Biomimetic gasotransmitter-releasing alginate beads for biocompatible antimicrobial therapy

Abstract

Hypothesis:

Alginate is widely used in biomedical applications due to its high biocompatibility as well as structural and mechanical similarities to human tissue. Further, simple ionic crosslinking of alginate allows for the formation of alginate beads capable of drug delivery. S-nitrosoglutathione is a water-soluble molecule that releases nitric oxide in physiological conditions, where it acts as a potent antimicrobial gas, among other functions. As macrophages and endothelial cells endogenously produce nitric oxide, incorporating nitric oxide donors into polymers and hydrogels introduces a biomimetic approach to mitigate clinical infections, including those caused by antibiotic-resistant microorganisms. The incorporation of S-nitrosoglutathione into macro-scale spherical alginate beads is reported for the first time and shows exciting potential for biomedical applications.

Experiments:

Herein, nitric oxide-releasing crosslinked alginate beads were fabricated and characterized for surface and cross-sectional morphology, water uptake, size distribution, and storage stability. In addition, the NO release was quantified by chemiluminescence and its biological effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus were investigated. The biocompatibility of the alginate beads was tested against 3T3 mouse fibroblast cells.

Findings:

Overall, nitric oxide-releasing alginate beads demonstrate biologically relevant activities without eliciting a cytotoxic response, revealing their potential use as an antimicrobial material with multiple mechanisms of bacterial killing.

Graphical Abstract

1. Introduction

Nitric oxide (NO) is a free radical gas that is endogenously produced in several areas of the body. It was first discovered in the endothelium as the endothelial-derived relaxation factor (EDRF) in 1980 [1]. Several years later, scientists discovered that NO is released by activated macrophages as an antimicrobial and wound healing cytokine [2] and is also capable of preventing blood platelet aggregation and activation [3]. As an antimicrobial agent, its high reactivity leads to the formation of reactive oxygen (ROS) and nitrogen species (RNS) that induce oxidative and nitrosative stress on microbial membranes, proteins, and DNA. The multiple mechanisms of microbial killing exercised by NO and its ROS and RNS intermediates have ushered NO into the biomedical world as a potent antibacterial molecule to combat the rise in antibiotic-resistant organisms. Further, research in general is trending toward biomimetic approaches to combatting infections [4]. S-nitrosothiols (RSNOs) are NO donor molecules that can be incorporated into various polymers and gels for localized delivery to the body for antimicrobial, wound healing, and blood compatibility applications. RSNOs demonstrate controlled release of NO upon exposure to heat, light, and metal ions [5]. This encapsulation of RSNOs in a polymer or gel delivery platform is a necessary fabrication step, as free RSNOs, such as S-nitrosoglutathione (GSNO), have shown toxicity to human cells in concentrations as low as 200 μg mL⁻¹ [6]. GSNO is an endogenous RSNO commonly used in hydrogels as it is water soluble and can easily be blended into aqueous gel solutions [7]. Therefore, the objective of this paper is to develop macro-scale GSNO-incorporated alginate beads with NO release for effective antimicrobial and pro-healing capabilities.

Alginate is a natural polysaccharide derived from brown seaweed and is widely used in the biomedical field due to its biocompatible and highly hydrophilic nature, leading to gel formation [8]. In fact, swelling of alginate lends mechanical properties similar to the extracellular matrix of tissues, highlighting its tissue interface potential. As a result, alginate gels are often used as wound dressings or drug delivery vehicles. On a structural level, alginate is a block copolymer composed of l-guluronate and d-mannuronate residues that vary slightly in number and arrangement depending on the source [9]. These polymer chains can be easily crosslinked with the addition of divalent cations (such as Ca²⁺) that bind to guluronate residues, increasing gel stiffness [10]. The facile crosslinking process affords numerous conformations of alginate to be used in biomedical applications, including bio ink, sheets, and microspheres, or beads, that are useful in tissue engineering, wound healing, and drug delivery platforms [9,11].

The formation of spherical beads from an alginate solution affords the highly biocompatible polymer with new avenues of utilization. One of the most common uses of beads formed from alginate and other polysaccharides is for drug delivery [17]. Drugs such as anti-inflammatory agents [18], enzymes and probiotics for livestock digestion [19–21], and antibacterial agents [22–29] can easily be stirred into aqueous alginate solutions prior to ionic crosslinking to yield drug-encapsulated alginate beads. Successful fabrication of alginate beads has been accomplished using dropwise extrusion into a CaCl₂ crosslinking solution [18–40], and electrospraying or electrospinning has also been employed to control the size of the spheres [33,41]. One study used oil-phase dispersion to create alginate beads crosslinked by CaCO₃ nanoparticles [42]. Alternative divalent crosslinking options such as Ba²⁺ or Al³⁺ have also been investigated, but it was found that crosslinking with Ca²⁺ provides beads with greater water uptake and more favorable biodegradation properties [43]. The fabrication technique utilized for this study involves the use of a superhydrophobic surface to form alginate beads and presents an inexpensive method without the use of harsh solvents or oils from emulsions, while also eliminating the necessity of complex syringe pump systems [44]. Different methods can be used to create solidified beads from the superhydrophobic surface, such as UV crosslinking or freezing of the beads before dropping into a CaCl₂ solution for crosslinking.

