Advances in Nitric Oxide-Releasing Hydrogels for Biomedical Applications

Abstract

Hydrogels provide a plethora of advantages to biomedical treatments due to their highly hydrophilic nature and tissue-like mechanical properties. Additionally, the numerous and widespread endogenous roles of nitric oxide have led to an eruption in research developing biomimetic solutions to the many challenges the biomedical world faces. Though many design factors and fabrication details must be considered, utilizing hydrogels as nitric oxide delivery vehicles provides promising materials in several applications. Such applications include cardiovascular therapy, vasodilation and angiogenesis, antimicrobial treatments, wound dressings, and stem cell research. Herein, a recent update on the progress of NO-releasing hydrogels is presented in depth. In addition, considerations for the design and fabrication of hydrogels and specific biomedical applications of nitric oxide-releasing hydrogels are discussed.

Graphical Abstract

1. Introduction

The discovery of the plethora of physiological functions of nitric oxide (NO) opened up vast new research fields and led to its crowned achievement as Molecule of the Year by Science in 1992.¹ The endogenously produced gaseous signaling molecule, or gasotransmitter, is responsible for several regulatory roles and host responses throughout the body, including vasodilation, infection control, and neurotransmission (Figure 1). Nitric oxide is produced by three isoforms of NO synthase (NOS): neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), all through the L-arginine – Nitric oxide pathway (Figure 2).^2-4

Figure 1. Physiological Roles of Nitric Oxide.

NO plays a crucial role in multiple parts of the body as it is released by three different nitric oxide synthase (NOS) enzymes, each correlating to various physiological responses in vivo.

Figure 2.

Nitric oxide synthase (NOS) pathway for endogenous NO production

Neuronal NOS is found within specific neurons in the brain, the spinal cord, and several other peripheral nerves and is responsible for maintaining synaptic plasticity, regulating central blood pressure, and causing vasodilation via peripheral nitrergic nerves.² NO-messaging is accomplished through hemoglobin (Hb) transport, a process previously though to inhibit NO signaling. Though there are multiple mechanisms, one of the most common is NO binding to the ferrous heme forming iron nitrosyl Hb with an affinity so high, it has a dissociation constant (K_d) of 10⁻¹⁰ – 10⁻¹¹ M.⁵ Signaling can also be accomplished by nitrosation of the reduced thiol of Hb by NO, though pH and Hb state play a key role.^{5, 6}

Also appropriately named, eNOS is primarily expressed in endothelial cells and plays a role in vasodilation, inhibition of platelet aggregation and adhesion, inhibition of leucocyte adhesion and vascular inflammation, control of vascular smooth muscle proliferation, activation of endothelial progenitor cells, and stimulation of angiogenesis.^{2, 7} Both nNOS and eNOS are Ca²⁺-dependent, whereas iNOS is Ca²⁺-independent and is upregulated by cytokines, microbial lipopolysaccharides, immune complexes, cell-to-cell contact, and various antibiotics.^{2, 8} Although primarily found in macrophages as part of the immune response, iNOS can be stimulated in a wide range of cells as long as inducing agents are present.² The release of NO as an immune response is utilized mainly as an antimicrobial agent, as the NO radical is highly effective at inducing nitrosative stress to microbial membranes, proteins, and DNA.⁹ Due to NO's potency, research into antimicrobial NO-releasing therapies is extensive. However, the use of NO in research and clinical applications requires the use of a delivery vehicle due to the instability of NO donor molecules in physiological conditions. Various platforms such as nanoparticles and polymeric delivery platforms provide a more stabilized release of NO and a more favorable material interaction with living cells and tissues. One such vehicle for NO release that has been used extensively in research is hydrogels.

Hydrogels are characterized by their highly hydrophilic nature, consisting of three-dimensional polymer networks that can be modified through methods such as crosslinking, freeze-drying, swelling, and degradation. Hydrogels can be classified in many ways, the most general distinction being natural versus synthetic hydrogels. Natural hydrogels such as hyaluronic acid (HA), collagen, fibrin, alginate, and chitosan are derived from naturally occurring components, making them exceptionally biocompatible, though with some source-dependent variations. On the other hand, synthetic hydrogels are constructed from synthetic monomer or polymer chains and are highly characterized and favorable for scale-up as batch-to-batch variation is minimal.^{10, 11} Examples of synthetic polymers include poly(vinyl alcohol) (PVA), poly (ethylene glycol) (PEG), poly(acryl amide) (PAA), poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), and many more.^{10, 11} Both natural and synthetic hydrogels can be crosslinked to enhance mechanical properties, which can be categorized in two ways: physical and chemical. Physical crosslinking is considered reversible as polymers are held together by secondary forces such as H-bonding, ionic bonding, or hydrophobic forces and are more susceptible to degradation.¹² Conversely, chemical crosslinking involves the formation of covalent bonds between polymer chains within the matrix.¹¹ Further characterization of hydrogels can include polymer charge, crystallinity, sensitivity to stimuli, hydrogel form, porosity, mechanical properties, and bioactivity, which are explored extensively in previous review papers.^10-15

The large water content of hydrogels is accredited to the functional groups of constituent networks, which affords them with a structure that is similar to extracellular matrix (ECM) in tissues, making them useful in biomedical applications like tissue engineering, cell encapsulation, drug delivery, and wound healing.^{15, 16} Furthermore, although hydrogels show great promise in multiple biomedical applications, the addition of active agents such as nitric oxide to the formulation endows a biologically active component, further enhancing the scope of use.

Comprehensive research about the antimicrobial effects of NO-releasing hydrogels and applications such as cardiovascular therapy, vasodilative and angiogenic potential, wound healing, and stem cell differentiation has been explored and will be discussed below. The tailoring of NO-releasing hydrogels for such applications shines a light on the novel advancements possible in the biomedical field when complex chemical solutions are substituted with scientists taking cues from human physiology on maintaining health and homeostasis. This review focuses on the release of NO from various hydrogel systems for biomedical applications and the design considerations that lead to such effective treatments.

2. Considerations When Fabricating NO Releasing Hydrogels

2.1. Hydrogel fabrication and modification techniques

The hydrophilic nature of hydrogels that imbues applicability in biomedical scenarios also requires careful consideration during fabrication due to the reduced mechanical integrity associated with high water swelling. Specific to individual hydrogel systems, the gelation mechanism of the material can directly impact the physical properties of the gel that can be adapted through further modification of the organization of polymer chains.¹⁷ In most cases, chemical or physical crosslinking methods are utilized to prevent the complete dissolution of the hydrophilic groups of the material in water and bodily fluids.¹⁸ Physical crosslinking methods such as through ionic interaction have an advantage of self-healing capabilities, as the hydrogel network can reform if the stress or interruption is removed. Chemical crosslinking can be associated with less biocompatible reagents, though it forms covalent bonds among polymer chains, which are much stronger and more permanent than physical interactions.¹⁹ However, the degree of crosslinking greatly determines other hydrogel properties that affect the material's efficacy, such as porosity, water uptake, biodegradability and degradation rates, biocompatibility, and viscosity (Figure 3). For instance, a greater degree of crosslinking can lead to higher viscosity gel with less biodegradability and biocompatibility. The NO release from hydrogels can also be indirectly affected by crosslinking, though through stabilization or decreased leaching of the NO-donor molecule within the hydrogel structure, rather than having a direct effect on the gasotransmitter itself. The choice of material and crosslinking method allows for specialized hydrogel designs fit for virtually any biomedical application.

