Date Published: April 23, 2018
Publisher: John Wiley and Sons Inc.
Author(s): Tao Yang, Alexander N. Zelikin, Rona Chandrawati.
Nitric oxide (NO) is a highly potent radical with a wide spectrum of physiological activities. Depending on the concentration, it can enhance endothelial cell proliferation in a growth factor‐free medium, mediate angiogenesis, accelerate wound healing, but may also lead to tumor progression or induce inflammation. Due to its multifaceted role, NO must be administered at a right dose and at the specific site. Many efforts have focused on developing NO‐releasing biomaterials; however, NO short half‐life in human tissues only allows this molecule to diffuse over short distances, and significant challenges remain before the full potential of NO can be realized. Here, an overview of platforms that are engineered to release NO via catalytic or noncatalytic approaches is presented, with a specific emphasis on progress reported in the past five years. A number of NO donors, natural enzymes, and enzyme mimics are highlighted, and recent promising developments of NO‐releasing scaffolds, particles, and films are presented. In particular, key parameters of NO delivery are discussed: 1) NO payload, 2) maximum NO flux, 3) NO release half‐life, 4) time required to reach maximum flux, and 5) duration of NO release. Advantages and drawbacks are reviewed, and possible further developments are suggested.
Nitric oxide (NO) is a highly potent two‐atom radical with a wide spectrum of physiological activities. In 1992, NO was crowned the “Molecule of the Year”1 and in 1998 Robert Furchgott, Louis Ignarro, and Ferid Murad shared the Nobel Prize in Physiology or Medicine for their significant discoveries on NO as a signaling molecule in the cardiovascular system. NO has been the focus of immense scientific and medical research, and is recognized as a versatile player in nearly every physiological system: cardiovascular,2 immune,3 central nervous system,4 and outflow physiology.5 In the body, NO at varied concentrations (nm–µm) is produced intracellularly by the enzymatic action of NO synthase (NOS) from amino acid l‐arginine. Several isoforms of NOS exist, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Healthy endothelial cells produce NO at a flux of 0.05–0.40 nmol min−1 cm−26, 7 (NO flux denotes the amount of NO flows in certain areas at a defined timeframe). NO can also be generated through non‐NOS pathways, i.e., via conversion of nitrite ions to NO. In the cardiovascular system, NO signals the surrounding smooth muscle to relax, leading to vasodilation (widening of blood vessels) and increasing blood flow.2 NO influences angiogenesis and vascular remodeling,8 transmits neural messages,9 and aids in the killing of various pathogens, i.e., bacteria and parasites.10 This radical affects an early step in the replication cycle of influenza viruses and NO has been shown to severely impair the replication of influenza A and B viruses.11 In the outflow physiology, NO has been reported to contribute to the physiological regulation of aqueous humor outflow and to lower intraocular pressure in various animal models and human patients.5, 12, 13, 14, 15
NO donors are pharmacologically active substances that carry NO and stabilize the radical until release is required. There are several classes of NO donors, depending on their chemical reactivity or the mechanism of NO release from the carrier. The release of NO from donor molecules can be triggered by factors such as light, heat, pH changes, or enzyme activity. On a different route, NO can be released via chemical reactions of the donors with acids, alkalis, metals, or thiols.30 Readers are referred to excellent reviews that cover detailed aspects of NO donors.31, 32, 33 Due to their distinct advantages, a number of NO donors to note include nitrates, diazeniumdiolates (NONOates), and S‐nitrosothiols (RSNOs). NONOate is one of the most investigated NO donors due to its capability to release two moles of NO per mole of donor at physiological conditions and its pH‐dependent decomposition property. Generally, NONOates can be synthesized through reacting secondary amines with gaseous NO under high pressure (usually 5 atm). Structures with cationic primary amines can electrostatically stabilize the anionic diazeniumdiolate groups, leading to a range of NO release half‐life from 2 s to 20 h.34 A number of compounds in this group include: spermine NONOate, diethylamine NONOate, diethylenetriamine NONOate, dipropylenetriamine NONOate, and proline NONOate. Unlike NONOates, laboratory generation of RSNOs require reactions between thiols and nitrosating agents, such as alkyl nitrite, dinitrogen trioxide, and nitrous acid. NO can be exhausted from RSNOs by multiple triggers (i.e., heat, light, and copper ions). Two relatively stable compounds in this group and the most commonly used for in vivo preclinical studies include S‐nitroso‐N‐acetylpenicillamine (SNAP) and S‐nitrosoglutathione (GSNO).31
To achieve functional NO‐releasing platforms, generally NO donors have to be available over the course of the envisaged therapeutic applications. NO donors must be stable enough at physiological conditions to reach the desired site, but are sufficiently labile under conditions unique to the target site. The clinical applications of low molecular weight NO donors have been restricted due to issues such as burst release and nontargeted delivery. Their encapsulation in carriers enabled controlled and sustained delivery of NO. A majority of studies on NO delivery from injectable materials to date have focused on their efficacy against bacteria and biofilms in vitro.