A review of recent literature in NO-releasing alginate and alginate bead research shows the potential for both concepts, but there are several limitations that must be addressed. In several studies, RSNOs were incorporated into alginate hydrogels (often in combination with synthetic hydrogel materials) and tested for antimicrobial and wound healing capabilities [12–15]. Ahonen et al. covalently modified alginate oligosaccharides with different amine precursors to release NO, but the maximum half-life of NO release was only ~ 40 min [16]. Wu et al. utilized GSNO nanoparticles encapsulated into alginate and chitosan microbeads [45]. Microscale alginate and chitosan particles were fabricated using a double emulsion method, with GSNO-loaded nanoparticles crosslinking into alginate or chitosan droplets for oral delivery of GSNO. However, NO release did not last more than 24 h and storage was limited to 15 days at −20 °C. Further, the low NO release would likely be ineffective in preventing or treating infection. In order for the technology to be commercially viable, the NO payload, storage stability, and cytocompatibility must be enhanced beyond that of previous work. The size of the beads must also be investigated, as high surface area to volume ratio does not afford long-term NO release.

Herein, we have developed NO-releasing alginate beads for antibacterial biomedical applications. The NO donor, GSNO, was incorporated in an alginate solution and spherical beads were formed via a superhydrophobic surface, followed by crosslinking by CaCl₂. Three bead types were fabricated: pure alginate, 10 mg mL⁻¹ GSNO in alginate (G10), and 20 mg mL⁻¹ GSNO in alginate (G20). The three bead types were first characterized in terms of surface and cross-sectional morphology, size distribution, swelling capacity, and storage stability. Additionally, the GSNO diffusion and NO release from the G10 and G20 beads was analyzed over 24 h. Then, the antimicrobial potential of the three bead types was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Finally, biocompatibility of the beads was confirmed against 3T3 mouse fibroblast cells using a viability assay and scratch assay.

2. Materials and methods

2.1. Materials

Sodium alginate, ethylenediaminetetraacetic acid (EDTA), calcium chloride (≤ 7 mm, ≥ 93.0 % purity), hydrochloric acid (37 %), sodium nitrite (≥ 99.0 % purity), phosphate buffered saline (PBS), LB broth, LB agar, Tryptic Soy Broth, and Tryptic Soy Agar were purchased from Sigma Aldrich (St. Louis, MO, USA). Reduced l-glutathione was purchased from Gold Biotechnology (Jersey City, NJ, USA). Acetone (≥ 99.5 % purity) was purchased from VWR (Radnor, PA, USA). Potassium bromide (KBr), FTIR grade was procured from Alfa Aesar. Escherichia coli (ATCC^® 25922^™) and Staphylococcus aureus (ATCC^® 6538^™) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). 3T3 mouse fibroblast cells (ATCC^® 1658^™) were also procured from ATCC. The cell counting kit (CCK-8) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Sigma Aldrich (St. Louis, MO, USA). Cell culture inserts were obtained from Ibidi (Fitchburg, WI, USA).

2.2. GSNO synthesis and characterization

GSNO was synthesized by first dissolving 2.7 g of glutathione in a 2 M HCl solution which was then chilled in an ice bath for 10 min. NaNO₂ was then added to the beaker and chilled again for 40 min. Chilled acetone was added while stirring for 10 min as the color changed from dark red to light pink. The precipitates were separated by vacuum filtration, rinsed, and dried in a desiccator in the dark overnight. Collected GSNO powder was stored at −20 °C until further use. ¹H NMR data of GSNO was characterized of its distinct functional groups (Figure S1) to ensure batch-to-batch consistency using a Varian/Agilent mercury spectrometer (300 MHz. D₂O, δ): 1.07–1.19 (SHCH₂CH-), 2.06 (CHCH₂CH₂C-), 2.32 (CHCH₂CH₂C-), 3.03 (ONSCH₂CH-), 3.28 (ONSCH₂CH-), 3.64 (-COCHNH₂CH₂-), 3.86 (-NHCH₂COOH), 4.55 (CH₂CHNHCHO-), 13.18 (-COOH). Fourier transform infrared spectroscopy (FTIR) was also completed using a Spectrum Two Spectrometer from PerkinElmer (Greenville, SC, USA). Infrared spectra were recorded from 4000 to 650 cm⁻¹ with 64 scans using 4 cm⁻¹ resolution (Figure S2). KBr loading method was used for the analysis. Approximately, 1 wt.% of the analyte was ground with anhydrous KBr. The ground mixture was subjected to 2-ton pressure for 5 min in a hydraulic press to create a translucent disk.

