Impact of a Reinforced Graphene Oxide and Alginate Hydrogel with an Oxygen Releasing System on the Biocompatibility of Pancreatic β‐Cells for Diabetes Therapy

ABSTRACT

Type 1 diabetes is characterized by autoimmune destruction of pancreatic β‐cells, resulting in insulin deficiency and impaired blood glucose regulation. Cell encapsulation using alginate‐based hydrogels offers immunoprotection but faces challenges due to low oxygen solubility, compromising cell survival to restore vascularization after implantation. To address this, we developed an alginate hydrogel reinforced with graphene oxide (ALGO) and integrated calcium peroxide (CPO) as an oxygen‐releasing system, stabilized with poly‐L‐lysine (PLL). Physicochemical and mechanical characterization confirmed incorporation of all components and improved elasticity with increasing CPO concentration. Hydrogels maintained structural stability for eight days and released oxygen throughout this period. Biocompatibility assays revealed that ALGO containing 0.25% CPO (0.25CPO) preserved cell viability and proliferation for 96 h, while 1% CPO negatively affected survival. Oxygen consumption analysis showed that 0.25CPO sustained mitochondrial respiration and enhanced maximal respiratory capacity. Glucose‐stimulated insulin secretion demonstrated that 0.25CPO maintained functional responsiveness under low and high glucose conditions. These findings indicate that 0.25CPO hydrogels provide controlled oxygen delivery, mechanical stability, and improved biocompatibility, making them a promising platform for pancreatic β‐cell encapsulation and future preclinical applications in type 1 diabetes therapy.

A combined alginate hydrogel with graphene oxide, reinforced with poly‐L‐lysine, and calcium peroxide to ensure the oxygen uptake and viability of pancreatic β‐cells, with enhanced elastic behavior, respiration, biocompatibility, and cellular functionality over 8 days, gives a scaffold to serve as a platform for future pre‐clinical studies targeting pancreatic β‐cell replacement in type 1 diabetes treatment.

1. Introduction

Type 1 diabetes is a chronic disease in which pancreatic β‐cells are attacked and destroyed by the immune system, impairing the ability to produce insulin [1, 2]. This condition prevents proper regulation of blood glucose levels, which can lead to serious complications if not adequately controlled [3, 4, 5, 6]. Currently, the main treatment for this disease involves administering insulin to compensate for the hormone deficiency [7, 8]. However, the use of exogenous insulin presents significant limitations, leading to the development of new therapies and approaches to insulin delivery aimed at improving patients’ quality of life and optimizing metabolic control [9, 10].

Natural and synthetic biomaterials have been used as immunoprotective devices to restore vascularization after implantation [11]. This is possible because cells can be encapsulated, creating a barrier that protects them from the immune system's response [11, 12], making this a relevant approach for regenerative therapy. Alginate is an anionic polymer extracted and prepared from dark and brown algae, is a linear, homogeneous, naturally derived material [11, 13, 14]. It exhibits high aqueous solubility, water retention through capillary forces, and a tendency to gel, enabling the development of biocompatible non‐cytotoxic gels [11, 13, 14] but with low mechanical properties. To reinforce alginate hydrogels, addition of graphene oxide (GO) significantly increases their mechanical stability due to its mechanical, electrical, optical, and thermal properties [15, 16, 17, 18, 19]. In addition, GO has been demonstrated in various biomedical applications with promising results, as it enhances cell viability, reduces the rate of apoptotic cells, and positively impacts cellular processes, such as a decrease in the immune response [19, 20, 21].

However, alginate hydrogels alone can bring adverse effects such as the foreign body response (FBR) [11, 12]. To reduce this risk and improve biocompatibility, the use of polycation coatings on the surface of hydrogels has been implemented. Poly‐L‐lysine (PLL) is the most used, providing stability to alginate hydrogels and preventing destabilization with chelators such as phosphate (PO₄ ^3‒) or citrate [11, 12]. However, the use of biomaterials like alginate hydrogels comes with some disadvantages, including the low solubility of oxygen, which affects encapsulated cells [22, 23, 24, 25]. Pancreatic β‑cells are characterized by exceptionally high metabolic activity and depend critically on continuous oxygen supply to sustain mitochondrial respiration and glucose‑stimulated insulin secretion. Under physiological conditions, β‑cells receive up to 15% of oxygen‑rich arterial blood to maintain their endocrine function; however, this condition is severely disrupted when cells are encapsulated within biomaterials due to limited oxygen diffusion and delayed vascularization following implantation [23, 26]. As a result, oxygen deprivation leads to metabolic dysfunction, impaired insulin secretion, and the formation of necrotic regions within three‑dimensional constructs [26, 27].

To address oxygen limitation, oxygen‑releasing compounds such as calcium peroxide (CPO) have been incorporated into biomaterials to locally generate oxygen via controlled chemical reactions [28, 29]. These studies present various applications in regenerative therapy for cells with high metabolic demands, such as pancreatic cells, hepatocytes, neurons, and muscle cells [29]. They highlight the importance of selecting appropriate oxygen‐releasing systems based on cell type and metabolic requirements [29]. CPO has been widely studied due to its high oxygen‑generating capacity and relatively predictable hydrolytic decomposition into oxygen and water [30]. CPO‑based platforms have demonstrated improved survival and metabolic activity of highly demanding cells, including pancreatic islets [31, 32]. However, many reported systems rely on non‑biodegradable carriers such as PDMS, require surgical removal, or induce adverse immune responses due to prolonged material persistence [29, 32]. Moreover, excessive or poorly regulated oxygen generation can result in hyperoxic stress, which is detrimental to pancreatic β‑cell viability and function [29, 33]. However, studies with pancreatic β‐cells have shown difficulties because the biomaterials designed providing oxygen exhibit little or no biodegradation [32]. Surgical intervention is needed to remove the implant, leading to complications since the presence of the biomaterial in the body triggers adverse immune responses [29, 33]. Despite these advances, important gaps remain in the design of oxygen‐releasing biomaterials for pancreatic β‐cell encapsulation. Most reported systems either rely on non‐biodegradable matrices, focus primarily on oxygen release or short‐term cell viability, or lack a comprehensive evaluation of β‐cell metabolic and endocrine function. In addition, the interaction between oxygen delivery, hydrogel mechanics, and hydration state is rarely addressed in an integrated manner, despite its relevance for β‐cell survival and performance. Ratzavi et al. [34] recently described that insulin‐producing cells are not easily incorporated into these three‐dimensional (3D) polymeric scaffolds because they include an oxygen‐releasing system in a non‐biodegradable biomaterial. This ultimately results in non‐uniform delivery of oxygen to the cells embedded in the material and subsequent cell death. Due to the require high metabolic activity of pancreatic β‐cells, the selection of the appropriate oxygen source, a suitable carrier material (biomaterial), and a controlled release method are required to establish successful oxygenation for consideration in translational applications [26, 29]. Nevertheless, the use of biocompatible natural biomaterials such as alginate in combination with calcium peroxide (CPO) are not thoroughly investigated and therefore may address the limitations in biomaterials providing oxygen to pancreatic β‐cells. The novelty of our work relies in addressing the limitations mentioned above by developing an alginate‐based hydrogel reinforced with graphene oxide and stabilized with PLL, incorporating CPO as a tunable oxygen source. Beyond physicochemical and mechanical characterization, we systematically evaluate how oxygen release modulates mitochondrial respiration and glucose‐stimulated insulin secretion of encapsulated pancreatic β‐cells. By directly linking material properties with β‐cell metabolic and functional data, this work provides a more comprehensive framework for the rational design of oxygen‐releasing hydrogels with potential translational relevance for type 1 diabetes therapy.