Figure 3.

Several design constraints and fabrication considerations must be taken into account when creating a material for a specific application. (A) When designing for ocular applications, the use of a NO photo donor allows for enhanced NO release upon irradiation with visible light. (B) NO therapy designed for co-transplantation of a vascular stent is facilitated by coating the metal stent with the NO-releasing nanofiber gel using an electrospinning technique. When tailoring a gel for wound healing applications, tissue adhesivity was one of the goals for a poly(ethylene) glycol-N-hydroxy succinimide (PEG-NHS) fibrinogen adhesive hydrogel that contained SNAP-embedded fibrin microparticles. (C) Crosslinking of the gel occurred as the NHS formed a stable amide bond with (D) reactive amines present in fibrinogen. Following (E) synthesis of the SNAP-fibrin microparticles, (F i-iii) each element of the adhesive hydrogel went through (F iv) in-situ curing that led to (F v) adhesion between the tissue and hydrogel and consequent NO release. A was reproduced/adapted from Seggio, M., et al. (2020). "A thermoresponsive gel photoreleasing nitric oxide for potential ocular applications." Journal of Materials Chemistry B 8(39): 9121-9128, with permission from the Royal Society of Chemistry, Great Britain. B was reproduced/adapted from Oh, B. and C. H. Lee (2014). "Nanofiber-coated drug-eluting stent for the stabilization of mast cells." Pharm Res 31(9): 2463-2478, with permission from Springer Nature. C-F was reprinted/adapted with permission from {Joseph, C. A., et al. (2019). "Development of an Injectable Nitric Oxide Releasing Poly(ethylene) Glycol-Fibrin Adhesive Hydrogel." ACS Biomater Sci Eng 5(2): 959-969.} Copyright {2019} American Chemical Society.

The highly tunable nature of hydrogels allows for countless design strategies and material properties. When creating a NO-releasing hydrogel, knowing which properties are ideal for the chosen application and which will lead to complications is essential. Some modifications are as simple as adding a plasticizer to a hydrogel formulation to increase flexibility or decrease the viscosity^{20, 21} or embedding the NO-releasing mixture into an already formed ointment or gel.^{22, 23} Other methods of enhancing the mechanical properties of a hydrogel system include casting the gel into an electrospun polymer mat^{24, 25} or combining multiple hydrogel types into a single formulation.^26-35 For example, Pluronic hydrogels are often combined with natural polymers such as alginate^{26, 36} or chitosan³² to improve mechanical strength and biocompatibility.

Modifications must be thoroughly considered in some scenarios, as strict design constraints exist for a particular application. For instance, when designing a NO-releasing hydrogel for ocular applications to decrease ocular pressure and fight bacterial infections, parameters such as light penetration and transparency, appropriate gas diffusion, and swelling properties, as well as corneal cell biocompatibility cannot be overlooked.^{34, 37} Similarly, this application might permit the use of a NO photo donor, allowing for NO release triggered by visible light traveling to the eye (Figure 3A). Other applications suitable for the implementation of an NO photo donor are those in which the hydrogel will be placed external to a patient in vivo, such as dermal vasodilation enhancement³⁸, utilization of a wound adhesive²⁷, and exterior infection treatment.³⁹ On the other hand, hydrogel treatment of blood vessel injury caused by stenting is more likely to use a NO donor that is thermally or hydrolytically degraded while being applied perivascularly^40-42 or as a stent coating³⁰ for localized NO therapy (Figure 3B). Although hydrogel wound dressings possess many ideal properties such as high swelling capacity, gas exchange permeability, and biocompatibility, they can be improved further with the addition of fibrin microparticles to a poly(ethylene) glycol-N-hydroxylsuccinimide (PEG-NHS)-fibrinogen adhesive hydrogel (Figure 3C-F).²⁷ The active primary amine groups present on the PEG-NHS and the S-Nitroso-N-acetylpenicillamine (SNAP)-loaded fibrin nanoparticles easily crosslink with the amine and thiol groups found in the fibrinogen, fibrin, and tissue extracellular matrix at the wound site, endowing adhesive properties to the hydrogel treatment. This adhesive nature allows the gel to conform to the local microenvironment and release NO locally for optimized antibacterial and wound healing properties. Another advancement dealt with hydrogel application as a powder that forms a gel upon exposure to wound exudates, allowing for an exact fit to irregular wound sites.²⁹

While some properties and characteristics of hydrogels, such as polymer type and NO donor, can be specifically chosen and modified to fit an application, many fabrication processes along the way can have lasting effects on hydrogel quality. The consequences of hydrogel fabrication and modification methods should be well understood when designing a hydrogel for specific applications. For instance, crosslinking of hydrogels can drastically affect almost all other hydrogel properties (Figure 4). A simple example of this is the relationship between cross-linking and the swelling capacity of hydrogels: increased crosslinking that is often performed to enhance mechanical properties or prevent donor leaching decreases the swelling potential of hydrogels due to the increased density of polymer chains within the matrix and limited space for water molecules to invade.⁴³ Additionally, the pore size within hydrogels can affect properties such as swelling capacity, as shown in a previous study.⁴⁴ For hydrogels with water-sensitive NO donors embedded into the bulk hydrogel, greater swelling capacity directly correlates to higher NO release from the hydrogel as water-polymer contact increases and hydrolytic degradation of the donor is enhanced. Therefore, NO release is altered and can be modified by transforming the pore size of the bulk hydrogel. Furthermore, porosity can often be affected by freeze-thaw cycles sometimes used to induce physical crosslinking of hydrogels.⁴⁵ The slight manipulations and alterations performed to achieve ideal crosslinking and mechanical properties can have lasting effects on the biological efficacy of NO-releasing hydrogels and should always be considered from all angles before final formulation.

Figure 4. Properties of NO Hydrogels Affected by Degree of Crosslinking.

The cross-linking of hydrogels allows for enhanced mechanical properties, though the effects can be seen in other essential hydrogel properties such as porosity, swelling capacity, biodegradability, NO release, biocompatibility, and resistance to deformation.