2.3. Hydrogel bead fabrication

The formation of alginate beads containing 0, 10, and 20 mg mL⁻¹ of GSNO followed a straightforward and facile synthesis process. Briefly, the alginate precursor solution was made by adding sodium alginate to DI water and stirring at 90 °C until all particles were dissolved to create a 3 wt.% alginate solution. The solution was then removed from heat and allowed to stir until it cooled to room temperature. Once the solution cooled, GSNO was added at either 10 mg mL⁻¹ (G10) or 20 mg mL⁻¹ (G20) concentrations and stirred for at least 30 min. Droplets (20 μL) of pure alginate, G10, or G20 were pipetted onto a silicone rubber tube cut in half and coated with a superhydrophobic coating [46]. The superhydrophobic coating formed spheres of alginate from the 20 μL droplets. The beads on the tubing were then stored at −80 °C for ~ 30 min. During that time, a 5 wt.% aqueous calcium chloride (CaCl₂) solution was prepared. After freezing, hydrogel beads were removed from the superhydrophobic tubing with a spatula and dropped in the 5 wt.% CaCl₂ solution for 5 min to allow for crosslinking of the alginate on the surface of the spheres. Pure alginate, G10, and G20 beads were crosslinked in separate CaCl₂ baths to prevent any solubilized GSNO in the crosslinking solution to interfere with later tests. Following 5 min of crosslinking, beads were removed from the CaCl₂ bath, rinsed three times in DI water, and placed on a microfiber wipe to dry for ~ 5 min. Fabricated hydrogel beads were used immediately for characterizations, bacteria and cell studies, or stored in the appropriate location for stability studies.

2.4. Hydrogel bead characterization

2.4.1. Scanning electron microscopy (SEM) and Energy-Dispersive X-ray spectroscopy (EDS) for surface analysis

In this study, pure alginate, G10, and G20 beads were imaged to determine differences in surface roughness and overall appearance. Hydrogel beads used for imaging were fabricated using the above technique, followed by 4 h of lyophilization. Prior to imaging, lyophilized beads were sputter-coated with 10 nm of gold–palladium using a Leica sputter coater (Leica Microsystems) and mounted on SEM stubs with double-sided SEM stickers. Images were acquired through a scanning electron microscopy (SEM, FEI Teneo, FEI Co.) setup employed at an accelerating voltage of 5.00 kV. Magnifications ranging from 400x to 600x were used for image acquisition. The porosity of samples was calculated using cross-sectional SEM images. Using ImageJ analysis, the pixel area of pores was compared to that of the entire image, and a percent porosity was calculated. Analysis was carried out on n = 4 images of different areas of the cross-section for each sample type.

The SEM setup was also equipped with an energy-dispersive X-ray spectroscopy system (EDS, Oxford Instruments) used for elemental surface mapping and analysis. Oxygen and carbon were used to confirm the alginate surface and sulfur was utilized to map the distribution of GSNO in the hydrogel beads. An accelerating voltage of 20.00 kV was used for EDS measurements.

2.4.2. Size distribution

Using a Mitutoyo IP65 digital micrometer, diameters for pure alginate, G10 and G20 beads were measured in mm. Measurements were taken directly after the rinsing and drying synthesis steps to avoid any potential water evaporation from the beads. No significant difference in the average diameter was expected between the sample types since all spheres were formed via 20 μL droplets on a superhydrophobic surface.

2.4.3. Swelling capacity

To investigate the hydrophilic nature of the beads, the swelling capacity of alginate, G10, and G20 beads was determined. Beads of each type were fabricated as mentioned above and dried in a desiccator in the dark for 1 h. After drying, each bead was weighed and placed in a separate container of PBS. Half of the beads were kept at room temperature in the dark while the other half were kept in a 37 °C incubator shielded from light. The beads were removed from PBS and weighed after 1 h of swelling, and Equation (1) was used to determine swelling capacity of the hydrogel beads, where W_H represents the weight of the bead after hydration and W_D represents the weight of the bead while dehydrated.

$$\mathit{\text{Swelling Capacity}}(\text{\%})=\frac{W_H-W_D}{W_D}\times 100$$ (1)

2.4.4. GSNO loading & storage stability

The loading of GSNO into the alginate beads was quantified and compared to the theoretical quantities calculated based on the concentration of GSNO in the precursor solutions. GSNO loading was determined by catalytically releasing all of the NO present in the beads and measuring NO release with Sievers Nitric Oxide Analyzers (NOAs) model 280i (Boulder, CO). NO was purged from the G10 and G20 beads using copper (II) chloride solution (CuCl₂) and ascorbic acid as catalyzers. One at time, beads were placed in an amber reaction chamber containing 2.8 mL phosphate buffered saline (PBS) with 200 μL of 0.1 M CuCl₂ and 100 μL of 0.1 M ascorbic acid. A nitrogen bubbler was placed in the solution containing the beads at a flow rate of 200 mL min⁻¹ to carry any NO being emitted to the NOA reaction chamber. Inside the reaction chamber, an incoming flow of oxygen allowed for the following reactions to take place:

$$\mathit{\text{NO}}+O_3\to {\mathit{\text{NO}}}_2^{\ast }+O_2{\mathit{\text{NO}}}_2^{\ast }\to {\mathit{\text{NO}}}_2+h\nu$$ (2)

The excited photon released from the reaction is converted to a (parts per billion) PPB or parts per million (PPM) reading and is recorded over time. The area under the NO release curve was calculated to determine the amount of GSNO initially present in each bead. NO release was standardized per mg of bead.

