This is the accepted version of the article: Carné-Sánchez A., Carmona F.J., Kim C., Furukawa S.. Porous materials as carriers of gasotransmitters towards gas biology and therapeutic applications. Chemical Communications, (2020). 56. : 9750 - . 10.1039/d0cc03740k. Available at: https://dx.doi.org/10.1039/d0cc03740k Porous materials as carriers of gasotransmitters towards gas biology and therapeutic applications Arnau Carné-Sánchez,a,c Francisco J. Carmona,a Chiwon Kim,a,b Shuhei Furukawa,a,b* The discovery of NO, CO, and H2S as gasotransmitters and their beneficial role in multiple physiological functions opened an era of research devoted to exogenously deliver them as therapeutic agents. However, the gaseous nature of these molecules demands new forms of administration that enable to control the location, dosage and timing of their delivery. Porous materials are among the most suitable scaffolds to store, deliver and release gasotranmistters due to their high surface area, tunable composition and reactivity. This review highlights the strategies employed to load and release gasotransmitters from different kinds of porous materials, including zeolites, mesoporous silica, metal-organic frameworks and protein assemblies. of CO, its role was supposed to be as a merely noxious 1. Introduction toxin.2_ENREF_9 Further research demonstrated that H2S is involved in many intracellular signaling pathways with Signaling molecules behave as messengers between cells, which important functions in cytoprotection, anti-oxidation, trigger subsequent physiological and chemical transformations vasodilation and angiogenesis.9, 10 that are vital for the correct function of our organism. Among The beneficial effects of gasotransmitters in various those, gaseous signaling molecules, also termed as physiological functions open the possibility to deliver them gasotransmitters, represent a unique class of signaling exogenously as therapeutic agents. However, all three molecules because their gaseous nature allows them to diffuse gasotransmitters identified so far behave as a double-edged through cell membrane and to trigger a broad range of catalytic, sword. They possess acute toxicity at high concentration while metabolic processes within a short time after their release. To they present the therapeutic effects at low concentration. In date, three main gasotransmitters have been identified: nitric addition, their activity is also dictated by where they are oxide (NO), carbon monoxide (CO) and hydrogen sulphide located. For instance, high accumulation of CO or NO in the (H2S).1-3 Chronologically, NO was the first gaseous molecule lungs might result in oxygen replacement in heme and severe identified as a signaling molecule. In 1987 NO was identified as hypoxia. On the other hand, vasodilation is induced when these the endothelium-derived relaxing factor (EDRF) responsible for gases are released into blood vessels, which is an effective vein and artery dilation.4 Since then, the interest in the treatment against coronary diseases. Another example is biological relevance of NO was increased and its biological provided by the anti-inflammatory effect of CO when delivered synthesis from L-arginine by a family of enzymes named as NO in the organs or tissue, which postulates CO as a promising non- synthases (NOS) was discovered.5 Unlike NO, the biosynthesis steroidal ant-inflammatory drug.11 of CO from haem degradation catalysed by haem oxygenase In addition, their gaseous nature implies that challenges (HO) was known long before and for a long time CO was related with their handling, storage and appropriate delivery considered as a toxic by-product of the metabolism.6 The have to be overcome before considering the use of discovery of the biological function of NO triggered a renovated gasotransmitters as drugs. In this context, the use of scaffolds research effort focused on the biological role of CO. These able to immobilize bioactive gas molecules in solid state (or in efforts yielded the identification of CO as the second solution) and liberate them on demand is highly desirable. gasotransmitter with an important physiological impact in a Molecules that release bioactive gases gradually or upon variety of processes such as vasodilation, anti-inflammation, external stimuli have been extensively studied and excellent anti-proliferation and anti-apoptosis.7, 8 H2S was included to the family of gasotransmitters in 2002 and is the last molecule added to this kind of signaling molecules thus far. The presence of H2S in the organism was already described and, as in the case a. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida, Sakyo-ku, Kyoto, 606-8501, Japan b. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan c. Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and Barcelona Institute of Science and Technology, Campus UAB, Bellaterra 08193, Barcelona, Spain. † Footnotes relating to the title and/or authors should appear here. Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/x0xx00000x reviews are available.12-16 However, the fast diffusion of molecules once administrated limit the scope of this strategy as they may cause toxicity to untargeted healthy tissues either by the action of the liberated gasotransmitters or by the release of by-products after gas release. Thus, structuring of gasotransmitters into functional scaffolds in the solid state is a promising strategy to achieve higher payloads of the bioactive gas in targeted areas because they are easier to localize. To date, many kinds of solid-state materials are utilized to release bioactive gases such as inorganic and silica nanoparticles,_ENREF_18 liposomes_ENREF_18 and organic polymers.17-19 However, in most cases these materials lack from low payloads, which limits their releasing capacity. This is due to the fact that the surface of materials is only available for drug loading (through surface functionalization). A promising strategy to overcome this issue is to use porous materials as functional scaffolds. The high surface area that these materials possess enables to incorporate gasotransmitters not only on their material surfaces but also within the pores, which guarantees a higher drug loading. Thus, in this review the current advances on gasotransmitters loading, and release, in porous materials are surveyed including zeolites, mesoporous Figure 1: Scheme of the different strategies to prepare gasotransmitter-releasing silica, metal-organic frameworks and protein assemblies. materials based on porous frameworks. The existing combinations between strategy and porous framework are indicated. Nitric oxide molecule is used as a gas model. 2. Strategies to load gases into porous materials To incorporate gasotransmitters or gas-donor molecules into 3. Zeolites porous materials one can chose among several strategies. However, the loading methodology will influence not only the Zeolites are a family of crystalline porous aluminosilicates with loading efficiency but will also affect the rate of release and general formula Mn+x/n[(AlO2)x(SiO2)y]x-. With their well- stimuli that will trigger the release. We categorized the existing organized and regular system of channels and pores, zeolites methodologies to incorporate bioactive gases into porous have been applied for ion exchange, catalysts and molecular materials as follows (Figure 1); sieves.20 The non-toxic behavior of zeolites combined with their (1) Use of open metal sites (OMSs) as binding moieties. All molecular storage property based on microporosity provides an porous materials listed here can incorporate metal ions into opportunity to be used as drug carriers.21 The presence of OMSs scaffolds and expose an unsaturated coordination site on metal in zeolites, upon guest removal, is advantageous towards ions as an OMS. The three gasotransmitters, NO, CO and H2S, trapping and releasing gasotransmitters. Likewise, the inherent have a strong coordination capability to metal ions, and thus, OMSs together with the feasibility of generating catalytic sites can be anchored on the pore walls of materials containing in their frameworks (e.g. acidic sites) could enable the OMSs. production of gasotransmitters from bioavailable substrates. (2) Use of the catalytic properties of the OMSs to trigger Even though one can find reports on the adsorption of H 2S and chemical reactions that produce gasotramsmitters as products. CO in zeolites,22, 23 they are solely focused in their adsorption OMSs can not only be used as an anchoring site, but also as a for removal application and not in their release for therapeutic catalytic site. In this case the porous material behaves similarly applications. Therefore, in this section the loading and release to an enzyme able to transform a donner molecule of of NO will be exclusively discussed. gasotransmitters or known as a pro-drug into a gasotransmitter. 3.1 Coordination on open metal sites (3) Immobilization of donor molecules of gasotransmitters on the pores by post-synthetic modification. In this strategy, The inherent or post-synthetically generated OMSs in zeolites coordination or covalent bonds are often used to fix donor can act as anchoring points, where gasotransmitters can be molecules on the pore walls after forming porous materials. coordinated. This induces a slower and more controlled release (4) Physical encapsulation of donor molecules of than when gasotransmitters are merely physisorbed. gasotransmitters in porous materials. By taking an advantage of Morris and co-workers demonstrated and quantified the porous materials, pro-drugs can be incorporated into the pores. high affinity of NO towards zeolites by means of gas sorption However, due to the low host-guest interaction, the molecules experiments. In these experiments, the Co-exchanged zeolite A can be leaked out of pores. Several strategies are available to (Co-LTA) showed an uptake of 1.7 mmol g-1 at 800 mmHg. More limit the extent of the leakage and will be discussed in this interesting is the fact that 76 % of NO was not released when review. the pressure was reduced, which demonstrated the strong appearance of synergistic effects. For example, the burst a) b) antibacterial effect of the NO-loaded Zn-LTA against Gram- negative and Gram-positive bacteria was coupled with the long lasting antibacterial effect of the slowly leached Zn(II) ions from the zeolite. Likewise, its hybridization with macroscopic materials could not only modulate the properties of the resulting materials, but also favor its development toward real applications. In this regard, Joshi et al., developed an ointment containing a 33 % of NO-loaded Zn-LTA. 