2.2. Nitric oxide incorporation strategies

Incorporating NO donors into a hydrogel system presents several challenges researchers have creatively overcome in recent years. One strategy is the direct physical inclusion of a NO donor molecule^{21, 23, 32, 36, 46-49}, such as S-nitrosoglutathione (GSNO), a water-soluble S-nitrosothiol (RSNO) molecule that releases NO in the presence of heat, light, and metal ions.⁵⁰ Interestingly, GSNO is simply the S-nitrosated derivative of glutathione, the most abundant cellular thiol that is also readily taken in and processed by several microbes, enhancing the antibacterial effects of NO therapy.^{9, 50} Although the solubility of GSNO seems promising, the high water uptake of hydrogels leads to excessive GSNO leaching, which can induce cellular toxicity if present in sufficient quantities.^{32, 46} Several other small molecules NO donors that have been utilized in hydrogels include S-Nitroso-N-acetylpenicillamine (SNAP)²⁷, S-nitrosocysteine (Cys-NO)^{41, 42, 51}, and nitroglycerin.²³ Rather than physical incorporation, several hydrogels have been indued with NO-releasing capabilities through ‘nitrosation’ of amines, a term that refers to the specific chemical reactions where a nitrosonium ion (NO+) is added to a nucleophilic group. The analogous term, ‘nitrosylation,’ is more specific and refers to the direct addition of NO to a reactant, though it is more appropriately used in biological/physiological contexts.⁵² Nevertheless, the nitrosation of amines through high-pressure NO gas leads to the formation of diazeniumdiolates (NONOates)^{22, 28, 31, 37, 40, 42, 51, 53-60} that spontaneously hydrolyze to NO in aqueous media.⁶¹ Nitrosation of thiol moieties is another technique for imparting NO donating capability to the hydrophilic polymer systems and generally provides a more stable and prolonged NO release compared to NONOates.^{20, 33, 43, 51, 62-64} A unique technique utilizes nitrosyl ruthenium species for NO release as they have increased thermal stability and release NO in a controlled manner via light irradiation.^{38, 65} The final method of endowing hydrogels with NO-releasing capabilities draws directly from endogenous environments, where a donor produces molecules that are utilized by cells found in the treatment site. In one study, L-arginine is cleaved from the donor molecule by peptidase enzymes that is then utilized by activated macrophages to produce NO.⁶⁶ Similarly, ammonia deposited as a result of gelatin crosslinking by microbial transglutaminase is partially oxidated through biosynthetic coupling with the urea cycle to continuously release NO.⁶⁷

Whatever the method of NO incorporation, the levels of NO released from fabricated hydrogels is of considerable interest.⁶⁸ In terms of target therapeutic applications, many studies aim to replicate the surface flux value of 0.5 – 4 (x10⁻¹⁰ mol cm⁻² min⁻¹) as calculated by Vaughn, et al. to be the NO release rate from endothelial cells.⁶⁹ However, lower conentrations in the picomolar range have also shown therapeutic effects in blood and antibacterial applications.^{70, 71} The upper threshold for NO release to prevent mammalian cell cytotoxicity may be slightly harder to determine. One study found levels around 1 μM NO to be toxic in glial cultures treated with cytokines, which was confirmed by another study revealing ~ 0.5 μM NO (cumulative dose of ~ 150 μM NO min⁻¹) leads to cell apoptosis.^{72, 73} Although, toxicity of NO can vary depending on cell type, culture conditions, cumulative NO dose, and release levels, as well as inherent toxicity of the NO donor rather than the gas itself.⁷⁴ The complexity of NO interactions and biological responses highlight the need for studying mammalian cell reactions to specific NO-releasing materials intented for biomedical use.

3. In vitro and in vivo applications of NO-releasing hydrogels

3.1. Cardiovascular therapy

The roles of NO in cardiovascular health are numerous and indispensable. NO is a signaling molecule for blood vessel dilation, stem cell differentiation, cardiac contraction, and immunomodulation.⁷⁵ The protective actions of NO are fulfilled through the prevention of platelet adhesion and aggregation, suppression of leukocyte and monocyte adhesion, and inhibition of endothelial cell apoptosis.⁷⁵ Another key characteristic of NO in the cardiovascular system is maintaining a balance between endothelial cell and smooth muscle cell proliferation and migration after injury to blood vessels. Oftentimes, mechanical, such as stent implantation, or immunological, such as overactive immune response, vessel injury can lead to neointimal hyperplasia, a condition characterized by rapid smooth muscle cell proliferation in the intima, increasing arterial wall thickness, and decreasing arterial lumen space. A decreased lumen area can cause further complications relative to blood flow and increase the risk of atherosclerosis and myocardial infarction.⁷⁶ Due to the high-risk nature of the imbalance of endothelial cell (EC) and smooth muscle cell (SMC) growth, many studies have been conducted to investigate exogenously delivered NO from hydrogels.

In all cases, incorporating an NO donor, usually through covalent linking or nitrosation, increased EC proliferation while decreasing SMC proliferation in vitro, without exhibiting toxicity.^{28, 41, 42, 51, 56, 77, 78} A co-culture study of ECs and SMCs on a collagen hydrogel treated with varying levels of GSNO revealed that in addition to inhibiting SMCs and enhancing the proliferation of ECs, supplementing 100 nM GSNO to the co-culture system significantly upregulated the production and deposition of extracellular matrix (ECM) proteins.⁷⁷ Increased matrix synthesis further enhanced eNOS (in ECs) and iNOS (in SMCs) expression, creating a positive feedback loop of NO release and vessel remodeling.⁷⁷ A PEG-based hydrogel with covalently linked Cys-NO showed similar results of increased matrix protein production after cell incubation with Cys-NO, a useful tool that must be carefully modulated as the overproduction of ECM proteins can lead to restenosis or blood vessel narrowing.⁴² Some studies also showed decreased platelet adhesion to a model thrombogenic surface after treatment with a NO-releasing hydrogel, highlighting NO ability to inhibit platelet adhesion and activation.^{40, 41, 51, 78}

When tested in vivo, NO gels were often applied after vessel injury by the expansion of an angioplasty balloon, followed by an analysis of reendothelialization in the following days and weeks. PEG-based NO gels that were applied perivascularly to injured vessels and then photo-crosslinked led to significant decreases in rat intimal thickening at 14 days post-injury in three studies from the West laboratory^40-42, with one study revealing significant inhibition of medial proliferation in as little as 4 days.⁴⁰ Cell marker analysis also revealed that treatment with PEG-Cys-NO hydrogels caused a significant reduction of medial cells staining positive for a proliferation marker, indicating successful reendothelialization without unwanted deeper vessel cell proliferation (Figure 5A-D).⁴¹ The inflammatory response in injured vessels was also investigated, but this time utilizing treatment with a self-assembling nanofiber gel containing two different NONOate NO donors, 1[N-(3-Aminopropyl)-N-(3-ammoniopropyl]diazen-1-ium-1,2-diolate (DPTA/NO) or disodium 1[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO).⁵⁶ Results showed that the PROLI/NO gel reduced the inflammatory response following injury, as characterized by positive staining for monocytes and leukocytes (Figure 5E-G). PROLI/NO also showed the least amount of cell proliferation, a favorable characteristic when attempting to prevent neointimal hyperplasia following arterial injury (Figure 5H). The inability of the DPTA/NO gel to reduce inflammation and cell proliferation once again suggests the complexity of the physiological effects of varying NO release levels and highlights the necessity of proper material tuning for specific applications.

Figure 5.