Polymer films are a great tool of delivery system as they can maintain a high local concentration of therapeutic agent at a specific site.81 Freestanding drug‐containing polymer films can be directly attached on the pathological site of tissues or they can be used as coatings deposited on medical devices such as implants or stents. Implantable medical devices are critical in restoring body functions, however their performance can be affected by undesirable blood clot formation and bacterial infection. Films or coatings containing NO with inherent antithrombotic and antibacterial properties offer a solution to these issues. The development of NO‐releasing polymer films with multiple antibacterial mechanisms was demonstrated by Pant et al. These films were developed through incorporation of NO donor SNAP in CarboSil polymer and sequential immobilization of antimicrobial molecules (benzophenone based quaternary ammonium (BPAM)) on top of the SNAP‐CarboSil films (Figure9a).82 Compared to pristine SNAP films, an increase in NO flux was observed for SNAP‐BPAM films over a 24 h period. This is not surprising since the presence of positively charged ammonium functional groups on BPAM topcoats increased the film hydrophilicity. Dual‐action SNAP‐BPAM films showed a 4‐log reduction in bacterial viability for S. aureus biofilms and a 3‐log reduction for P. aeruginosa biofilms, compared to control CarboSil films. On the other hand, single‐action BPAM films reduced the viability of S. aureus by 3 log units and single‐action SNAP films reduced the viability of P. aeruginosa by 2 log units. These data clearly implied that the combination of SNAP and BPAM is favorable in eradicating bacterial biofilms.
An approach that draws inspiration directly from nature is to generate, synthesize NO specifically where and when it is needed. Radical NO species are short‐lived. For this reason, controlled delivery of precise amounts of NO with spatiotemporal resolution is highly challenging. Even for the fine‐tuned systems where such control is exerted, reservoir‐type depots are disadvantages in terms of the deliverable payload by the finite pool of the NO donor. Nature offers a straightforward approach to deal with these problems—to perform localized synthesis of NO. Several isoforms of NOS enzymes are known, most notably nNOS, eNOS, and iNOS types.91 Enzymatic synthesis of NO is highly localized and can generate continuous and steady amounts of the product. It relies on endogenous, nutritional precursors such as l‐arginine and is therefore “limitless” in terms of the deliverable payload. Enzymatic output and the amount of NO generated is tightly controlled and can be up/downregulated, specifically through variation of concentration of the NOS cofactors (nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, and (6R‐)5,6,7,8‐tetrahydrobiopterin (BH4)).91 These attributes of a localized pool of NO are highly appealing and inspired engineering of synthetic implantable biomaterials with capacity to achieve localized synthesis of NO. Two strategies emerge from the knowledge of natural, enzymatic NO synthesis: 1) implementation of “enzyme‐prodrug therapy” (EPT) based on natural enzymes and custom‐made synthetic prodrugs of NO, and 2) design of enzyme mimics that produce NO using endogenous donors. These developments are quite recent, with the first reports dating back by only two decades and a considerable surge of interest observed in the last few years (judging by the number of publications on the subject).
Recent progress in the delivery of NO is highly inspiring. In this review, we highlighted NO release parameters (NO payload, maximum NO flux, NO release half‐life, time required to reach maximum flux, and duration of NO release), however at times it is still challenging to compare the parameters between studies due to irregular reporting. Thorough characterizations must remain at the forefront of NO efficacy studies. Importantly, the actual NO levels achieved within a biological system or native tissues should be addressed to facilitate a more rapid clinical translation of NO‐based therapeutics. This leads to an aspect that needs academic attention, i.e., quantification of NO at the desired site in vivo. Fluorescent dyes may assist in visualization of NO and provide a simple absolute quantification of cellular NO concentration in vitro132 but to our knowledge, are not available as yet for in vivo quantification of NO. Chang et al. recently reported a peptide‐based NO sensor that can resolve nanomolar concentrations of NO while providing information within biological systems (detecting NO in response to physiological levels of shear stress).133 This sensor may be introduced intravenously to assess NO levels in the circulation or at specific tissues. This example demonstrates the type of sensors that stand to make impact for the development of personalized NO therapies.
The authors declare no conflict of interest.