A preliminary study was made with alginate hydrogels with GO (ALGO) at different concentrations to establish their effective synthesis with which there was greater biocompatibility. The condition with 25 µg/mL of GO (ALGO25) was the most promising candidate among the conditions evaluated since its advantage in biocompatibility and cellular functionality compared to other concentrations was demonstrated in a previous study by us². In that sense, 1.87% alginate hydrogels were developed, and 25 µg/mL of GO (ALGO25) was added, then CPO was incorporated as an oxygen‐releasing system at a concentration of 0.25% and 1% (0.25CPO and 1CPO). These are polymerized in a CaCl₂ bath for ionic gelation and finally, the developed hydrogel was reinforced with 0.3% PLL. Physicochemical and mechanical characterization of the developed hydrogels was performed. Subsequently, the effect on biocompatibility (viability, proliferation, and cytotoxicity), respiration, and cellular functionality was analyzed. Our study shows that these oxygen‐releasing biomaterials were not only capable of improving the biocompatibility of pancreatic cells but also hold the potential to be used a promising platform for future pre‐clinical studies.

2. Materials and Methods

2.1. Cell Culture

Pancreatic β‐cells (BRIN‐BD11 cells, 10033003, ECACC) were cultured in RPMI‐1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) antibiotic/antimycotic solution (Sigma–Aldrich, USA) at 37°C in an atmosphere of 5% CO₂. Cells were passaged when they reached 85% confluence with 0.25% (w/v) trypsin–EDTA solution and seeded at 2 × 10⁵ cells/cm².

2.2. Hydrogel Synthesis

Alginate hydrogels reinforced with PLL, containing GO and CPO, were fabricated under sterile conditions (Figure 1). A 1.87% (w/v) alginate solution (Sigma–Aldrich, USA) was prepared in distilled water at a temperature of 37°C and filtered [15, 16, 35]. GO was obtained and characterized in a preliminary study by our group [2]; a 1 mg/mL solution was prepared and sonicated with a Fisherbrand Model 120 Sonic Dismembrator (Fisher Scientific, USA) at 50 Hz for 1 h. We have previously determined that maintained physicochemical, microstructural, mechanical, biocompatibility, and adequate functionality characteristics was a concentration of 25 µg/mL of GO with alginate (ALGO25) [2]. Thereafter, CPO (Sigma–Aldrich, USA) at 0.25% and 1% (w/w) was treated for 5 h with PBS 1× (Sigma–Aldrich, USA) [1] and added to ALGO25. 1 × 10⁶ cells/mL were added to the gel‐forming solution, and to promote ionic gelation of the hydrogels with GO and 0.25 or 1% CPO, they were extruded in a bath of CaCl₂ solution (50 mM, Sigma–Aldrich, USA) under constant agitation (200 rpm) for 7 min. They were placed in contact with 0.3% (w/v) PLL (Sigma–Aldrich, USA) for 2 min as a polycationic reinforcement. The spheres were transferred to a 24‐ or 48‐well plate for assays and incubated at 37°C with 5% CO₂ in RPMI‐1640 medium.

FIGURE 1.

Schematic representation of reinforce alginate hydrogel with an oxygen‐releasing system fabrication. Alginate was prepared at 1.87%, and GO was added at 25 µg/mL. CPO was treated with PBS 1× before addition to the solution. Pancreatic β‐cells were encapsulated, and hydrogels were reinforced with PLL. The oxygen‐releasing hydrogels were then analyzed for their physicochemical, stability, and oxygen delivery properties, and biocompatibility parameters.

2.3. Physicochemical Characterization

2.3.1. Fourier Transformed Infrared (FTIR) Spectroscopy

To analyze the chemical composition of hydrogels, an FTIR assessment was carried out [2]. Briefly, hydrogels were dried and placed under a CARY 630 FTIR spectrophotometer (Agilent, California, USA) with attenuated total reflectance (ATR) in transmittance mode, with eight scans performed at 4000–600 cm⁻¹.

2.3.2. Rheological Evaluation

To assess the flow of the hydrogels depending of the strain applied [2]. Briefly, after the gelation process, acellular hydrogels were placed under a 20 mm diameter parallel plates of an MCR 302 ANTON PAAR rheometer (Anton‐Paar, Graz, Austria) with a Peltier system for temperature control at 37°C. This allowed to determine the storage modulus (G′) and the loss modulus (G′′) for amplitude (deformation: 0.01%–300%) and frequency sweeps (angular frequency: 10 rad/s).

2.3.3. Dynamic Mechanical Analysis (DMA)

A compression test was performed to assess the maximum deformation and obtain the Young's modulus [2]. Briefly, hydrogels were placed on a DMA Q800 (TA Instruments, New Castle, USA) with a preload force of 0.0010 N, a force ramp rate of 0.5 N/min on a round disk at a temperature of 37°C. To obtain the Young's modulus, the slope of the linear region of the stress–strain curves was obtained.

2.3.4. Stability Tests

To evaluate the stability of hydrogels over time, we measured the diameter, swelling, and degradation percentage [2]. Briefly, upon synthesis, hydrogels were measured to determine their diameter for 8 days. To determine the swelling ratio, we weighed the initial mass (m_i) of the hydrogels and incubated over time at 37°C. We obtained the final swollen mass (m_s) and final dry mass (m_f) measured at different timepoints (0, 1, 2, 4, 6, and 8). Equation 1 was used to calculate the swelling mass:

$$q=\frac{m_s}{m_f}$$ (1)

The water content [36] (%) is defined by Equation 2:

$$Watercontent\left(\%\right)=\frac{m_s-m_f}{m_s}\times 100$$ (2)

In addition, the final swollen mass (m_f) was weighted, and the following Equation (3) was used to calculate the degradation percentage:

$$Degradation\%=\frac{m_i-m_f}{m_i}\times 100$$ (3)

2.3.5. Oxygen Release

To analyze the oxygen release over time, the oxygen‐releasing biomaterials were in contact with a physiological medium, and the O₂ was measured with an oxygen sensor. We used an Oxygraph‐2k equipment (Oroboros Instruments, Innsbruck, Austria), which is an instrument enabling the measurement of oxygen concentration under a confined and isolated chamber using a polarographic oxygen sensor. Prior to analysis, hydrogel beads were gently rinsed and transferred into a sealed 2 mL chamber containing fresh culture medium. The chamber was initially equilibrated with atmospheric oxygen, providing a known starting oxygen concentration as previously suggested [33, 35, 37], and subsequently sealed to prevent flow‑mediated oxygen exchange with the external environment. Under these conditions, changes in oxygen concentration reflect oxygen released from the hydrogel beads into a defined, non‐renewed volume rather than bulk culture conditions. Biomaterial samples were incubated at 37°C for 8 days with measurements taken every 2 days under agitation at 350 rpm, and oxygen was measured for 30 min and expressed as O₂ release in µM. The oxygen solubility factor was 0.89 for RPMI‐1640, and the local barometric pressure was 91 kPa.

2.4. Biocompatibility

2.4.1. Cell Viability

To assess the biocompatibility capacity of oxygen‐releasing biomaterials, we performed 3‐(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenyltetrazolium bromide (MTT) and live/dead assays. Encapsulated cells in CPO‐containing hydrogels were incubated for 48 and 96 h, and the MTT reagent was added to the culture medium at 0.5 g/L concentration for 5 h. The absorbance was measured at 570 nm using a microplate reader Synergy H1 (Agilent Technologies, Santa Clara, CA, USA), and values were expressed as relative percentage with respect to Cell Culture Plastic (CCP). In addition, we stained the cells for 1 h at room temperature with 5 µg/mL calcein AM and 5 µg/mL ethidium homodimer‐1 (Invitrogen, ThermoFisher Scientific, USA) and visualized by fluorescence imaging using an Olympus microscope BX53 (Olympus America Inc., NY, USA)

2.4.2. Proliferation

We analyzed the proliferation in encapsulated cells using the Quant‐iT PicoGreen dsDNA Kit following the manufactures instruction and as stated previously [2]. Briefly, hydrogels samples were collected after 48 and 96 h of incubation and treated with proteinase K (2 mg/mL) overnight. A standard DNA curve was used to extrapolate DNA concentration. The sample was mixed with 100 µL of DNA‐binding fluorescent dye solution and measured at an excitation of 480 nm and an emission of 520 nm in a Synergy H1 (Agilent Technologies, Santa Clara, CA, USA).