For storage stability studies, G10 and G20 beads were stored at −80 °C, −20 °C, and 4 °C shielded from light. Beads were stored immediately following fabrication, in their swollen state. The amount of GSNO remaining in the beads was measured at various timepoints over a 28-day period and again after a total of 6 months. During NO measurements, the amber reaction chamber was maintained at 37 °C using a water bath. The percent of GSNO remaining was relative to the values found on day 0 post-fabrication.

2.4.5. GSNO diffusion

The hydrophilic nature of alginate allows for water uptake and subsequent GSNO diffusion from the beads. This release was quantified via UV–vis Spectroscopy due to the signature peak of GSNO at 340 nm (Figure S3). Beads were placed in 1 mL PBS and stored at 37 °C in the dark. At several time points, the PBS was removed, and the absorbance was taken. Following readings, fresh PBS was added to the beads, and they were returned to storage conditions. Cumulative GSNO diffusion was calculated using a standard curve of varying GSNO concentrations (Figure S4).

2.4.6. Nitric oxide release

The release of NO from the alginate beads was also quantified in real time using chemiluminescence detection methods over a 24 h period. Beads were weighed and placed in 3 mL of PBS in the sample chamber following the determination of a baseline reading. The sample chamber was protected from light and maintained at 37 °C using a water bath. NO released from the beads was purged from the sample chamber with 200 mL min⁻¹ N₂ gas. Inside the reaction chamber, NO reacts with ozone (O₃, converted from an O₂ inlet gas) to form NO₂* in an excited state. As NO₂* drops back down to ground state, a photon is released, measured by the instrument, and converted to a PPB reading. Utilizing an NOA constant (mol PPB⁻¹ s⁻¹), this PPB reading is then converted to an NO release value (x10⁻¹¹ mol min⁻¹ mg⁻¹). Once a stabilized NO release was reached, the beads were removed from the sample chamber and stored at 37 °C in PBS and shielded from light between measurements. The release of NO was standardized per mg of beads.

2.5. Alginate bead biological activity

2.5.1. Antimicrobial activity – 24 h bacterial viability assay

The antimicrobial activity of the NO releasing alginate beads was investigated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) using a 24 h viability assay. To begin, a single bacterial colony was inoculated into LB (E. coli) or TSB (S. aureus) media and incubated at 37 °C in a shaking incubator (150 rpm) until the log phase of bacterial growth was reached. The bacteria were collected by centrifugation, rinsed with PBS, and diluted to ~ 10⁸ CFU/mL in PBS. All three types of beads, prepared as mentioned above, were weighed, UV sterilized, and exposed to 1 mL of ~ 10⁸ CFU/mL bacteria in PBS. Wells with only bacteria present were utilized as a control for comparison to wells treated with the three bead types. The well plate was sealed with parafilm and placed in a shaking incubator (150 rpm, 37 °C). After 24 h, solutions were diluted and plated on LB (E. coli) or TSA (S. aureus) agar plates. Viable CFUs were counted following 18–24 h of incubation of the agar plates. The reduction of viable bacteria was calculated using Equation (3).

$$\mathit{\text{Reduction in Viable Bacteria}}(\text{\%})=\frac{{\mathit{\text{control CFU mL}}}^{-1}-{\mathit{\text{treatment CFU mL}}}^{-1}}{{\mathit{\text{control CFU mL}}}^{-1}}\times 100$$ (3)

2.5.2. Cell cytotoxicity

A cell viability assay was performed following ISO 10993 standards. NIH 3T3 mouse fibroblast cells were cultured in a T-75 flask containing DMEM media with 10 % FBS and 5 % penicillin–streptomycin (complete DMEM) at 37 °C with 5 % CO₂. Once the cells reached 80 % confluency, the cells were transferred to a 96-well plate at a seeding density of 1.5 × 10⁴ cells mL⁻¹. Simultaneously, leachates of the alginate beads were prepared by soaking the samples in complete DMEM for 24 h. After 24 h, the cells in the 96-well plates were exposed to the leachates from the samples in complete DMEM and incubated in a CO₂ incubator at 37 °C with 5 % CO₂. After 24 h exposure, the leachates were replaced with complete DMEM containing 10 % CCK-8 solution and incubated for another 2 h to develop a yellow-orange colored end product, formazan, which is formed via the reduction of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) by dehydrogenase activity of viable cells. The amount of formazan, detectable at 450 nm, is directly proportional to the number of viable cells. The results are reported as percent viability according to Equation (4).

$$\mathit{\text{Cell viability}}(\text{\%})=\frac{\mathit{\text{Absorbance of test samples}}}{\mathit{\text{Absorbance of control}}}\times 100$$ (4)

2.5.3. Scratch assay

In vitro scratch assay was performed to assess the GSNO leaching effects on mammalian cells, as high levels of NO donors and NO have proven toxic in previous studies [6]. Further, low levels of NO have shown to enhance cell migration in several wound healing studies [47–50]. 3T3 mouse fibroblast cells were seeded into the cell culture inserts (Ibidi, Fitchburg, Wisconsin) at a 3 × 10⁴ cells mL⁻¹ density. The inserts contain a divider at the center, which ensures a linear zone of no cells. After 24 h of incubation in a humidified atmosphere with 5 % CO₂ at 37 °C, the divider was removed, and media was replaced with the leachates from the samples. Migration of cells was monitored using an EVOS-XL microscope at 0, 6, and 18 h timepoints. The experiment was performed in triplicates, and representative images were chosen. ImageJ was used to analyze the reduction in scratch gap area from hours 0 to 6.