Following a rat model of balloon angioplasty, perivascular treatment with (A) control PEG hydrogels led to unwanted medial cell proliferation, whereas (B) Cys-NO hydrogels revealed enhanced vessel reendothelialization without medial cell growth, as well as (C) reduced intimal thickness and (D) 77% lower intimal area to medial area ratio. Arrows mark the internal elastic lamina. (E) A similar rat carotid artery injury model was utilized to demonstrate the decreased inflammatory response in the vessel following treatment with two different NO-releasing hydrogels. Red staining reveals positive markers and green staining indicates the elastic lumina. PROLI/NO gels revealed decreased (F, G) inflammatory markers and (H) cell proliferation compared to control and alternative treatment groups, except the nanofiber gel showed similarly low levels Of leukocytes. A-D were used with permission of Elsevier Science and Technology Journals from [Localized delivery of nitric oxide from hydrogels inhibits neointima formation in a rat carotid balloon injury model, Lipke, E. A., West, J. L., Vol 1, Edition 6, Copyright 2005]; permission conveyed through Copyright Clearance Center, Inc. E-H were reprinted from [Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia. Vol 47/Edition 1. Kapadia, M. R., et al. 173-182, Copyright (2008)], with permission from Elsevier.

In a different study, a novel design of a drug-eluting stent was created by electrospinning a formulation of PLGA, GSNO, alginate, and Edaravone (EDV), a ROS scavenger, onto a metal stent that would be used to treat a blocked artery or vessel.³⁰ The addition of alginate to the mix endowed ideal mechanical properties for the expansion of the metal stent while crosslinking the alginate prolonged the release of NO and EDV. The combination design also stabilized mast cells, a migrant immune cell that, once activated, can release cytokines and ROS that prevent uniform reendothelialization after stenting. While this study considers possible immune responses to stenting, a consequence often overlooked in in vitro and in vivo studies, it only touches the surface of the intricate cascade of events caused by blood vessel injury that NO-releasing hydrogels can carefully regulate.

3.2. Enhancement of vasodilation and angiogenesis

The vasodilative properties of NO, utilized to regulate systemic blood pressure, can be harnessed to treat peripheral vascular diseases such as Raynaud’s syndrome⁷⁹, diabetic microcirculatory disorders, and chronic ulcers characterized by endothelial dysfunction.⁸⁰ In addition to providing localized treatment, NO is a water-soluble molecule that exhibits passive diffusion through tissues⁸¹, unlike many commercial nonsteroidal anti-inflammatory drugs (NSAIDs) used in topical applications to treat pain and inflammation.⁸² Efficient increases in dermal blood flow have been exhibited in several studies, with one study revealing vasodilative effects in less than 10 mins and lasting for more than 4 hours as measured by Laser Doppler flow monitors.⁴⁵ In a nitrosated PVA model, there was an almost linear dose and time-dependent response in the ratio of blood flow increase for films nitrosated for 5-, 7-, and 10-mins following application times of 2, 5, and 10 mins (Figure 6A).⁶³ Additional studies revealed the relationship between increased irradiation of an NO photo donor hydrogel and rapidly increased dermal blood flow^{38, 62}, allowing for immediate treatment and results if necessary. A different dose-dependent trend was observed when comparing blood flow increases after dermal application of Pluronic F-127 hydrogel with either S-nitroso-N-acetylcysteine (SNAC, referred to as Cys-NO in a prior study) or GSNO embedded.⁸³ However, rather than changing doses, the two donor molecules were compared to observe if the higher degradation rate of SNAC (3.6 times greater than GSNO) influenced dermal blood flow and, if so, in what way. An interesting trend was observed where although SNAC has a 3.6-fold higher decomposition rate constant, it only achieved a 1.2-fold increase in maximum blood flow. Additionally, measurements of dermal nitrite concentrations through dialysate collection revealed a strong correlation between blood flow and nitrite concentration, but only at blood flow values of less than 250 units. At levels higher than 250 units, such as in the application of the SNAC hydrogel and rapid initial degradation, intradermal nitrite levels are paradoxically lower than expected. This trend can be explained by oxyhemoglobin scavenging of excess NO when blood flow reaches a physiological threshold when the body attempts to downregulate vasodilative effects to prevent vessel stretching.⁸³ In addition to general studies aiming to increase dermal blood flow for the treatment of vascular disorders, a Pluronic F-127 – GSNO hydrogel formulation was employed to examine NO’s analgesic potential and revealed a similar mechanism to morphine. After diffusing through the stratum corneum (top layer of skin), NO induces vasodilation through the relaxation of smooth muscle cells and increased blood flow. Further diffusion leads to the opening of a K+ channel that increases the nociception threshold, thereby reducing experienced pain.⁸⁴

Figure 6.

(A) Nitrosated PVA gels revealed a time- and dose-dependent response in the rate of blood flow when placed topically on the forearm skin of the authors. Further, after 10 minutes of application, the skin revealed a hyperemic response as shown by the inset photographs. (B) In a rat hindlimb ischemia model, results showed that co-transplantation of MSCs with a CS-NO gel allowed for enhanced neovascularization and functional recovery of ischemic hindlimbs compared to each treatment alone as well as no treatment. A was used with permission of Elsevier Science and Technology Journals, from [Nitric oxide-releasing poly(vinyl alcohol) film for increasing dermal vasodilation, Marcilli, R. H. and de Oliveira, M. G., 116, Copyright 2014]; permission conveyed through Copyright Clearance Center, Inc. B was used with permission of Elsevier Science and Technology Journals, from [A nitric oxide-releasing hydrogel for enhancing the therapeutic effects of mesenchymal stem cell therapy for hindlimb ischemia, Zhang, K., et al., 113, Copyright 2020]; permission conveyed through Copyright Clearance Center, Inc.

Taking a further look at the cellular level of NO treatments, angiogenic models for examining NO’s effect have been investigated. In vitro application of NO vehicles has confirmed the angiogenic potential of NO via the tube formation assay, where endothelial cells (HUVECs) are treated with NO and control hydrogels and examined for lumen-filled capillary structure formation in as little as two hours.^{58, 60} NO-treated samples typically display larger branches and more nodes forming a capillary network structure, a result is also displayed in a chick-embryo model after treatment with a chitosan-PVA-SNAP hydrogel.³⁵ The potential of in vitro angiogenesis is promising, but small animal models give a more holistic look into the angiogenic potential of NO hydrogels. One study used mice to investigate the potential of NO-releasing Fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF-SNAP) hydrogels in treating renal ischemia/reperfusion injury, which can occur following kidney transplantation, hemorrhage, trauma, and burns.⁸⁵ Self-assembly of the peptide hydrogel in physiological conditions created a nanofibrous shear-thinning hydrogel fabricated with SNAP concentrations of 15, 30, and 60 μM. When tested in vivo against free-SNAP at the same concentrations, FmocFF-SNAP gels showed significantly improved histopathological scores as well as decreased levels of oxidative stress biomarkers and enhanced eNOS expression, revealing repairs to the injured tissue. Moreover, in two separate rat hindlimb ischemic model studies, both using chitosan hydrogels but with slightly different treatment methods, NO release enhanced vascular recovery of ischemic hindlimbs by promoting microvessel growth and increasing capillary density in the affected tissues, where many untreated and control groups loss complete vascular function. Zhao et al. implemented a chitosan hydrogel with a galactose-protected NONOate donor that releases NO after enzymatic degradation by galactosidase, significantly reduced necrosis, and stimulated recovery of hindlimb perfusion in NO-therapy-treated rats.⁷⁸ The NO donor group of the polymer was chemically protected by galactose due to the instability of NONOates and introduced the possibility of fine-tuned NO release by controlling the degradation of the CS-NO polymer. Similar success was reported when the chitosan-NONOate protected gel (CS-NO) was co-transplanted with mesenchymal stem cells (MSCs). The co-transplantation allowed for 40% survival of the MSCs after 7 d, decreased amputation rates, the largest area of normal muscle function, and the best functional recovery of ischemic hindlimbs (Figure 6B).⁶⁰ The successful implementation of NO-releasing hydrogels leading to complete recovery of ischemic limbs holds great promise for application in larger animal models and, eventually, human clinical trials.