2.4.3. Cytotoxicity

To evaluate the cytotoxic influence of CPO‐containing hydrogels, we evaluated the Lactate Dehydrogenase (LDH) enzyme activity release in the medium with a LDH activity kit following the manufacturer instructions. In short, supernatants were collected and mixed with the LDH substrate and measured at 450 nm for 30 min at 37°C in a spectrophotometer (Multiskan GO, Thermo Scientific, USA). We used a standard curve to interpolate to the adequate concentrations.

2.5. Oxygen Uptake

To analyze the mitochondrial respiratory capacity of encapsulated cells, we performed a high‐resolution respirometry assay using an Oxygraph‐2 k (Oroboros Instruments, Innsbruck, Austria) as reported before [36]. In brief, encapsulated cells (2×10⁶ cells/mL) in hydrogels were placed in two chambers of 2 mL at 37°C under gentle agitation (750 rpm). Different respiratory states were analyzed: “basal” denotes oxygen consumed without the presence of inhibitors or uncouplers. “Leak” is respiration occurring in the presence of 2.5 µM oligomycin. The “uncoupled” state refers to the maximum oxygen consumption in the presence of 1 µM FCCP. Nonmitochondrial respiration was obtained after adding 2.5 µM rotenone and 2.5 µM antimycin, and it was subtracted from respiration. The results were obtained as oxygen flow per cell [pmol/(sec*1 × 10⁶ cells)] and expressed as a relative value in respect to CCP.

2.6. Glucose‐Stimulated Insulin Secretion Assessment

We evaluated the cell functionality by the glucose‐stimulated insulin (GSIS) assessment as previously reported [36]. Briefly, encapsulated cells were incubated with Krebs–Ringer Bicarbonate (KRB) buffer (125 mM NaCl, 3 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 1 mM NaH₂PO₄, 22 mM NaHCO₃, 10 mM HEPES, and 0.1% BSA, Sigma–Aldrich, USA) at low (2.8 mM) and high (28 mM) glucose concentration for 1 h, respectively. Supernatant was collected and insulin levels were detected using a Rat Insulin ELISA kit (Invitrogen, Thermo Scientific, USA). Finally, insulin secretion was normalized to DNA content (µg/mL) using the Quant‐iT PicoGreen assay.

2.7. Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM), and the statistical analysis was performed using GraphPad Prism, Version 7 (GraphPad Software Inc., USA). One‐way analysis of variance (ANOVA) was used, followed by post hoc Tukey's test to compare multiple groups. With a 95% confidence level, a statistical value of p < 0.05 is considered statistically significant.

3. Results

3.1. Physicochemical Characterization of the Hydrogel

3.1.1. Components in the Chemical Structure

To evaluate the integration of the components in the chemical structure of the hydrogel of ALGO25 with 0.25CPO and 1CPO, an elemental analysis by FTIR was carried out in order (Figure 2). Hydrogels of ALGO25, 0.25CPO, and 1CPO presented similar wavenumbers because the proportions of GO and CPO within the samples were relatively small. Similarly, OH groups were present due to their vibration in 3100–3500 cm⁻¹ [2, 38]. The absorption band at 2914 cm⁻¹ showed a weak signal that corresponds to the symmetric vibration of CH─ groups [2, 39]. Similarly, the presence of the absorption bands at 1400–1600 cm⁻¹ may be associated with the elongations of COOH groups [2, 39, 40]. The ALGO25 presented a band at 1400 cm⁻¹ but a slight shift was seen in the 0.25CPO and 1CPO conditions at 1406 cm⁻¹ and the presence of a new peak at 1470 cm⁻¹ which may be due to the O─Ca─O group of the CPO [41]. The bands of ALGO25 were common to those published recently by our group [2]. Based on the CPO spectrum obtained, a peak can be seen at 711 cm⁻¹ that only appears in the 0.25CPO and 1CPO conditions which have been assigned to the O─O group of the CPO [41, 42]. On the other hand, the 1010–1200 cm⁻¹ bands are associated with the stretching vibration of the C─O groups, while the 890 and 810 cm⁻¹ bands are assigned to the C─O─C stretching vibrations and polysaccharide structure [43]. There may be an overlap in the GO groups in the 1600 cm⁻¹ bands assigned to carboxyl groups, and epoxides at 1020 cm⁻¹ [40, 44].

FIGURE 2.

Fourier transform infrared spectroscopy (FTIR) of ALGO25 hydrogels with 0.25 and 1% CPO and PLL.

3.1.2. Rheological Analysis

We performed an amplitude and frequency sweep for rheological analysis to understand how ALGO 25 hydrogels with CPO respond to applied stresses (Figure 3). In Figure 3A, the amplitude sweep for the hydrogels of ALGO 25 with CPO can be observed. The storage or elastic (𝐺′) and loss or viscous (𝐺″) moduli depend on the applied deformation. In all hydrogels, the elastic modulus was higher than the viscous one (𝐺′ > 𝐺″) and the linear viscoelastic range (LVR) was higher for hydrogels with CPO than in the ALGO25 condition. Higher CPO concentration allowed higher modulus 𝐺′and 𝐺″ of hydrogels compared to ALGO25 hydrogels. It was found that the gc, which is the critical strain point corresponding to the maximum strain value where the elastic modulus remains constant, was higher for hydrogels with 0.25CPO and 1CPO than the ALGO25 condition. This value corresponded to 0.74% for both conditions, while for the ALGO25 hydrogel, it was 0.58%. In addition, the yield stress, which is the point where the sample loses elasticity, was higher in hydrogels with CPO in comparison to those without this component. The viscoelastic behavior of the hydrogels was later confirmed with the oscillatory test (Figure 3B), revealing a constant behavior where the storage modulus did not undergo significant changes despite the applied angular frequency.

FIGURE 3.

Mechanical characterization of hydrogels. (A) Amplitude and (B) frequency sweep for the rheological analysis of hydrogels. (C) Young's modulus (MPa) of ALGO25, 0.25CPO and 1CPO hydrogels. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01 vs. ALGO25.

3.1.3. Dynamic Mechanic Analysis

To evaluate the stress and strain, we performed a DMA analysis, and we obtained the Young's modulus (Figure 3C). The compression modulus found for the ALGO25 hydrogel was 1.338 ± 0.145 MPa, whilst 0.25CPO had an elastic modulus of 1.841 ± 0.023 MPa. However, the 1CPO condition had a high elastic modulus of 2.276 ± 0.206 MPa, which was significantly statistical than ALGO25 (p = 0.0039). These results suggest that CPO concentration influences the elastic capacity of ALGO hydrogels.

3.1.4. Evaluation of Swelling Rate, Water Content, and Degradation Percentage

The swelling index and the percentage of degradation over time were determined to find out the water absorption capacity and mass loss of the hydrogels (Figure 4). On day 1, it was found that ALGO25 hydrogel had a swelling index of 42.517 ± 1.953, which was significantly higher than the hydrogel with 0.25CPO (p = 0.0153) and 1CPO (p<0.0001) (Figure 4A). The 0.25CPO hydrogel had a higher swelling index with a statistically significant higher corresponding value of 34.238 ± 1.412 (p = 0.0166) than hydrogels with 1CPO. On day 4, this swelling capacity increased for the 1CPO hydrogel to a value of 33.910 ± 3.12 (p = 0.1289), and it was not significant with respect to the ALGO25 and 0.25CPO conditions. Additionally, these two conditions (ALGO25 and 0.25CPO) together showed very similar values on day 4.