2.5.4. Statistical analysis

Reported data is the mean ± standard deviation if not stated otherwise. All statistical analyses were carried out using Prism 9.1 (GraphPad Software, San Diego, CA USA). For characterization studies, statistical comparisons were determined using student’s t-test. For biological studies, statistical comparisons were determined using ordinary-one-way analysis of variance (ANOVA) for multiple comparisons between means of the sample groups. For antibacterial studies, analysis was performed on the logarithmic calculations. Values of p < 0.05 were deemed significant. Samples of n = 3 were used for each sample type in each experiment unless otherwise noted.

3. Results and discussion

3.1. Alginate bead characterization

This study represents the first investigation of macro-scale nitric oxide-releasing alginate beads. The fabrication process (Scheme 1) yields alginate beads with tunable GSNO content, encapsulated in the beads by the external crosslinking of the guluronate blocks of the polymer by the divalent cations (Ca²⁺) on the surface, characterized as the egg-box model [9,51]. The spherical structure of the beads, as well as the gradient of a pink hue, characteristic of GSNO, can be seen in Fig. 1c.

Scheme 1.

Alginate beads were formed by GSNO dissolution in an alginate solution, followed by bead formation via a superhydrophobic surface, freezing at −80 °C, and external crosslinking in CaCl₂ solution.

Fig. 1.

(a) Alginate beads (with and without GSNO) were fabricated and imaged using SEM, showing similar surface morphologies and porous cross-sections. Scale bars for surface images represent 200 μm and scale bars for cross-sectional images represent 50 μm. (b) Percent porosity of each sample type was calculated using cross-sectional images, with no significant difference between samples (n = 4). (c) Alginate, G10, and G20 beads exhibited comparable size distribution with greater GSNO content visualized by an increase in pink hue.

3.1.1. Morphology via scanning electron microscopy (SEM)

The surface and cross-sectional morphology of freeze-dried alginate, G10, and G20 beads were investigated using Scanning Electron Microscopy (SEM, Fig. 1a). Surface crosslinking of the alginate beads and subsequent lyophilization leads to the shrunken outer shell of all three bead types. Interestingly, G10 and G20 reveal a slightly more textured surface, potentially due to the presence of the GSNO crystals within the polymer matrix. Cross-sectional cuts of the beads reveal the highly delicate porous nature of the natural polymer, crosslinked by the uptake of the CaCl₂ solution during submersion. Quantification of cross-sectional porosity revealed no statistically significant difference between sample types with alginate beads displaying 46.9 ± 6.2 %, G10 showing 38.9 ± 4.1 % and G20 revealing 42.9 ± 2.3 % porosity (Fig. 1b). Elemental mapping and sulfur detection within cross-sections of the alginate beads confirmed the presence of GSNO, with G20 beads revealing about twice the amount of sulfur compared to G10 beads, 3.7 ± 0.1 % and 6.9 ± 0.1 % respectively (Fig. 2a). This displays the highly tunable nature of the beads, with adjustable GSNO content specific to the application.

Fig. 2.

(a) Energy-Dispersive X-ray Spectroscopy (EDS) imaging of the cross-sections of alginate, G10, and G20 beads display equivalent oxygen and carbon concentrations attributed to the alginate base-polymer. An increase in the GSNO concentration was confirmed in G10 beads with 3.7 % sulfur surface elemental analysis and 6.9 % sulfur in G20 beads. (b) Size distribution revealed similar bead diameters among bead types (n = 25). (c) Swelling capacity of the beads at room temperature and physiological temperature revealed no significant difference among sample types, though all bead types showed an increase in swelling at physiological temperature (n = 4). (d) Alginate, G10, and G20 beads were stored at 4 °C, −20 °C, and −80 °C for up to six months and displayed enhanced stability at lower temperatures, determined by GSNO content remaining (n = 3).

3.1.2. Size distribution

The size distribution of the alginate beads was calculated by measuring the average diameter of each bead type. The 20 μL volume used to form beads was chosen to increase the NO longevity, as micro or nano beads have a higher surface area to volume ratio and would likely exhibit a large burst release of NO, depleting the GSNO reservoir. [45] As expected, no statistical difference between the bead types was found (pure alginate: 2.42 ± 0.19 mm, G10: 2.35 ± 0.19 mm, G20: 2.35 ± 0.14 mm, Fig. 2b).

3.1.3. Swelling capacity

Alginate’s exceptional biocompatibility is largely attributed to the high water swelling capacity of the material, forming a swollen polymeric material with properties resembling those of extracellular matrices in tissues. This property endows immense potential for biomedical use of alginate but must be thoroughly investigated to properly characterize a material. Therefore, the swelling capacity of the fabricated alginate beads with and without GSNO was evaluated at room temperature and physiological temperature (Fig. 2c). At room temperature, each bead type showed remarkable swelling capacity (%) at 421.01 ± 69.0, 410.28 ± 117.6, and 481.63 ± 92.3 for alginate, G10, and G20, respectively. There was a slight rise in swelling for all bead types at 37 °C with alginate at 523.06 ± 136.7 %, G10 at 607.58 ± 108.0 %, and G20 at 550.83 ± 75.0 %. The increase in swelling at physiological temperature is due to the increase in flexibility between the polymer chains, creating greater space for water to absorb. It is also important to note that there was no significant difference in swelling capacity between any of the bead types, revealing that the inclusion of GSNO into the alginate structure does not affect the hydrophilicity of the material. This is most likely due to the fact that the GSNO within the alginate is water soluble, allowing for uninterrupted absorption of the fluid. High swelling capacity bestows the material with applicability in many biomedical applications such as wound healing, as exudate absorption aids the wound healing process and in turn enhances NO release.