3.3. Antimicrobial Applications

The antimicrobial potency of NO lies in the highly reactive nature of the nonpolar, uncharged, free radical molecule. Once produced, NO readily diffuses through microbial membranes, reacting rapidly with oxygen (O₂), thiols, and metals, producing an array of reactive nitrogen (RNS) and oxygen species (ROS), each uniquely capable of inducing nitrosative and oxidative stress to foreign pathogens. Peroxynitrite (OONO⁻) is a highly reactive and toxic molecule produced by the reaction of NO with O₂⁻ known to cause oxidative damage to multiple cellular targets.^{8, 9, 86} Other ROS and RNS such as nitrogen dioxide (NO₂^•), dinitrogen trioxide (N₂O₃), dinitrogen tetroxide (N₂O₄), hydrogen peroxide (H₂O₂), and hydroxide (^•OH) species can modify proteins and lipids as well as deaminate DNA.^{9, 86} NO's reaction with thiols can lead to S-nitrosation of proteins, resulting in the creation of temporary NO reservoirs and altering protein function through disulfide bond formation.⁸⁶ In terms of metal reactions, NO can deplete cellular iron stores and inactivate essential enzymes through the reaction with cellular iron or iron-sulfur complexes.⁸⁶ Although these antimicrobial strategies of NO have been previously identified, the exact critical targets responsible for microbial death have yet to be discovered.⁹

NO-releasing hydrogels for antimicrobial applications should have a stable and prolonged release of NO, a facile synthesis process, and bactericidal efficacy. Through various modifications, hydrogel systems can utilize these characteristics to eradicate microbes in vitro and in vivo.^{21, 55} Often, the NO-releasing hydrogel can be functionalized to carry either synthetic or natural antimicrobial agents in addition to its inherent antimicrobial properties. For example, when combined with a NO donor, S-nitroso-mercaptosuccinic acid, silver metal nanoparticle-loaded hydrogels can achieve potent anticancer and local antimicrobial effects without systemic toxicity.²⁰ Natural antimicrobial agents extracted from plants and animals, such as chitosan, have gained increasing attention for their inherent biocompatibility and bioavailability. Chitosan is a polycationic polysaccharide biopolymer that gained popularity for its known activity to promote wound healing and encourage surface endothelialization. One recent study highlighted a synergistic enhancement in chitosan’s anti-biofilm ability against methicillin-resistant Staphylococcus aureus (MRSA) when prepared as a NO-releasing hydrogel with GSNO.²¹ The hydrogel's in vivo performance was evaluated on diabetic mouse models and demonstrated excellent dispersal of MRSA biofilm and wound healing efficacy within 15 days.⁸⁷ Comparatively, an injectable and self-healing chitosan hydrogel was imbued with NO-releasing capabilities through covalent attachment of N-acetyl-cysteine, followed by nitrosation and crosslinking with HA using aldehyde-modified polyethylene glycol (PEG).⁸⁸ When tested for antimicrobial efficacy, the chitosan-NO gel showed a 2-log reduction in E. coli after only 1.5 h and a 1-log reduction in Staphylococcus epidermidis (S. epidermidis) after 4 h exposure. Hence, chitosan is a common polymer of choice for NO-releasing hydrogels as it enhances antimicrobial effects. Natural-derived carboxymethyl cellulose (CMC) is a water-soluble polymer attractive for its biocompatibility, ability to increase solution viscosity, and adhesiveness to tissue surface.⁵⁴ CMC derivatives modified with NONOates previously developed by Feura et al. can adhere to periodontal pocket tissue proteins and concomitantly reduce common periodontopathogens such as Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans).⁵⁴ Besides naturally derived antibiotic agents, bio-inspired bactericidal substances have also been utilized in NO-releasing hydrogels. Silver nanoparticles synthesized from green tea extract were incorporated into an alginate hydrogel with NO donor, S-nitroso-mercaptosuccinic acid (S-nitroso-MSA), for topical antibacterial applications. The combination had a synergistic effect against common infectious pathogens E. coli, S. aureus, and S. mutans.²⁰

During the synthesis process of NO-releasing hydrogels, Pluronic or Poloxamer are copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) that are often used as the polymer matrix due to their tunable and thermoresponsive characteristics. PEO segments are more hydrophilic, whereas PPO segments are more hydrophobic.⁸⁹ The PPO segments provide a desirable microenvironment for the incorporation of lipophilic molecules such as NO. Therefore, the NO release stability, duration, and photochemical delivery in hydrogels can be tuned by adjusting the number of the PEO/PPO segments in the polymer.⁴⁷ Pluronic F127 (PEO-PPO-PEO) hydrogel has been used as a NO delivery vehicle for various antimicrobial studies. In some cases, GSNO was blended into a Pluronic F127 – chitosan hydrogel matrix where a 3-log reduction in CFUs against MRSA and multidrug-resistant P. aeruginosa (MDRPA) was achieved.³² A similarly designed hydrogel utilized Pluronic F-127 combined with GSNO and alginate.²⁶ Since the PEO segment of the Pluronic has weak mechanical strength and rapid erosion toxicity, alginate was added for its bio-adhesive property and increased cell viability. The resulting hydrogel exhibited potent bactericidal activity against Gram-positive MRSA and Gram-negative MDRPA with sustained NO release for up to 7 days. Other studies also linked tri-block polymer Pluronic F-68 with branched polyethyleneimine (BPEI) for conjugation of NONOates for a sufficient release profile that induces bactericidal action against S. aureus, MRSA, and E. coli.³¹ NO-releasing low molecular weight alginate oligosaccharides have reported the unique ability to alter biofilm morphology and mucin assembly and, therefore can be used as an adjuvant therapy to conventional antibiotics. Moreover, low-molecular-weight HA has shown promising results in wound healing in vivo murine models due to its intrinsic tissue remodeling properties. The combination of HA and NO-releasing biopolymer reduced the bacteria viability of E. coli, P. aeruginosa, S. aureus, and E. faecalis by 3-logs over 4 h.⁸⁷