FIGURE 4.

Stability of hydrogels and oxygen kinetics. (A) Swelling index, (B) percentage degradation, and (C) water content for ALGO 25 hydrogels with 0.25 and 1% CPO. (D) Diameter of hydrogel hydrogels ALGO25 hydrogels with 0.25 and 1% CPO during 8 days of incubation. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 vs. ALGO25; ^# p <0.05, ^## p <0.01, ^### p <0.001 vs. 0.25CPO. (E) O₂ release from ALGO25 hydrogels with 0.25 and 1% CPO over time. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Tukey's test, (a) vs. 0.25CPO, (b) vs. 1CPO, p <0.0001.

Interestingly, on day 6, this capacity was sustained for ALGO 25 at a value of 39.540 ± 3.024, and for the 0.25CPO condition, it was 35.153 ± 1.087. However, the index for hydrogels with 1CPO drastically decreased to 24.400 ± 2.812 compared to ALGO25 (p<0.0001) and 0.25% (p = 0.0032). At the end of the incubation time, ALGO25 and 0.25CPO hydrogels had an index of 28.277 ± 2.035 and 29.410 ± 0.433, respectively. In contrast, 1CPO hydrogels further decreased statistically significant this index to a value of 14.92 ± 1.449 compared to ALGO25 (p = 0.0003) and 0.25CPO (p = 0.0001). It can be seen in Figure 4B that the hydrogels started with their mass at 100% on day 0. As time passed, there were no significant results between groups (p>0.05) as they had a decrease of less than 10% at the end of the incubation period. Therefore, at 8 days, the remaining mass percentage for the ALGO25, 0.25CPO, and 1CPO hydrogels was 95.610 ± 0.291, 96.273 ± 0.859, and 93.910 ± 0.498%, respectively. The water content percentage was also measured (Figure 4C), and on day 0 it was over 90% for all conditions (p>0.05). This behavior was sustained for ALGO25 and 0.25CPO after 8 days on incubation with no statistical significance. However, as time went by, 1CPO hydrogels lost significant water content and on day 8 of incubation, reaching 84.425 ± 0.753 compared to ALGO25 (p = 0.0366) and 0.25CPO (p = 0.0411), respectively.

3.1.5. Diameter

To further verify the stability of the developed alginate hydrogels over time, the diameter (mm) was measured as shown in Figure 4C. On day 0, this analysis showed that the developed biomaterials had an approximate diameter of 4.181 ± 0.109, 4.239 ± 0.134, and 4.154 ± 0.121 mm for ALGO25, 0.25CPO, and 1CPO hydrogels (p>0.05), respectively. As time passed, the hydrogels had a low tendency to shrink, and on day 8, this measurement for ALGO25 hydrogels was 3.853 ± 0.081 mm, for the 0.25CPO condition was 3.955 ± 0.038 mm, and for the 1CPO condition was 3.823 ± 0.119 mm.

3.1.6. Oxygen Release

We evaluated the capacity of the systems with CPO to generate oxygen via an analysis of oxygen release over time (Figure 4D). On day 0, it can be observed that hydrogels with 0.25% CPO started with an oxygen release of 39.188 ± 2.611 µM (p<0.0001) with respect to the 1CPO condition. On the same day, the 1CPO condition had an oxygen release capacity of 104.429 ± 3.015 µM. However, the release gradually decreased as the days progressed to a value of 5.023 ± 0.132 and 12.333 ± 3.050 µM for the 0.25CPO and 1CPO conditions, respectively on day 8. These results demonstrate the ability of alginate hydrogels with CPO to release oxygen for up to 8 days.

3.2. Biocompatibility

3.2.1. Cell Viability

To analyze the impact of ALGO25 hydrogels with 0.25 and 1% CPO on cell viability, an MTT assay was carried out and further confirmed using the live/dead assay over time (Figure 5). ALGO25 and 0.25CPO hydrogels at 48 h obtained a non‐significant values of 104.385 ± 1.811% (p = 0.5122) and 109.467 ± 3.585 % (p = 0.1324), respectively, compared to CCP. However, the 1CPO condition had a drastic decrease in cell viability of approximately 19% (p = 0.0157) with respect to CCP. Furthermore, when statistically compared to ALGO 25 (p = 0.0107) and 0.25CPO (p = 0.0016) the decrease was also found to be significant. At 96 h, it was found that 0.25CPO maintained cell viability at a non‐significant value of 108.017 ± 4.132% (p = 0.2933) with respect to CCP. However, the ALGO25 condition presented a non‐significant decrease in cell viability with a value of 99.190 ± 0.746 (p = 0.9250). 1CPO hydrogels affected cell viability at 96 h as they had an average viability of 78.873 ± 5.802. This decrease was statistically significant when compared to CCP (p = 0.0120), ALGO25 (p = 0.0238), and 0.25CPO (p = 0.0005). These quantitative results found by MTT assay were confirmed by the qualitative findings (Figure 5B and Figure S1) of the live/dead assay. It can be observed that pancreatic β‐cells encapsulated in hydrogels at 46 and 96 h of ALGO 25 and with 0.25CPO did not present significant affectations on cell membrane integrity during this incubation time. However, hydrogels with 1CPO presented an increase in the presence of dead cells (red) throughout the entire construct as well as a decrease in the density of viable cells (green) at both 48 and 96 h.

FIGURE 5.

Effect of ALGO25 hydrogels with 0.25CPO and 1CPO on cell biocompatibility. (A) Effect of alginate, GO and CPO on viability of pancreatic β‐cells encapsulated in hydrogels at 46 and 96 h. (B) Fluorescence micrographs of pancreatic β‐cells encapsulated in hydrogels at 46 and 96 h, confirming qualitative cell viability by the live/dead assay. (C) Effect of ALGO25 hydrogels with 0.25CPO and 1CPO on the proliferation of encapsulated pancreatic β‐cells after 48 and 96 h of incubation. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Fisher's LSD test, ^* p <0.05, ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 vs. CCP; ^# p <0.05, ^## p <0.01, ^### p <0.001, ^#### p <0.0001 vs. ALGO25; ⁺ p <0.05, ⁺⁺ p <0.01, ⁺⁺⁺ p <0.001, ⁺⁺⁺⁺ p <0.0001 vs. 0.25CPO. (D) Cytotoxicity of ALGO 25 hydrogels with 0.25CPO and 1CPO on encapsulated pancreatic β‐cells demonstrated through LDH activity after 48 and 96 h of incubation. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 vs. Positive control.

3.2.2. Cell Proliferation

A PicoGreen assay was performed to evaluate the effect of ALGO25 hydrogels with 0.25 and 1% CPO on the proliferation of encapsulated pancreatic β‐cells (Figure 5C). Initially, at 48 h it can be observed that in ALGO25 hydrogels a significant increase in cell proliferation was obtained since they presented a DNA content of 414.466 ± 16.124 ng/mL (p = 0.0494) with respect to CCP. Similarly, hydrogels with 0.25CPO obtained a significant increase with a higher DNA content of 496.049 ± 17.575 ng/mL than CCP (p<0.0001) and the ALGO25 condition (p = 0.0032). However, hydrogels with 1CPO drastically affected cell proliferation in terms of DNA content as a concentration of 158.637 ± 29.076 ng/mL (p<0.0001) was obtained with respect to CCP. This result also decreased significantly when compared to ALGO25 (p<0.0001) and 0.25CPO (p<0.0001). At 96 h it can be observed that the hydrogel with 0.25CPO had the capacity to maintain a significant increase in DNA content with an average value of 421.050 ± 1.357 ng/mL (p = 0.0309) with respect to CCP. Likewise, upon statistical comparison with ALGO25 (p = 0.0382), a significant increase was found. However, it was found that in the ALGO25 base hydrogel, the DNA concentration was 370,650 ± 8,999 ng/mL, which was comparable to CCP (p = 0.9980). Finally, in the 1% hydrogel at 96 h there was no induction of cell proliferation since the DNA concentration was 153,150 ± 12,731 ng/mL. This value presented statistical significance with respect to CCP (p <0.0001), ALGO25 (p <0.0001), and 0.25CPO (p <0.0001).