3.1.4. GSNO loading & storage stability

The loading efficiency of GSNO into the alginate beads was determined by catalytically depleting the entire supply of NO present in the beads. The value was then compared to the theoretical quantity of NO from the concentration of GSNO in the precursor solutions. For G10, 0.2 mg of GSNO was expected per bead due to the precursor concentration (10 mg mL⁻¹) and 20 μL bead volume whereas 0.4 mg was the theoretical load of GSNO in G20 beads. Following catalytic NO release and depletion of the GSNO reservoir within the beads, the loading efficiency was found to be 28.31 ± 2.1 % for G10 and 23.89 ± 4.2 % for G20 (Figure S5). This low loading level is likely due to GSNO lost during the Ca²⁺ crosslinking and DI water washing steps during synthesis. However, NO release in physiological conditions (discussed later) reveals sufficient NO impregnation into both bead types.

For storage stability studies, beads were stored at −80 °C, −20 °C, and 4 °C. The amount of GSNO present within the beads was measured intermittently for 28 days and then after 6 total months in storage (Fig. 2d). At each time point, the GSNO content of the beads was compared to the initial GSNO loading study at day 0. The GSNO within the beads was unstable at 4 °C, with merely 5.07 ± 1.5 % of the initial GSNO for G10 and 2.13 ± 0.5 % for G20 remaining after 5 days. GSNO within the beads was more stable at −20 °C as after 28 days in storage, 69.92 ± 4.9 % for G10 and 77.55 ± 5.9 % for G20 remained, a significant improvement over other NO-releasing alginate beads at the microscale. [45] However, the −80 °C storage condition yielded the most stable environment for GSNO in the beads, with 104.93 ± 12.2 % for G10 and 116.57 ± 8.2 % for G20 remaining after 28 days. A GSNO content greater than 100 % is due to the variability among the alginate beads and their comparison to the batch tested at day 0. Furthermore, after 6 months in storage at −80 °C, the GSNO remaining for G10 dipped to 72.90 ± 8.9 % and G20 to 90.54 ± 16.5 %. The slight dip in GSNO content of the beads after 6 months of storage is likely due to the degradation of alginate over time leading to a less stable polymer matrix as well as the slow degradation of GSNO in the water-rich alginate beads. Although it is not uncommon for antimicrobial therapeutics to be stored at −80 °C prior to use, these storage conditions are not ideal. One potential way to improve storage stability and NO release over longer periods of time is to lyophilize the alginate beads immediately following fabrication. As GSNO releases NO through hydrolytic degradation, removal of the water from the polymer matrix may prolong NO release lifespan. Further, water uptake studies revealed the potential of the lyophilized bead to uptake large quantities of water upon immersion. This modification may allow for easier storage of the beads at room temperature or −20 °C to prevent heat-activated degradation and subsequent NO release, though more thorough studies must be conducted to confirm.

3.1.5. GSNO diffusion

Owing to the high water-uptake of alginate and the water solubility of GSNO, the GSNO that diffused out of the alginate beads was investigated, as it provides an additional mechanism of NO release from the material (Fig. 3a). After soaking in PBS and measuring the absorbance of the solution, the GSNO release was determined over a 24 h period. For both G10 and G20, GSNO diffusion ceased at ~ 8 h, cumulatively releasing 24.21 ± 0.36 % and 18.03 ± 0.4 % of the total encapsulated GSNO, respectively (Fig. 3b). After 24 h, the percentage of cummulative GSNO released from the beads did not increase substantially, with 24.61 ± 0.12 % released for G10 and 18.22 ± 0.03 % for G20. The fact that the majority of GSNO diffusion occurred in the first 8 h is not surprising due to the previously mentioned hydrophilic nature of alginate combined with the water solubility of GSNO. Further, as the alginate becomes more hydrated, the polymer chains become more separated, increasing water permeability and consequent diffusion. Any GSNO released after the 8 h time point was more deeply embedded in the beads and diffused out following further water absorption. The favorable levels of GSNO diffusion from the alginate beads will be investigated further for antibacterial efficacy and cytocompatibility.

Fig. 3.

(a) Degradation of GSNO via heat, light, metal ions, or hydrolysis leads to the release of 2 mol NO per 2 mol of GSNO followed by the formation of a disulfide bond between two glutathione molecules. (b) GSNO diffusion in PBS measured over 24 h for G10 and G20 beads (n = 3). (c) Instantaneous NO release profiles of representative samples of G10 and G20 beads measured at 0 h (n = 1). (d) Stabilized NO release from G10 and G20 beads measured over 24 h (n = 3). Statistical significance denoted by * (p < 0.05) and ** (p < 0.01).