In addition to the aforementioned processes to synthesize NO-releasing hydrogels, the self-assembling peptide-functionalized hydrogel can also provide similar antibacterial characteristics and properties. Antimicrobial peptide MSI-78, or pexiganan, has a broad spectrum of antimicrobial activity against microbes and a high likelihood for self-assembly due to its hydrophobic and aromatic-rich peptide sequence.^90-92 The self-assembled hydrogen can be triggered by the addition of sodium hydroxide, which induces an immediate phase transition to translucent gel. The combined system of self-assembled hydrogel and NO donor NONOate can induce the slow kinetic release of NO for up to 15 days.^{53, 93} In addition to pH shift triggers, light-induced self-assembly supramolecular hydrogel with bactericidal action has also been developed. Spontaneous self-assembly of the hydrogel can be achieved by interaction between poly-β-cyclodextrin polymer, hydrophobically modified dextran, and NO photo-donor bearing an adamantyl appendage. The resulting gel network prevented the leaching of the NO photo-donor compound upon visible light excitation while maintaining bactericidal actions.³⁹ A hyaluronic acid nanogel was contrived to be NO-releasing following a crosslinking process with divinyl sulfone followed by the physical incorporation of SNAP and a specifically designed antimicrobial peptide. The combination of antimicrobials led to the successful killing of E. coli, S. aureus, and P. aeruginosa in planktonic and biofilm form, though NO release, measured by UV-Vis Spectroscopy only lasted ~24 h.⁹⁴

Another appealing class of NO-releasing hydrogel is injectable S-nitrosothiolated gelatin (GelSNO) with gelatin-based hydrogels formed by horseradish peroxidase/H₂O₂ reaction. Upon thermal, light, and oxidizing agent-driven stimulus, this hydrogel generates OONO⁻ in situ from released NO and H₂O₂ residues for up to 14 days.⁴³ Prolonged storage of NO-releasing hydrogels can be achieved by in situ hydrogel-forming/NO-releasing powder dressings (NO/GP) developed by Yoo et al. The powder was fabricated by blending and micronizing GSNO, pectin, alginate, and polyethylene glycol (PEG).²⁹ The NO/GP powder remained stable for more than four months when stored at 4 or 37 °C and displayed sustained NO release for 18 hours upon gel formation. Moreover, incubation with NO/GP resulted in a 6-log reduction in colony-forming units (CFUs) of MRSA and P. aeruginosa. NO-releasing hydrogels possess impressive antimicrobial potential and can be fabricated through a variety of techniques depending on the application conditions and environment (Table 1).

Table 1.

Previously studied antibacterial NO-releasing hydrogels

Material	NO Donor	Antibacterial Capability	Model	Ref.
Silver nanoparticle-loaded alginate hydrogel	S-nitroso-MSA	Complete bacterial killing after 2 h for S. mutans UA159, S. aureus (ATCC 25920), and E. coli (ATCC 25922)	In vitro time-kill curves	20
Chitosan	S-nitrosoglutathione (GSNO)	> 3 log reduction in colony forming units (CFUs) against MRSA (USA300)	In vitro anti-biofilm crystal violet staining and in vivo biofilm assay	21
Chitosan and PEG-modified Hyaluronic acid	S-nitroso-N-acetyl-cysteine (SNAC)	> 2 log reduction in E. coli (ATCC 25922) after 1.5 h and > 1 log reduction in S. epidermidis (ATCC 12228) after 4 h	In vitro time-kill curves	88
Carboxymethylcellulose (CMC) derivatives	Diazeniumdiolates (NONOates)	3 log reduction in CFUs against planktonic P. gingivalis (ATCC A7436) and A. actinomycetemcomitans (ATCC 43717)	In vitro minimum bactericidal concentrations (MBC)	54
Pluronic F-127 -chitosan hydrogel	GSNO	Minimal inhibitory concentration of 0.5 μg·mL−1 against P. aeruginosa (ATCC 27853)	In vitro minimal inhibitory concentration (MIC) and MBC	32
Pluronic F-127-alginate hydrogel	GSNO	> 3 log reduction in CFUs against MRSA (KNRRB 3089) and MDRPA (KNRRB 2200)	In vitro modfied Oxford cup method and in vivo bacterial burden reduction	26
Pluronic F-68 with branched polyethyleneimine (BPEI)	NONOates	3 log reduction against E. coli (KCCM 25922), S. aureus (KCCM 29213), and MRSA (KCCM 33591)	In vitro MIC	31
Self-assemble peptide N-Fmoc-Pexiganan (MSI-78)	NONOates	Complete bacterial killing after 2 h for E. coli	In vitro time-kill curves	53
Poky-β-cyclodextrin polymer with hydrophobically modified dextran	Tailored NO photo-donor bearing an adamantyl appendage	> 3 log reduction in CFUs against antibiotic-resistant E. coli DH5α	In vitro bacteria exposure with irradiation	39
Hyaluronic acid nanogel with antimicrobial peptide	SNAP	Minimum inhibitory concentrations of 1.6 mg/mL (E. coli), 0.4 mg/mL (P. aeruginosa), and 0.8 mg/mL (MRSA ATCC BAA 1683)	In vitro MIC, and catheter biofilm eradication	94
Gelatin-based hydrogel	S-nitrosothiolated gelatin	Almost 50% inhibition of E. coli (ATCC 11775) and S. aureus (ATCC 14458) with 0.28 μmol/mL of NO-hydrogels. Eradication of S. aureus at 0.58 μmol/mL of NO-hydrogels	In vitro zone of inhibition and viability test	43
Alginate-pectin-PEG powder dressing	GSNO	6-log reduction in CFUs of MRSA (USA300) and P. aeruginosa (PA01)	In vitro 24 hr exposure and LIVE/DEAD staining	29
Poly-ε-lysine (pεK) gel	NONOates	5-, 4-log reduction in CFUs against P. aeruginosa (PA01) and S. aureus (ATCC 25922), respectively	In vitro 24 hr expore and plating using the Miles and Misra method	37
Hyaluronic acid hydrogel	NONOates	3 log reduction in CFUs against E. coli, P. aeruginosa, S. aureus, and Enterococcus faecalis	In vitro time-based planktonic bacteria assay, biofilm eradiaction assay, and in vivo infected murine wound	87

3.4. Wound healing

In wound healing, the role of nitric oxide is multifaceted and crucial in ensuring damaged tissues are successfully healed, reorganized, and regrown to recover full function. In a perfect scenario, a wound goes through the four phases of healing without much deviation.⁹⁵ The first phase is bleeding, where injured blood vessels constrict and the clotting cascade begins, forming a blood clot from crosslinked fibrin fibers. The inflammatory phase follows next and lasts for about 3 days to establish an immune barrier against invading microorganisms. The third phase of wound healing, the proliferative phase, lasts from about 3 – 17 days where the tissue is repaired and rebuilt through fibroblast proliferation, migration, and production of ECM proteins that replace the provisional fibrin network of the blood clot, collagen deposition, and angiogenesis. Once the tissue structure has begun forming, the fourth phase of the remodeling begins and continues for weeks to months to years depending on the severity of the wound. During this time, new endothelium and scar tissue are developed, and balance is formed between degrading disorganized collagen deposited in early stages and replacing it with organized, aligned collagen fibers. Macrophage and fibroblast density decreases as the tissue return to a more normal and healthy state.