3.2.3. Cytotoxicity

An evaluation of the released LDH enzyme activity was carried out to determine the cytotoxic effect of ALGO 25 hydrogels with 0.25 and 1% CPO on the encapsulated cells (Figure 5D). Initially, after 48 h of incubation, we found that ALGO25 and 0.25CPO hydrogels had a relatively low LDH enzyme activity of 4.203 ± 0.049 (p<0.0001) and 0.907 ± 0.041% (p<0.0001), respectively, compared to the positive control. In addition, it was shown that the enzyme activity in the 1% condition had a value of 13.051 ± 1.391% (p<0.0001) with respect to the positive control. After 96 h, LDH activity was found to have a slight change for ALGO25 and 0.25CPO conditions at an average relative value of 5.746 ± 1.625 (p<0.0001) and 1.407 ± 0.182% (p<0.0001), respectively. Similarly, LDH activity for the 1CPO condition had a higher value of 15.793 ± 1.144% (p<0.0001) with respect to the positive control.

3.2.4. Oxygen Uptake

An analysis of oxygen consumption was performed to evaluate the respiration capacity of pancreatic β‐cells encapsulated in ALGO25, 0.25CPO, and 1CPO hydrogels over time (Figure 6 and Figure S2). After 48 h of incubation, we found that in the basal state, the respiration capacity for ALGO25 and 0.25CPO hydrogels was 59.853 ± 10.060 (p = 0.0706) and 79.354 ± 4.238% (p = 0.3192), respectively, when compared to CCP. However, for the hydrogel with 1% CPO, there was a significant reduction in respiratory capacity of approximately 58% (p = 0.0281) with respect to CCP. Then, we analyzed the leak state with respect to CCP and the conditions showed no significant effect on this state as they had a respiration percentage of 74.104 ± 14.959 (p = 0.4852), 83.796 ± 9.592 (p = 0.8287) and 73.450 ± 15.611% (p = 0.4652) for the cells in ALGO25, 0.25CPO and 1CPO hydrogels, respectively. In contrast, in the state of higher respiratory capacity, that is, the uncoupled state, we found that the hydrogels of the ALGO25 and 0.25CPO conditions had no significant effects on the cells, with average values of 113.627 ± 9.398 (p = 0.2842) and 111.285 ± 0.467% (p = 0.4060), respectively and compared to CCP. The 1CPO hydrogel had a drastically significant effect in the oxygen consumption capacity when compared to CCP (p = 0.0302), ALGO25 (p = 0.0051), and 0.25CPO (p = 0.0099) by having an average value of 67.165 ± 5.426%.

FIGURE 6.

Effect of ALGO25 hydrogels with 0.25 and 1% CPO on the respiration of encapsulated pancreatic β‐cells over time. Cell respiration was determined in the absence of inhibitors and uncouplers (basal state), in the presence of oligomycin (leak state), and in the presence of FCCP (uncoupled or uncoupled state). The results were expressed as a percentage with respect to the control. Values correspond to mean ± SEM, n≥3, one‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01, ^*** p <0.001 vs. CCP; ^# p <0.05, ^## p <0.01, ^### p <0.001 vs. ALGO25; ⁺ p <0.05, ⁺⁺ p <0.01, ⁺⁺⁺ p <0.001 vs. 0.25CPO.

After 96 h, the basal respiratory capacity of the cells was maintained for the 0.25CPO condition with an average value of 112.212 ± 0.452% (p = 0.5654) with respect to the CCP. However, the ALGO25 condition had a value of 59.690 ± 0.937%, which showed a significant decrease in the respiratory capacity of the basal state of the cells with respect to the CCP (p = 0.0076) and the 0.25CPO condition (p = 0.0021). The 1CPO condition had a drastic decrease in the basal state because the percentage of respiration was 59.937 ± 3.549% when compared to the CCP (p = 0.0079) and to the 0.25CPO (p = 0.0021) condition. The leak state, the ALGO25 and 0.25CPO conditions had no significant effect when compared to CCP (p>0.05). However, the 1CPO condition had an inhibitory effect as it exhibited a significant decrease of approximately 56% (p = 0.0015) in respiratory capacity with respect to CCP. In contrast, the hydrogel with 0.25% CPO induced a significant increase in maximal respiratory capacity in the uncoupled condition at 96 h, whose value was 122.658 ± 0.282 when compared to CCP (p = 0.0202) and to the ALGO25 condition (p = 0.0099). ALGO25 condition in the uncoupled state exhibited a non‐significant percentage of 94.661 ± 6.857% (p = 0.5236) with respect to the CCP. The 1CPO condition in the uncoupled state induced a significant decrease when compared to the 0.25CPO condition of approximately 38% (p = 0.0015), but no significant differences were found with respect to CCP (p = 0.0795).

3.2.5. Cellular Functionality

To evaluate the effect of ALGO25 and the oxygen release system hydrogels (0.25CPO and 1CPO) on cell functionality, an analysis of glucose‐stimulated insulin secretion (GSIS) was performed by an ELISA assay (Figure 7). In Figure 7A, cells under a low glucose stimulus in the ALGO25 condition secreted insulin levels of 0.704 ± 0.002 µIU/mL×µg DNA (p = 0.0001), which was significantly higher than the CCP. Similarly, the 0.25CPO condition had a significant increase in insulin secretion of 0.702 ± 0.009 µIU/mL×µg DNA (p = 0.0001) compared to CCP. However, it could be observed that there was a drastic reduction in insulin secretion of the 1% CPO condition when compared to CCP (p<0.0001), ALGO 25 hydrogels (p<0.0001), and the 0.25% CPO condition (p<0.0001). On the other hand, upon putting a high glucose stimulus to the cells, the cells produced a significantly higher level of insulin in the ALGO 25 condition (p = 0.0200) than the CCP. Similarly, the secretory capacity of cells in the hydrogels with 0.25% CPO was significantly increased in a high glucose stimulus as the insulin concentration was 0.857 ± 0.034 µIU/mL×µg DNA (p = 0.0007). However, the hydrogel with 1% CPO was not stimulated by the high glucose concentration and had a rather low average value compared to the CCP (p<0.0001), ALGO25 (p<0.0001), and 0.25CPO (p<0.0001) conditions. On the other hand, Figure 7b shows the stimulation rate in the experimental conditions. These results showed that the ALGO25 (1.157 ± 0.057, p = 0.0950) and 0.25CPO (1.220 ± 0.059, p = 0.3182) conditions had a non‐significant rate with respect to CCP. However, the 1CPO condition had a significant decrease with respect to CCP, as its average index was 1.113 ± 0.047 (p = 0.0276).

FIGURE 7.

Effect of ALGO 25 hydrogels with 0.25 and 1% CPO on the functionality of encapsulated pancreatic β‐cells. (A) Insulin secretion in response to low glucose (2.8 mM) and high glucose (28 mM) stimulus expressed as (µIU/mL×µg DNA) after 48 h of incubation. Values correspond to mean ± SEM, n≥3, two‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01, ^*** p <0.001, ^**** p <0.0001 vs. CCP; ^# p <0.05, ^## p <0.01, ^### p <0.001, ^#### p <0.001 vs. ALGO25; ⁺ p <0.05, ⁺⁺ p <0.01, ⁺⁺⁺ p <0.0001, ⁺⁺⁺ p <0.001 vs. 0.25CPO. (B) Stimulation index of pancreatic β‐cells encapsulated in ALGO25 hydrogels with 0.25 and 1% CPO. Values correspond to mean ± ESM, n≥3, one‐way ANOVA, Tukey's test, ^* p <0.05, ^** p <0.01 vs. CCP.