3.1.6. Nitric oxide release

NO released from the alginate beads was measured over a 24 h period with the beads submerged in PBS with EDTA during the entire study. The quantitative NO release combines the release from GSNO diffused from the beads as well as that embedded in the polymer matrix. The instantaneous release profiles of G10 and G20 reveal a burst release of NO upon introduction to the sample chamber, with G10 reaching ~ 0.5 x10⁻¹¹ mol min⁻¹ mg⁻¹ and G20 reaching ~ 2.5 x10⁻¹¹ mol min⁻¹ mg⁻¹ within the first 5–8 min (Fig. 3c). The burst of NO release is due to the GSNO molecules on the outer layers of the alginate beads, as well as any initial GSNO diffusion that leads to NO release in the 37 °C environment. After ~ 30 min, both bead types had a stabilized release of NO that remained relatively unchanged over the 24 h period. G10 released 0.075 ± 0.04 x10⁻¹¹ mol min⁻¹ mg⁻¹ at hour 0 (after stabilizing) and 0.053 ± 0.02 x10⁻¹¹ mol min⁻¹ mg⁻¹ at hour 24 (Fig. 3d). G20 displayed a release roughly twice that of G10: 0.186 ± 0.04 x10⁻¹¹ mol min⁻¹ mg⁻¹ at hour 0 and 0.156 ± 0.03 x10⁻¹¹ mol min⁻¹ mg⁻¹ after 24 h. It is expected that G20 NO release is twice as high as G10 NO release as twice the GSNO was loaded into the precursor alginate solutions. Further, the GSNO embedded into the beads led to the longevity of NO release, while the GSNO diffusion enhanced initial NO release, a favorable release strategy to combat bacterial infections. Taking the two modes of NO release into account, this fabrication approach allows for tunable NO release characteristics depending on the amount of GSNO loaded into the precursor alginate solution.

3.2. Alginate bead biological activity

3.2.1. Antimicrobial activity

Antimicrobial activity of the alginate beads with and without GSNO was investigated against Gram-negative E. coli and Gram-positive S. aureus. Greater GSNO content was expected to be associated with higher bacterial reduction, as NO is a potent antimicrobial gas that has shown effectiveness against bacteria, fungi, and viruses. [52] The NO released from GSNO reacts with environmental oxygen (O₂), generating reactive oxygen (ROS) and nitrogen species (RNS) such as peroxynitrite (OONO⁻) which is exceptionally toxic due to high oxidation potential. [52,53] These free radical gases are then able to penetrate and disrupt the bacterial membrane and wreak havoc within the microorganism. Thiol nitrosation by NO can modify proteins, inhibiting their functions. [54] NO also actively attacks DNA through deamination and strand breaks, as well as impairs DNA repair systems, causing irreparable damage. [55] NO and its ROS and RNS counterparts exhibit such specific and unique reactivity and antimicrobial strategies, bacterial lipids, proteins, and DNA are indefensible, enabling NO to obliterate microbes and thwart infections. [56]

The antibacterial efficacy of GSNO containing alginate beads showed a promising trend over a 24 h period. For E. coli there was no significant bacterial reduction between the untreated bacterial solution and pure alginate beads, whereas G10 and G20 beads showed an 88.03 ± 4.0 % and 98.99 ± 0.6 % bacterial reduction compared to controls, respectively (Fig. 4a). S. aureus treatment followed the same trend of enhanced bacterial killing with greater GSNO incorporation into the beads. Pure alginate beads showed negligible bacterial inhibition while G10 displayed 99.4 ± 0.3 % greater killing and G20 revealed 99.92 ± 0.02 % (Fig. 4b). As previously mentioned, the improved killing of G20 beads compared to G10 is expected, as greater GSNO incorporation leads to higher NO release and therefore more oxidative and nitrosative stress exerted on the bacteria. The gaseous nature of NO allows rapid and effortless penetration of bacterial membranes to initiate killing externally as well as from within. These multiple mechanisms of killing leading to enhanced bacterial eradication display the potential benefit of nitric oxide antimicrobial treatments compared to currently utilized antibiotics and should be further investigated for their clinical potential.

Fig. 4.

The antibacterial activity of all bead types was tested against (a) E. coli (n = 4) and (b) S. aureus (n = 4) via colony counting. Statistical significance is represented as *** (p < 0.005) and **** (p < 0.001) when comparing the connected columns.

3.2.2. Cell cytotoxicity

The antimicrobial activity of NO releasing alginate beads shows promise for biomedical applications, but the biocompatibility must first be examined. As alginate is a naturally derived polysaccharide it is not expected that there will be any toxicity against 3T3 mouse fibroblast cells. Mammalian cells are also comparatively more resistant to NO than bacteria, as there are internal antioxidant mechanisms to prevent the conversion of NO into harmful peroxynitrite species. Similarly, GSNO is simply the nitrosated derivative of glutathione (GSH), the most abundant cellular thiol, therefore no harmful effects on mammalian cells of any GSH byproducts are expected from the beads as NO is exhausted. The viability of mouse fibroblast cells, as determined by a CCK-8 assay, was compared to untreated cells following treatment with leachates from alginate, G10, and G20 beads. No cytotoxicity was detected due to any of the treatments. Relative to untreated cell viability, alginate beads showed 90.71 ± 2.9 %, G10 showed 101.04 ± 9.6 %, and G20 displayed 112.42 ± 5.3 % viability (Fig. 5a). Endogenously, NO promotes cell proliferation at low flux values such as those displayed by the alginate beads. The slow release of NO over the 24 h incubation time led to increased cell proliferation and therefore greater relative cell viability.