However, wounds are rarely perfect and are often burdened by microbial infection, irregular damage, and excessive inflammation that can lead to chronic wounds or poorly reconstructed tissues. To combat this, research in wound healing is intensive on ensuring the injury follows a timely progression through the healing process and providing a favorable environment for it to do so. Hydrogels are ideal materials for wound healing as they maintain sufficient moisture within the wound while absorbing exudates, allow for proper gas exchange, and are easily modified for the co-delivery of therapies such as NO. Pluronic hydrogels have long been of interest to wound healing research and, with the addition of GSNO, have shown augmented wound healing in all aspects, revealing improved wound contraction and re-epithelialization when the GSNO-containing gel was applied during the inflammatory phase⁹⁶, proliferative phase⁹⁷, and a more significant effect when applied during both phases⁹⁷. Administration of NO during the inflammatory phase is highly crucial as neutrophils and macrophages endogenously produce it to prevent microbial invasion and control the inflammatory process. Several studies have shown that NO-hydrogel treatment has decreased the microbial burden compared to control and non-NO-releasing hydrogel treatment and alleviated the excessive inflammatory wound reaction when an infection is present.^{21, 26, 29, 55, 96, 98-100} This is especially advantageous in cases of diabetic or ischemic wounds, where endogenous NO production is limited and if left untreated, has a high probability of forming a chronic wound. Nonetheless, NO-hydrogel treatment overcame these limitations and prevented hyperactive inflammation while allowing for organized collagen fiber formation in diabetic and ischemic wounds.^{23, 99, 101} NO administration in the proliferative phase upregulates fibroblasts and therefore collagen deposition and wound closure.⁵³ This trend has been displayed in various in vivo wound models where NO-hydrogel treatment leads to faster wound contraction and greater granulation tissue organization^{101, 102} compared to control wounds and those treated with non-NO releasing gels (Figure 7A).^{21, 22, 24, 33, 96, 97, 103-105} Within the formed wound, NO treatment has been shown to enhance blood vessel density and thickness^{21, 24, 33, 101, 103, 104} and even increase the expression of growth factors such as VEGF and CD34 that further propagate angiogenesis.⁹⁸ One study showed the heavy infiltration of neo-vascularization during the wound-healing process, followed by a decrease in angiogenesis once the wound was healed and in the remodeling phase.²² Enrichment in wound healing through treatment with a NO-releasing hydrogel provides invaluable benefits, but additional factors can also speed up healthy wound healing. Nie et al. combined NO-hydrogel treatment with asiaticoside (AC), an extract from dry grass used for medicinal purposes in China with exceptional wound healing properties.⁹⁸ The combination treatment proved highly effective and paired well together as AC can prevent scar formation associated with excess collagen deposition from fibroblasts, sometimes caused by high NO levels.

Figure 7.

(A) Chitosan (CS) and CS-NO hydrogels were used to treat wounds on diabetic and non-diabetic mice with dressings changed every 3 days. Untreated wounds were unable to overcome MRSA biofilm and showed little wound closure over 15 days. CS-treated wounds were still plagued by biofilm, but CS appeared to assist in relative wound closure. CS-NO treated wounds killed the infection and enhanced collagen deposition and thus, wound closure. (B) A biomimetic approach was utilized when L-Arginine and H₂O₂ were added to a PVA hydrogel to induce low levels of NO release for antimicrobial effects as well as enhancement of macrophage chemotaxis, cell proliferation, collagen deposition, and angiogenesis. A was used with permission of Elsevier Science and Technology Journals, from [Chitosan-based nitric oxide-releasing dressing for anti-biofilm and in vivo healing activities in MRSA biofilm-infected wounds, Choi, M., et al., 142, Copyright 2020]; permission conveyed through Copyright Clearance Center, Inc. B was reproduced/adapted from Yu, J., et al. (2022). "Injectable Reactive Oxygen Species - Responsive Hydrogel Dressing with Sustained Nitric Oxide Release for Bacterial Ablation and Wound Healing." Advanced functional materials: 2202857, with permission from Wiley.

The delivery of NO from SNAP-loaded fibrin microparticles created a supportive composite wound dressing. The fibrin delivery (an ECM protein) to the wound site and the covalent bond formation between the PEG-NHS hydrogel vehicle and amine and thiol groups found on tissue ECM induced a strong adhesive connection between wound and dressing.²⁷ The increased adhesiveness allowed for extremely localized NO delivery as well as the creation of a solid boundary layer to protect internal elements from bacterial colonization or contamination.

Rather than relying on donor molecules for NO treatment at a wound site, several studies have incorporated L-Arginine into biocompatible hydrogels to enhance localized NO release by mimicking physiological actions.⁶⁶ One such study added L-arginine and low amounts of H₂O₂ to a PVA hydrogel, where H₂O₂ acted to assist in NO production from L-arginine and degraded the gel over ~ 14 days (Figure 7B). Low levels of NO and H₂O₂ imparted antimicrobial activity as well as enhanced the wound healing process in a 12-day mice wound model.

Precise control over NO dosage in treating wounds is essential, important as NO release characteristics determine many biological functions in physiological systems. There have been several examples of the detrimental effects of excessive levels of NO release, often leading to increased inflammation and delayed wound closure, even compared to untreated control wounds.^{33, 35, 98} In several studies, NO-releasing hydrogels effectively closed the wound and regrew high-quality, complex tissues, but there was an excess of scar tissue formation due to the enhanced collagen deposition from fibroblasts, attributed to an overload of NO signaling.^{42, 102} While scar tissue deposits are trivial in some areas of wound healing, they could cause severe complications if used in a cardiovascular setting where vessel elasticity would be severely hindered. In the case of treating periodontal disease, one study showed a dose-dependent response to polyvinylpyrrolidone (PVP) – GSNO treatments where 100 nmol GSNO significantly reduced alveolar bone loss, and inflammatory and oxidative stress markers while improving several other parameters. In contrast, the 500 nmol GSNO treatment had results similar to saline and control groups, eliminating all positive effects of the controlled NO therapy.¹⁰⁶

Physiological factors at the hydrogel treatment site must also be considered during the design stage. For instance, a GSNO-infused Pluronic F-127 hydrogel that showed remarkable improvements in wound contraction and re-epithelialization in a standard rat wound model was ineffective in a rat ischemic wound model.⁹⁹ The GSNO concentration had to be doubled to achieve promising wound healing results. The discrepancy lies in the ischemic wound environment where blood flow is hindered, and endogenously available NO levels are much lower. In another case, three different NO donors were covalently linked to a PEG hydrogel, and it was found that their degradation and corresponding NO release was inhibited in acidic pH.⁵¹ Though this interaction provides options for long-term storage, it can also be a limiting factor in applicability, as the slightly acidic environment in chronic wounds would impede their therapeutic effects. The field has shown great promise, yet numerous combinations and innovations are on the horizon.