4. Discussion

Biomaterials have been increasingly studied as a resource for the isolation of cells using encapsulation processes [11]. However, there is a problem to supply the necessary nutrients such as oxygen for cell survival, leading to the development of oxygen‐releasing systems that can recover the hypoxia generated by encapsulation in hydrogels [28, 29]. Therefore, the coupling of oxygen delivery systems with alginate‐based hydrogels has been explored as a promising strategy for different types of pathologies [1, 35, 45]. Our group has previously developed an alginate hydrogel with GO [2], where we suggested that the alginate condition with 25 µg/mL of GO (ALGO25) was the optimal condition that best maintained the physicochemical characteristics and adequate biocompatibility and functionality of insulin‐producing cells. However, the study of alginate hydrogels incorporating GO and an oxygen‐releasing system such as CPO for the treatment of TD1 has not been studied. Therefore, our study aims to develop an alginate‐based oxygen‐releasing system with 0.25 and 1% CPO incorporating GO and reinforced with PLL that can sustain biocompatibility and cellular functionality.

Initially, the chemical composition of the hydrogels was analyzed by FTIR. This allowed the observation of similar signals present in the alginate hydrogels with GO and CPO due to the relatively small proportions of these components in the samples. Therefore, the presence of some backbone groups of the alginate overlapped with the reactive groups of GO (carboxyls and epoxides), as previously observed [2, 40]. The broad band observed between 3000–3500 cm⁻¹ of OH groups can be attributed to water adsorbed by the hydrogel [2, 38]. Meanwhile, the presence of symmetric and asymmetric elongations of COOH groups in the range 1590–1600 cm⁻¹ is strongly related to the structure of the alginate polymer [2, 39, 40]; these peaks are useful to recognize the crosslinking caused by calcium ions in alginate [39]. Also, it was observed that there was a stretching vibration between 1010–1020 cm⁻¹ corresponding to the C─O groups [2, 40, 43]. It has been reported that the addition of calcium ions forms an “egg‐box” structure due to the interaction between ─COO⁻ and Ca²⁺ [39, 40, 46], which can be observed by the presence of the band at 1400 cm⁻¹ in the ALGO25 condition. However, due to the presence of CPO in the 0.25CPO and 1CPO condition, this band had a slight shift to 1410 cm^−1, which, together with the appearance of the new peak at 1470 cm⁻¹ is associated with the vibration of the O─Ca─O groups of CPO [41, 42]. It is important to add that the appearance of the absorption band at 2914 cm⁻¹ was weak compared to that reported in the literature for pure alginate [2, 39]. This may be due to the “egg‐box” structure achieved by ionic gelation, which limits the stretching vibration of CH groups and reduces the dipole moment [39, 40, 46].

We performed a rheological analysis to evaluate the response of ALGO25 hydrogels with CPO to applied stress in terms of flow. Our results allowed us to associate the CPO concentration with the response of the biomaterial to the applied deformation, especially those with CPO had a higher response in the 𝐺′ and 𝐺″. This shows the prevalence of the elastic character of the samples over the viscous, which was reflected in the maximum deformation (gc) in which the elastic modulus remained constant. The gc was higher in the samples with CPO than those with only GO. Similarly, this was verified with the oscillatory test showing that hydrogels behave as a viscoelastic solid, which allows them to hold their shape and are not easily deformed [47]. Furthermore, we hypothesized that adding CPO enhances elasticity of hydrogels, which was confirmed by the DMA analysis showing that 0.25 CPO and 1CPO conditions showed an increased Young's modulus. Several reports have found that combinations of alginate with CPO have positively influenced the elastic behavior of the sample [35, 48]. Meanwhile, other studies did not find that CPO particles influenced the stiffness and elasticity of the biomaterial [47]. However, the findings in that study may be related to the method of hydrogel development, which was by 3D bioprinting involving a high concentration of alginate to be extruded [47]. This translates into a little effect on the deformation of the developed scaffolds by the CPO particles added [47]. Similarly, the incorporated GO has been found to provide stability to the alginate hydrogel structure [2]. Moreover, the PLL coating constitutes a tool that provides stability to alginate hydrogels and does not allow destabilization with chelators such as phosphate (PO₄ ^3‒) or citrate [11]. Taken together, these findings highlight the distinctive rheological and mechanical properties of ALGO25 hydrogels with CPO and their ability to resist and adapt to applied stresses and forces during analysis. Although both CPO‐containing hydrogels exhibited similar LVR, indicating comparable stability under low, physiologically relevant deformations, the dose‐dependent increase in Young's modulus reflects differences in network stiffness that may influence cell mechanosensing in vitro; however, correlations with in vivo implant performance or immune response could not be addressed in the absence of implantation studies, which constitutes a limitation of the current study.

Next, the swelling capacity and biodegradation of the developed hydrogels were evaluated. It was found that alginate hydrogels with CPO showed a lower swelling rate compared to hydrogels without CPO, especially in the case of hydrogels with 1% CPO. This is consistent with previous findings where the swelling capacity decreases as the CPO concentration increases in alginate hydrogels with CPO [48, 49]. This result may be due to the saturation of the alginate structure with CPO molecules. One other possible explanation is the significant changes in pH, which have been reported to influence the behavior of water absorption capacity. The increase in pH (data not shown) may be produced by high concentrations of CPO due to its decomposition into Ca(OH)₂(s), an alkaline compound; however, the mechanism by which this decrease in water absorption happens within the alginate network is still poorly understood [50, 51]. Nevertheless, the swelling capacity of the developed biomaterials is in agreement with typical values found in tissues and in the literature [50, 51, 52]. The sharp decrease in swelling observed for the 1% CPO hydrogels after day 6 was further analyzed using complementary parameters derived from existing data. Although direct measurements of cross‐link density, elemental distribution, or microstructural imaging were not performed, analysis of water content revealed that the 1% CPO hydrogels exhibited a pronounced reduction in their hydration compared to the 0.25% CPO condition over time. This behavior is consistent with the higher Young's modulus observed at increased CPO loading, suggesting the formation of a denser and stiffer polymer network that limits water accommodation rather than bulk material degradation, as overall mass loss and bead diameter remained stable throughout incubation. Together, these findings support the interpretation that high CPO content alters the hydrogel microenvironment by increasing stiffness and reducing hydration, which may contribute to the observed decline in swelling capacity. Nevertheless, there is a need of a definitive elucidation of the underlying mechanism, which would require additional structural and chemical analyses.

The percentage degradation and diameter measurement of the developed hydrogels were also evaluated. Degradation analysis is an important parameter in regenerative therapy because low degradation allows the encapsulated cells to be implanted and establish in the new tissue [49, 52]. Our results show that all hydrogels maintained their mass at a value above 90% during all days of incubation. Similarly, alginate hydrogels with 0.25 and 1% CPO sustained this stability, which is in agreement with different studies [45, 48, 49, 51]. In contrast, we have reported that ALGO hydrogels diminish up to 25% of their mass during the first 24 h [2]; however, the developed hydrogels were not coated with PLL. Other studies have used polycation coatings such as PLL, which have reinforced the alginate matrix and avoided loss of stability and shape of hydrogels [16, 53]. This is because polymer‐polymer interactions between PLL chains and the alginate matrix are critical to avoid membrane detachment and exposure of immunogenic groups, which could affect implant survival [53]. Additionally, degradation also induces conformational and structural changes in hydrogels, which impacts the cell‐polymer matrix interaction sites [49, 52]. These results were corroborated by the measurement of the diameter of the hydrogels, which confirmed the stability found in the degradation test. However, one limitation of the present study is the absence of a direct quantitative determination of the absolute cross‐linking degree of the composite hydrogels. The hydrogels were ionically cross‐linked through Ca²⁺–alginate interactions and further stabilized by physical reinforcement with graphene oxide and PLL, therefore, this hybrid network does not readily allow direct calculation of an absolute cross‐linking degree using classical chemical or network theory approaches [54, 55, 56]. Instead, the relative extent of cross‐linking was assessed indirectly through complementary physicochemical parameters commonly applied to ionically cross‐linked hydrogel systems, including mechanical stiffness and hydration behavior. The increase in Young's modulus together with the reduced swelling and lower water content observed at higher CPO concentrations indicate a higher effective cross‐link density, consistent with a denser polymer network arising from increased calcium availability. The high‐water content shown by 0.25CPO hydrogel highlights their potential to mimic the physical properties of the native tissue and providing a supportive microenvironment where cells can settle and proliferate [57]. Nevertheless, future studies incorporating direct structural or chemical analyses will be necessary to fully characterize network cross‐linking.