27 The hybrid system showed a slower NO release than the pristine NO-zeolite, when c) suspended in phosphate buffered saline (PBS). The higher hydrophobic character of the ointment hindered the NO- exchange with water molecules at the OMSs. This more sustained NO-release resulted in a significant microbicidal activity, and during longer times, against common wound pathogens. Likewise, the topical application of the hybrid ointment demonstrated to enhance the healing of cutaneous wounds in rats (Figure 2c). These examples bring to light the feasibility of modulating the release of bioactive agents from zeolites (NO, Zn(II) ions), which determines the biological Figure 2: NO-releasing systems prepared by the chemisorption of NO to open response. They show the potential of NO-zeolites systems to be metal sites in zeolites and their beneficial effects: a) Structure of cobalt−NO incorporated in wound dressing towards the treatment of complex in zeolite-A as elucidated by single-crystal X-ray diffraction. Reprinted infected wounds. with permission from Ref. 24. Copyright 2006 American Chemical Society. b) SEM images of electrospun polylactic acid fibers containing a 13 % of cobalt-Zeolite A. Beside the naturally occurring LTA-type of zeolites, The hybridization of the zeolite into a macroscale material allows to prepare titanosilicates have also shown their potential in storing and humidity-triggered NO-releasing bandages with gradual gas release. Reprinted releasing NO. These materials possess stoichiometric amounts with permission from Ref. 25. Copyright 2009 American Chemical Society. c) of OMSs in their structures, which, largely affect the adsorption enhanced wound-healing due to the topical application of an ointment containing and release of NO. Rocha and co-workers focused on the OMSs a 33 % of NO-loaded Zn-zeolite-A in comparison with the application of the pristine ointment Reprinted with permission from Ref. 27. Copyright 2014 Microbiology of titanosilicates, ETS-4 ([Na9Si12Ti5O38(OH)·xH2O]), which are Society. based on pentacoordinated Ti(IV).28 In order to systematically study the impact of the OMSs on the performance of affinity of NO towards the OMSs present in the structure (Figure titanosilicates, ETS-4 was compared with ETS-10 2a).24 However, the release of NO could be triggered by water [(Na,K)2Si5TiO13·xH2O]), which possesses only fully coordinated molecules, which have stronger affinity towards open metal (hexacoordinated) Ti(IV). As expected, the uptake of ETS-4 sites than NO. Indeed, 1.0 mmol·g–1 of NO was released from largely outperformed ETS-10 (11 wt% and 5.5%, respectively). NO-loaded Co-LTA by flowing wet gas (11% of relative The further partial replacement of extraframeworks Na(I) humidity). In addition, the releasing kinetic could be tuned by cations by Cu(II) increased slightly the NO loading. However, the varying the relative humidity of the wet gas. This degree of post-functionalization led to an increase in the toxicity of the control of the NO delivery from the zeolite was exploited to material, probably because of the leaching of the probe the biological effect of exogenously delivered NO in cells extraframework copper cations.29 Accordingly, they explored by testing its inhibition in platelet aggregation. A key step the effects of the isomorphic substitution of framework silicium towards the use of NO releasing materials in real world groups in ETS-10, by the less toxic Al(III) and Ga(III) cations.30 The applications is their integration into shaped, easy-handling exchanged materials offered higher NO payloads than the macroscopic materials. In this context, Balkus et al hybridized unexchanged ETS-10, due to the chemisorption of the gas to the NO-loaded Co-LTA crystals with polymeric fibers of polylactic exchanged cations, being also higher than the previous acid (PLA) with bandage shape (Figure 2b).25 Interestingly, the reported Cu(II)-exchanged ETS-4. Furthermore, the Al- and Ga- hybridization induced a more prolonged release by preventing exchanged materials exhibited a significant lower toxicity than water to come into contact with the dispersed crystals. Such the Cu-ETS-4 analogs and no metal leaching was observed when hybrid nature offers the opportunity to attain additional control incubated in cell culture medium.31 Although the properties of over the NO releasing rate. titanosilicates as NO-storing solids are promising, the Zeolites can be easily functionalized by exchanging with assessment of their ability to regulate biological functions is transition metal ions in their structure. Thus, in addition to Co- essential toward their evolution to real therapeutic LTA, other transition metal ion-exchanged LTA were evaluated applications. With this aim in mind, Pinto and co-workers as NO delivery vehicles, such as pristine LTA, nickel,_ENREF_25 studied the biocompatibility, chemical stability and NO-release copper, manganese24 or_ENREF_27 zinc26._ENREF_29 This in biological medium of the above-mentioned materials. They tunability in the zeolite composition has an impact on the selected unexchanged ETS-4 matrix as the most promising maximum loading capacity, release kinetics and also the material in terms of amount and sustained NO-release, toxicity and stability. NO-loaded ETS-4 proved to elicit biological responses due