3.5. Stem cell response to NO hydrogels

Though wound healing and angiogenic studies provide insight into the tissue-level effects of NO-hydrogel treatment, some researchers took a closer look at how NO release affects cells in their most opportunistic form as stem cells. Several studies focused on exposing mesenchymal stem cells (MSCs) to NO-releasing hydrogels to investigate possible differentiation effects, revealing that NO-treated MSCs trended toward endothelial differentiation. Using LH-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a molecule that inhibits the NO-cGMP pathway and, therefore NO production, it was shown that NO was solely responsible for MSC proliferation, human umbilical vein endothelial cell (HUVEC) migration in a scratch assay, and activation of the VEGF/VEGFR2 pathway in MSCs rather than the hydrogel structure itself.⁵⁹ When MSCs were co-transplanted with a CS-NO hydrogel into a hindlimb ischemia model, the gel ensured prolonged survival of the cells for up to 49 days, compared to cells implanted without support that died after less than 24 h.⁶⁰ Additionally, CS-NO treatment upregulated angiogenic genes, which is not surprising as in vitro results revealed increased expression of eNOS in cells cultured in the CS-NO environment.⁶⁰ When MSCs were co-transplanted into a myocardial infarction mouse model with a NO-hydrogel, there was improved VEGF secretion in vivo, increased vascular density, and upregulation of pro-angiogenic cytokines due to the NO release. There was also decreased collagen deposition after co-transplantation, but after comparing all treatment types, it was discovered that the hydrogel structure was responsible and appeared to have a depressive effect on fibroblast collagen production.⁵⁹ Embryonic stem cells (ESCs) displayed similar trends as MSCs when cultured on a CS-NO hydrogel; Flk-1, an early marker for endothelial cells, was significantly enhanced in cells exposed to NO. On the other hand, both Nanog and Oct-4, two of the most important transcription factors for remaining in an undifferentiated state, were down-regulated in a time-dependent manner after exposure, even in the presence of leukemia inhibitory factor (LIF), used to maintain pluripotency of ESCs (Figure 8A-D).⁵⁷ Further exploration of the differentiation pathway led to the discovery that endothelial differentiation of ESCs is due to an interdependent relationship between NO and Akt phosphorylation, as actively inhibiting one or both abolished endothelial commitment of the cells.⁵⁷

Figure 8.

(A, C) NO release from a chitosan hydrogel down-regulates the expression of pluripotent genes, where β-Gal corresponds to NO release, as the hydrogel contains a caged NONOate group that is cleaved by β-Gal, leading to consequent NO release. (B, D) Down-regulation of both genes occurred in a time-dependent manner over a 48-h period. (E) A 14-day in vitro NO gel culture revealed that ADSCs exhibit endothelial cell characteristics, whereas BMSCs act more like pericytes. Red arrows indicate a tube-like formation. (F) Angiogenesis genes were also modulated in each cell type following NO culturing. A-D were used with permission of Elsevier Science and Technology Journals, from [Nitric oxide releasing hydrogel promotes endothelial differentiation of mouse embryonic stem cells, Nie, Y., et al., 63, Copyright 2017]; permission conveyed through Copyright Clearance Center, Inc. E-F were taken with permission from Kang, M. L., et al. (2020). "Hydrogel cross-linking-programmed release of nitric oxide regulates source-dependent angiogenic behaviors of human mesenchymal stem cell." Sci Adv 6(9): eaay5413, Copyright (2020).

A recently completed study investigated if the proangiogenic actions of NO and stem cells were universal or if there was a source-dependent relationship. By comparing bone marrow-derived stem cells (BMSCs) and adipose-derived stem cells (ADSCs), it was discovered that although NO inhibition significantly decreased the angiogenic activities of both, NO exposure via a gelatin hydrogel had vastly different effects on cell-specific characteristics. ADSCs exhibited endothelial cell characteristics as demonstrated by higher gene expression for endothelial cell markers, higher VEGF release, enhanced vessel density in a gel plug assay, and two-dimensional conformation.⁶⁷ BMSCs, on the other hand, responded to NO treatments by featuring pericyte-like characteristics; pericytes are protective multi-functional cells that wrap around endothelial cells that line capillaries. Pericyte characteristics displayed by BMSCs included expression of pericyte cell markers, increased secretion of a signaling molecule that promotes recruitment of pericytes to endothelial cell networks, and tube stabilization rather than tube formation (Figure 8E-F). Co-culturing of the two cell types with HUVECs further confirmed their separate roles as ADSCs migrated to the surface of the gel to form a two-dimensional endothelial cell-like layer while BMSCs became localized in contact with HUVECs.⁶⁷ The unique responses to NO exposure provide insight into the importance of utilizing stem cells from different sources and how they can affect overall treatment.

4. Commercial NO Products

The commercialization of NO-releasing hydrogels has had some success in various health sectors. NO-containing products for skincare and supplement purposes are popular in the form of liquid and adhesive patches.^{107, 108} However, these products are neither FDA approved nor regulated. Moreover, the exact release mechanism and concentration of NO release are not verified. One commercial NO nasal spray, Enovid, clamis to reduce up to 99% of viral load against COVID-19 in a Phase III Clinical Trial.¹⁰⁹ This product is approved and registered as a Class I medical device in Singapore, Nepal, and European Union.

Moreover, several hydrogel-based NO delivery systems are undergoing clinical trials. One such gel under investigation is composed of berdazimer sodium that has been synthesized to carry a NONOates NO donor on its polysiloxane backbone and was developed by Novan Inc. (NC, USA). Recently, the gel has shown success in Phase III clinical trials for the treatment of mulluscum contagiosum, a skin condition, and FDA approval is expected sometime in 2024.^{55, 110} In addition, there is an NO gel being studied for promotion of hair follicle growth in men with androgenetic alopecia.¹¹¹ Further, preclinical trials are in motion for a patented NO-releasing gel for the treatment of burn wounds.¹¹² The clinical translation from current benchtop research of NO-releasing hydrogels is promising, as its therapeutic potential has been proven in an increasing amount of publications. Previously mentioned studies in this review direct a progressive impact on hydrogel-based drug delivery systems. In attempts toward commercialization, the future goal of hydrogel research should focus on improving the shelf stability and ease of clinical usage, as many NO-donors utilized in hydrogel designs are sensitive to temperature, light, and pH. More expansive products with varying kinetic release profiles would also be beneficial to cover different clinical applications. Breakthroughs on the abovementioned challenges will significantly progress the commercial development of NO-releasing hydrogel designs.

5. Conclusions

The utilization of NO-releasing hydrogels spans various research areas within the biomedical field, for a good reason. Relatively simple and highly innovative designs alike have shown successful implementation of NO hydrogels for treating cardiovascular disorders, owing to NO’s ability to promote endothelial cell growth and prevent smooth muscle cell proliferation, leading to reduced blood flow area and further complications. They have also enhanced vasodilation in human volunteers and angiogenesis in ischemic animal models. Furthermore, wound healing hydrogels that release NO have proven highly effective at preventing microbial infection, decreasing the inflammatory response, increasing the proliferation of fibroblasts and granulation tissue formation, and displaying quicker wound closure. Lastly, the differentiation potential of several types of stem cells has been investigated to get a better look into NO’s role in endothelial cell differentiation. The capacity of NO-releasing hydrogels to further advance treatment options for patients in all sectors of medical care lies in the fine-tuning possibilities of hydrogel systems, along with the numerous and potent biological functions of NO. Though the results presented are quite impressive, innovative and creative minds are already preparing the next generation of NO-releasing hydrogels that will use the stepping stones of previous research to build more effective biomedical therapies.