The morphology and structural characteristics of GO were previously presented by our group and therefore were not repeated in the present study [2]. In that study, the typical sheet‐like morphology and layered structure of graphene oxide incorporated into alginate hydrogels were confirmed. The microstructure of the composite hydrogels during oxygen release and incubation was evaluated indirectly rather than through direct imaging techniques. Bead‐scale morphology and structural stability were confirmed by diameter measurements and mass retention, microstructural features were inferred from swelling behavior, water content, and mechanical properties. In particular, the reduced swelling and increased Young's modulus observed at higher CPO concentrations suggest a denser and stiffer polymer network, indicative of microstructural reorganization rather than bulk degradation. Nevertheless, the absence of direct microstructural visualization (e.g., SEM, elemental mapping) represents a limitation of the present study, and future work incorporating spatially resolved imaging will be required to fully characterize hydrogel microstructure as a function of oxygen release and incubation time.

Evaluation of the oxygen release kinetics suggested that the alginate hydrogels with CPO had a controlled release of oxygen for 8 days, and a higher CPO concentration showed a significant increase in oxygen release in the first day. This agrees with other findings that reported stable oxygen release over time in alginate hydrogels in which CPO was incorporated [1, 35, 47, 48, 49, 58]. It has been reported that the ability of CPO to be decomposed into oxygen over time is relatively slow, therefore, the addition of catalase as a reaction accelerator is not necessary [47]. Thus, we propose the encapsulating method of pancreatic β‐cells in an alginate‐based biomaterial with GO and CPO in the current study. Furthermore, to attenuate the hydrolytic activity of CPO, this was treated with PBS 1× before encapsulation method as this procedure was reported to have reduced the release of CPO upon contact with alginate and coating the CPO particles with calcium phosphate from the treatment done [1]. However, the exact mechanism by which the stability of the sudden release of CPO is influenced is not completely known. The observed oxygen release profile, characterized by an initial burst followed by sustained release, is consistent with a self‐limiting hydrolysis mechanism of CPO [1]. PBS pre‐treatment may contribute to this behavior by inducing partial surface stabilization of CPO particles through the formation of poorly soluble calcium phosphate species, which can reduce water accessibility and attenuate reaction kinetics. This mechanism would explain why oxygen release was mitigated but not fully suppressed, particularly at higher CPO loadings. While this interpretation is chemically plausible and consistent with prior reports [1, 59], the formation of a calcium phosphate surface layer was not directly demonstrated in this study and therefore remains a mechanistic hypothesis. However, the dose‐dependent oxygen release kinetics and the sustained biological functionality observed for the 0.25% CPO condition, discussed in the next paragraph, support this interpretation.

The effect of the developed hydrogels on cell viability was evaluated, and we determined ideal concentration of CPO that sustained cell viability during 96 h of incubation. It was observed that cell viability was sustained throughout the incubation time (evaluated at 48 and 95 h) in the 0.25CPO condition. These results may be due to the oxygen supply in the local area provided by the CPO incorporated in the alginate hydrogel [47]. We suggest that a self‐recovery effect of the encapsulated cells in the 0.25CPO condition is observed because they could maintain cell viability at 48 and 96 h after the stress carried by the gelation process [47]. Reports suggest that adequate ratios ranging from 0.05 to 0.5% CPO are able to sustain cell viability in different cell models and without depleting oxygen supply at the same time which agrees with our study [1, 45, 47, 51, 58]. The 1CPO condition of our study had a deleterious effect on the encapsulated cells as they were unable to recover, which may be due to the high oxygen concentrations to which they were exposed; this is in agreement with several investigations [45, 47, 51]. The quantitative cell viability findings of our study were confirmed with the live/dead assay, showing a decrease in viable cells in the 1CPO condition; while 0.25CPO maintained cell viability up to 96 h, showing no apoptotic/dead (red) cells. ALGO25 condition showed that the stability of the cell membrane at 96 h was affected since dead cells were present. Therefore, these qualitative results allow microscopic confirmation of the quantitative viability results found in the MTT assay.

The effect of ALGO25 hydrogels with CPO on cell proliferation was analyzed, showing a significant increase in the ALGO25 hydrogels compared to the control in the first 48 h. A higher DNA content was shown, indicating a greater amount of cells and higher proliferative activity, which is in agreement with previous studies [2]. However, this DNA content decreased at 96 h displaying similar cell proliferation to the 2D culture which may be due to the lack of oxygen and nutrient flow in the construct and an increase in cell stress leading to cell death [26]. 0.25CPO hydrogels showed at 48 h a significant increase in DNA content compared to CCP, suggesting a positive stimulation in cell proliferation. Moreover, after 96 h of incubation, it was shown that this condition could maintain a significant increase in DNA content compared to the other conditions, indicating a continuous stimulation of cell proliferation. Nevertheless, it is important to note that 1CPO hydrogels at 48 and 96 h had a dramatic effect on cell proliferation, showing significantly lower DNA content compared to all experimental conditions. These results agree with the studies reported by Lu et al. [47] and Pedraza et al. [31] showing that cell proliferation was positively influenced by optimal CPO concentrations. Additionally, we suggest that the inclusion of GO within the 0.25CPO constructs may have a synergistic effect of CPO as an oxygen generating system together with GO. The latter adsorbs extracellular matrix (ECM) proteins via hydrophobic interactions, electrostatic forces and hydrogen bonds, which promotes cell adhesion to GO and thus inducing cell proliferation [60, 61, 62]. Previously, our team presented a study with alginate hydrogels and GO displaying the benefit of adding this component at a concentration of 25 µg/mL strengthening the physical and chemical structure of the biomaterial, and also favoring the viability and survival of pancreatic β‐cells [2].

An assessment the LDH activity release provided important information of the cytotoxic effect of ALGO25, 0.25CPO, and 1CPO hydrogels on the encapsulated cells. At 48 and 96 h, we observed that the ALGO25 hydrogels and 0.25CPO condition had a decreased in relative LDH activity compared to the positive control, indicating that the cytotoxic effect was not significant on the encapsulated cells. Furthermore, the 1CPO hydrogels did not show an increased LDH activity. Our results agree with the findings found in the MTT and live/dead viability and cell proliferation assays of the present study. Pedraza et al. [31] showed that LDH activity was related to optimal CPO concentration, which would indicate that this effect would be dose dependent.

