hydrogels designed to survive gastric transit and degrade in the intestinal environment. , This degradation profile suggests that the 15-BSAg-Tris-HCl formulation may be a suitable platform for the controlled delivery of therapeutic agents in the gastrointestinal tract. However, further in vitro studies, such as cytotoxicity and controlled degradation assays, are required.

3.8. BSA Hydrogels Do Not Produce Seeding with BSA Monomers

A primary concern in the development of amyloid-based biomaterials is the risk of seeding and cross-seeding. The seeding phenomenon, traditionally associated with proteopathies, refers to the capacity of small, ordered cross-β assemblies to act as nucleation templates that accelerate the aggregation of native monomers by lowering the energetic barrier of nucleation. , As noted by Kumar et al., the structural hallmarks shared by functional and pathological amyloids make it imperative to assess whether exogenous matrices can recruit monomeric proteins, potentially posing a risk of iatrogenic amyloidosis.

Although albumin is not typically associated with misfolding diseases, the intentional induction of intermolecular β-sheet structures during hydrogel fabrication necessitates the rigorous validation of its seeding potential. To address this, monomeric BSA (BSA_m) was incubated with BSA hydrogels (10 or 15 mg mL^–1) at 37 °C under mild agitation for 168 h. Seeding activity was monitored via ThT fluorescence and second-order light scattering (Figure A,B). Our results demonstrate that BSA-based hydrogels are conformationally stable and do not act as propagation-competent templates. Under these conditions, BSA_m alone remained stable without spontaneous aggregation. More importantly, the presence of hydrogel scaffolds did not induce any detectable increase in the ThT signal or light scattering over 168 h compared to the BSA_m control. Even after the sequential addition of hydrogel “seeds,” the concentration of native monomers remained unchanged, confirming the absence of a nucleated polymerization process. A transient increase in scattering was observed at early time points (1 and 6 h) in the BSA_m + 15-BSAg-PBS condition; however, this effect was not statistically significant (p ≤ 0.05) and was consistent with the presence of residual particulate material rather than de novo aggregate formation.

8.

Native BSA (BSA_m) seeding assay using BSA-based hydrogels. Thioflavin-T (ThT) fluorescence (A) and second-order light scattering (B) were measured over 168 h at 37 °C to evaluate the potential of BSA hydrogels to induce aggregation of native BSA_m. The reaction mixtures contained 10 mg mL^–1 BSA_m alone or with BSA hydrogels (10 or 15 mg mL^–1), with hydrogel additions at 0, 72, and 120 h. Data are expressed as mean ± SEM; *p ≤ 0.05.

This behavior agrees with the reports of Simpson et al. for stable fibrillar hydrogels, where the supramolecular network restricts the availability of reactive oligomeric species capable of initiating nucleated polymerization. In this context, the absence of detectable seeding activity suggests that the assembled BSA hydrogel network represents a structurally stable end state that does not recruit soluble albumin monomers under physiological conditions.

4. Conclusions

This study demonstrates that BSA can be effectively aggregated under mild thermal conditions to form self-standing hydrogels, whose distinct morphological, structural, and functional properties are strongly modulated by environmental conditions. Hydrogels prepared in Tris-HCl exhibited a more compact and organized fibrillar architecture compared to their PBS counterparts, as evidenced by TEM and SEM analyses. These matrices demonstrated high fluid retention capacity and distinctive molecular release profiles governed by both the supramolecular hydrogel architecture and solute chemistry, highlighting their potential as versatile drug delivery platforms.

Notably, the 15-BSAg-Tris-HCl formulation exhibited superior stability both in vitro and in vivo without eliciting inflammatory or adaptive-immune responses in murine models. Furthermore, the absence of seeding activity indicates that these hydrogels have a conformationally stable end state that does not induce the aggregation of native BSA monomers under physiological conditions.

Collectively, these findings establish BSA hydrogels as a biodegradable and biocompatible platform with remarkable bioinertia. Future investigations should focus on the mechanical characterization, cytocompatibility assessment in human-derived cell lines, and release kinetics of therapeutic agents to accelerate the biomedical translation of these proteinaceous scaffolds.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11084.

• Additional FTIR spectra and table showing the relative content of secondary structures (α-helix, β-sheet, and cross-β) in BSA hydrogels compared to native BSA; SEM-based pore size dispersion analysis of PBS- and Tris-HCl-based BSA hydrogels. (PDF)

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.