Fig. 5.

(a) Relative cell viability was measured using a CCK-8 assay following incubation of 3T3 mouse fibroblast cells with leachates from alginate, G10, and G20 beads. Statistical significance is represented by ** (p < 0.01). (b) An in vitro scratch assay was utilized to investigate the role of diffused GSNO and released NO in fibroblast cell migration across the scratch gap. Blue arrows represent areas of cell migration, and the red star signifies non-uniform cell distribution in the control treatment. These images are representative from n = 3 plates. (c) Reduction in wound gap area was quantified, revealing no significant difference in fibroblast migration at hour 6 between treatment groups (n = 3).

3.2.3. Scratch assay

Endogenous NO has many physiological roles, such as acting as a cellular messenger to promote fibroblast proliferation and migration during wound healing and tissue reconstruction. This is demonstrated in the mammalian cell cytotoxicity data. Additionally, an in vitro cell migration scratch assay was utilized to determine if fibroblast migration was inhibited by the relatively high GSNO leaching from the beads. The scratch assay is an inexpensive and straightforward assay designed to accurately mimic the migration of cells in vivo and can be used to directly compare cell migration rates among treatment groups. [57]

At 0 h, a gap between the confluent monolayer of mouse fibroblast cells is visible and uniform among the various treatment groups (Fig. 5b). After 6 h, NO release from G10 and G20 beads led to slightly enhanced cell migration, though not statistically significant. Blue arrows represent groups of cells in migration toward the opposite edge. Control beads showed 50.3 ± 3.3 % reduction in wound gap area, followed by alginate with 47.6 ± 15.1, G20 with 54.6 ± 18.4 %, and G10 with 56.0 ± 11.2 % reduction in area (Fig. 5c). After 18 h, the cell gap on all treatments closes completely. Alginate, G10, and G20 cells are fully confluent displaying a uniform monolayer, whereas control cells grew together with less homogeneity, shown by the red star in Fig. 5b. Although the NO releasing beads only showed a moderate increase in the rate of cell migration, as displayed at hour 6, it is important to note that the high GSNO leaching values did not have a detrimental impact on the cell migration of mammalian fibroblast cells, revealing high biocompatibility for use in medical applications.

4. Conclusion

In summary, we fabricated the first macro-scale S-nitrosogluta thione-incorporated alginate beads through external ionic crosslinking with stable and tunable nitric oxide-releasing capabilities. The beads demonstrated a spherical shape with a porous internal morphology. All three bead types (alginate, G10, and G20) shared a comparable size distribution averaging between 2.35 and 2.42 mm in diameter, which is comparable to other hydrogel beads used for skin regeneration and bioactive treatments. [58,59] The beads also displayed high water uptake potential ranging from 410.3 % to 481.6 %, with an increase in swelling at physiological temperature. Chemiluminescent nitric oxide detection methods revealed stable nitric oxide release of ~ 0.4 (x10⁻¹¹ mol min⁻¹ mg⁻¹) for G10 beads and ~ 0.12 (x10⁻¹¹ mol min⁻¹ mg⁻¹) from G20 beads at 24 h, a significant increase in longevity of nitric oxide release compared to covalently modified alginates [16] and micro-scale GSNO-alginate beads [45], and without the necessity of catalytic agents to induce release of NO from the beads. [60] The total percentage of nitric oxide donor diffused from the beads after 24 h was 24.6 % for G10 and 18.2 % for G20, and there was no cytotoxic response from 3T3 mouse fibroblast cells. In fact, S-nitrosoglutathione leachate levels led to a slight increase in cell viability for the G10 and G20 treated cells. These levels of S-nitrosoglutathione diffusion and consequent nitric oxide release led to a 1-log reduction against Gram-negative E. coli for G20 beads and a 2- and 3-log reduction in Gram-positive S. aureus for G10 and G20 beads, respectively, following 24 h treatment. Overall, the fabricated material has strong antimicrobial characteristics with enhanced mammalian cell viability and migration. The combination of the highly biocompatible properties of alginate with the antimicrobial properties of nitric oxide-releasing S-nitrosoglutathione gives a novel nitric oxide-releasing alginate material that shows potential for various biomedical applications. Future applications of the material may include in vivo wound healing studies for external infected wounds or drug delivery studies utilizing nitric oxide donors in combination with other treatments. Additionally, the S-nitrosoglutathione content and size of the beads may also be modified to tune the nitric oxide release profile to fit specific applications.

HIGHLIGHTS.

• Sustained nitric oxide release for 24 h.

• 1-log reduction of E. coli and 3-log reduction of S. aureus after 24 h treatment.

• Exceptional biocompatibility with no effect on fibroblast proliferation or migration.

Data availability

Data will be made available on request.