We performed an oxygen consumption flux analysis, which provides information on the respiratory capacity of the pancreatic β‐cells encapsulated in the different hydrogels developed. We could verify that the respiratory capacity in the basal state in the first 48 h for the ALGO25 and 0.25CPO conditions was not significantly affected. However, the 1CPO hydrogel showed a significant reduction in basal respiratory state, indicating that high concentrations of CPO have an inhibitory effect on the electron transport chain and ATP production. Several factors in the findings of the present study could explain the lack of basal respiratory capacity in this condition, such as decreased cell viability and proliferation. We suggest that this may be influenced by the high pH level of the medium given by the CPO high concentration and the deleterious effect of the presence of high concentrations of H₂O₂ [29, 63, 64]. In the leak state, there were no significant alterations in the respiratory capacity for any of the conditions with respect to the control. However, in the uncoupled state the hydrogel with 1% CPO had a drastic decrease in respiratory capacity compared to all other conditions. In contrast, the ALGO25 and 0.25CPO conditions did not present changes that indicated that there was no inhibition in the mitochondrial respiratory chain, which agrees with previous studies [2, 31, 47].

After 96 h of incubation, the 0.25CPO condition maintained a similar respiratory capacity to control in the basal state. However, the ALGO25 hydrogel showed a significant decrease in basal respiratory capacity compared to the control and the 0.25CPO condition. As time passes, the encapsulated cells in hydrogels are not able to meet the nutritional needs, especially oxygen deprivation, which could explain these results [26, 28]. 1CPO condition presented a significant decrease in the leak state while ALGO25 hydrogels and the 0.25CPO condition did not present any significant alterations. Maximum respiratory capacity showed that 0.25CPO hydrogels had a significantly higher respiratory capacity than the control and the ALGO25 condition. This is beneficial because the 0.25CPO condition shows a great potential to be suggested as a prototype for pre‐clinical studies due to its capacity to sustain cell respiration for up to 96 h. Nonetheless, the 1CPO condition suggests that there is a negative effect on maximal respiratory capacity compared to the 0.25CPO condition. This correlates with the significant decrease in cell viability and proliferation as well, which would confirm the deleterious effects of this condition.

Although spatial oxygen gradients within the hydrogel beads were not directly mapped, oxygen release was quantified under confined conditions using a sealed Oroboros chamber containing a small, defined volume of fresh medium [33, 35, 37]. This setup provides a localized measurement of oxygen exposure rather than a bulk culture approximation. The hydrogel beads had an average diameter of approximately 4 mm, a size that permits oxygen diffusion through alginate‐based matrices. Consistent with this, live/dead staining revealed no evidence of necrotic cores, indicating that encapsulated β‐cells remained viable throughout the construct. While this qualitative assessment does not provide spatial oxygen mapping or absolute oxygen concentrations at the cellular microenvironment, the absence of central cell death, together with improved mitochondrial respiration and functional outcomes observed for the 0.25% CPO condition, suggests that oxygen released from the hydrogel system was functionally accessible at the bead scale. However, intrabead oxygen gradients were not resolved and future studies incorporating microscale oxygen sensing will be required for a more precise characterization of oxygen distribution.

Cell functionality analyzed by GSIS assay showed that in response to a low glucose stimulus, both the ALGO25 hydrogel and 0.25CPO condition promoted a significant increase in insulin secretion compared to the control. These results indicate that these conditions enhance the secretory response of pancreatic β‐cells under low glucose conditions, that is, a basal stimulus. However, the hydrogel containing 1CPO exhibits a significant decrease in insulin release compared to all other conditions. This suggests that the 1% CPO‐containing hydrogel has a negative impact on cellular functionality and the ability of pancreatic β‐cells to secrete insulin in response to low glucose levels. A significant increase in insulin secretion is observed under ALGO 25 and 0.25% CPO conditions compared to control, indicating that these hydrogels can enhance the secretory capacity of pancreatic β‐cells in the presence of high glucose concentration. These results are not only coupled to sustaining cell viability and proliferation under these two conditions, but also to sustaining respiratory capacity for both groups [1, 2]. GSIS is determined by the degree of ATP production, this translates into sustaining cellular respiration [65, 66]. This agrees with our study as there was a higher percentage of respiration in the ALG025 and 0.25CPO conditions. However, the 1CPO hydrogel does not possess a stimulated response to high glucose, indicating a significant decrease in the ability of the cells to secrete insulin compared to all other conditions. This suggests that there is a dose‐dependent effect by CPO influencing cell functionality, which has been reported previously [31]. Finally, the stimulation index confirms the findings of secreted insulin concentration. Pancreatic β‐cells encapsulated in alginate hydrogels show that ALGO 25 and 0.25CPO have no significant difference in this rate compared to the control. Therefore, it is suggested that these conditions do not negatively affect the responsiveness of pancreatic β‐cells to stimulation. However, the 1CPO condition shows a significant decrease in the stimulation rate compared to the CCP, indicating a negative impact on the functionality of the cells and a decrease in their responsiveness to glucose stimuli. Taken together, the unique strength of this study lies in its integrative design strategy and comprehensive functional evaluation, which provide mechanistic insight into how oxygen release, hydrogel mechanics, and hydration jointly regulate β‐cell survival and function. To our knowledge, such a combined approach using alginate–graphene oxide hydrogels with controlled oxygen release has not been previously reported for pancreatic β‐cells, apart from our earlier studies [2] that established the individual components required for the development of the present platform.

Cumulatively, these results showed the successful development of alginate hydrogels with GO and CPO coated with PLL. Thus, it can be suggested that the characteristics of the alginate‐based biomaterial with CPO that allow oxygen release and preserve the biocompatibility and functionality of insulin‐producing cells are the chemical and mechanical stability of the hydrogel with CPO demonstrated by FTIR and rheology assays. Also, preservation of swelling and degradation capabilities over time without significant loss of size with sustained oxygen production and improved survival, viability, and functionality of pancreatic β‐cells.

5. Conclusions

In conclusion, a reinforced‐PLL alginate containing GO hydrogel was designed, and CPO was incorporated as an oxygen release system (ALGO25, 0.25CPO, and 1CPO). Analysis of their chemical composition revealed backbone groups of the alginate, GO, and CPO. Rheological and dynamic mechanical results showed that hydrogels with CPO had a higher elastic response and higher stiffness compared to hydrogels without CPO. In contrast, the swelling capacity of hydrogels decreased with increasing CPO concentration, while degradation remained stable in all hydrogels. The CPO‐containing hydrogels release oxygen for up to 8 days, depending on the loaded concentration. Evaluation on biocompatibility revealed that the 0.25CPO condition improved viability with continuous stimulation of cell proliferation during 96 h of incubation, while the 1CPO condition had a detrimental effect on the encapsulated cells. Evaluation of oxygen consumption flux and functionality revealed that the 0.25CPO condition maintained the respiratory capacity over time and the responsive capacity of the cells, that is, insulin secretion under a high glucose stimulus. Based on these results, it is suggested that the appropriate condition to promote the viability, proliferation, respiratory capacity, and functionality of encapsulated pancreatic β‐cells and maintaining adequate physicochemical characteristics is 0.25CPO. These findings provide relevant information for the development of biomedical applications in diabetes therapy with encapsulated insulin‐producing cells.

Author Contributions

Conceptualization, E.C.‐G., N.M.‐C.; methodology, E.C.‐G., N.M.‐C., and O.V.‐C.; formal analysis, E.C.‐G., N.M.‐C., and O.V.‐C.; resources, N.M.‐C.; Writing – original draft preparation, E.C.‐G., N.M.‐C., and O.V.‐C.; project administration, N.M.‐C.; funding acquisition, N.M.‐C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by “Ministerio de Ciencia, Tecnología e innovación de Colombia‐Minciencias” under registration code: 110284466876 and announcement 844‐2019, Universidad Industrial de Santander, Universidad EIA, Hospital Pablo Tobón Uribe, and the International Mobility Program of the “Vicerrectoría De Investigación of the Universidad Industrial De Santander”, Colombia.

Ethical Approval

This Study Was Approved by the Ethics Committee of the Universidad Industrial De Santander (Code 4110) On 28 August 2020.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: mabi70227‐sup‐0001‐SuppMat.docx.

Data Availability Statement

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