to NO-release by reversibly inhibiting mitochondrial oxygen consumption in HeLa cells and promote cell migration, highlighting its potential application in wound healing.31 3.2 Catalysis As explained in the previous section, great advances have been carried out on the develop of zeolites for storing and releasing NO towards therapeutic applications. However, this strategy shows two main drawbacks: i) the stored gas is finite and only suitable for short-term applications and ii) the materials usually need to be conserved under special conditions (e.g. away from moisture). One alternative is the chemical production of the gas from a bioavailable substrate. It is known that nitrite reductase enzymes in bacteria transform NO2- to NO mediated by Cu(I) metal sites.32 Based on Figure 3: Scheme of the generation of NO from nitrites mediated by Cu(I) ions and this, Morris and co-workers prepared Cu(II)-Zeolite-X and Cu(II)- acidic sites in zeolites. Bioavailable compounds like cysteine (RS-H) are able to ZSM-5 by extraframework cation exchange. 33 The thermal regenerate the Cu(I) sites, allowing a continue release after the addition of activation of the materials led to generation of Cu(I) sites by substrate (e.g. NaNO2). In contrast, the acidic groups in zeolites are not self-reduction reaction.34 Although the materials generated a regenerated. Therefore, the repeatedly addition of substrate does not result in a significant amount of NO when suspended in nitrite aqueous continue release of NO when the active sites are consumed. Adapted from Ref. 37 with permission from The Royal Society of Chemistry. solution, the NO production lasted only for few hours. The concurrent action of both water and dioxygen molecules led to addition of substrate was inhibited. Conversely, the Cu(I) sites the re-oxidation of the Cu(I) centers to Cu(II), inhibiting the were regenerated by the oxidation of the cysteine present in further transformation of nitrites to NO. More interesting is the the media, allowing the continuous conversion of nitrite (Figure fact that the pre-adsorption of NO resulted in a constant 3). Among the different Cu-zeolites, Cu-SSZ-13 shows the best production of NO during 10 h. The exposition of the materials biocompatibility, making it a promising zeolite to use in the to gaseous nitric oxide results in the chemisorption of NO to the body. Cu(I) centers. In the presence of water, only a small fraction of the loaded NO is released, while the significant amount of NO is 4. Mesoporous silica maintained in the material. The irreversibility of chemisorbed NO hindered the interaction between both water and dioxygen Mesoporous silica are porous inorganic solids formed by with the Cu(I) sites and inhibited their re-oxidation, and amorphous silicon dioxide containing periodic arrays of therefore, favoring the sustained conversion of nitrite to nitric channels and cavities. They have been widely used as drug oxide. This demonstrated the cooperative effect of NO- delivery platforms due to their 2 to 50 nm pore size and adsorbed on the NO-production process. biocompatibility.38, 39 Compared to zeolites (described in The acidic transformation of nitrites is an alternative route Section 3) that store gasotransmitters within their small to produce NO. Morris and co-workers studied the effect of the cavities, mesoporosity offers new strategies to accommodate pore size of acidic zeolites on the production of NO from nitrites gasotransmitters. For example, one can encapsulate gas-donor by testing four different zeolites: Al-UTL, Al-IPC-2, Al-IPC-4 and molecules, much bigger than the gas itself, which can trigger the Al-IPC-6. To this end, they employed the ADOR (Assembly- release of gasotransmitters by different stimuli such as light, Disassembly-Organization-Reassembly) method,35 which allows heat or ultrasounds. This strategy can be enriched by the fact to selectively control the pore size of the zeolite by altering the that the pore surface can be covalently modified by silanol linkers between silicate layers. They observed that the higher chemistry.40 In the following sub-sections, representative works pore size, the larger amount of NO generated and the faster of these two strategies are summarized. kinetics, demonstrating the feasibility of tuning the NO- generation according to the targeted application.36 In another 4.1 Encapsulation of gas-donor molecules work, Morris et al. prepared several acidic and copper- The encapsulation of molecules into the pore of mesoporous exchanged zeolites based on mordenite, ferrierite, ZSM-5 and silica is basically driven by physisorption; the physical SSZ-13.37 Although all materials were able to produce NO from interaction between guest molecules and the pore surface of nitrite sources, the copper-loaded materials showed a higher host materials. Consequently, encapsulated guest molecules activity. In addition, given the known presence of cysteine in the can escape from the pores by diffusion due to the lack of strong body, the ability to generate NO of the materials in mixture of chemical bonding. Therefore, the extent of leaching is the most nitrite/cysteine at biologically relevant concentration was important issue in this strategy. assessed. As the acidic sites were consumed during NO- To prevent the escape of guest molecules, Rickus and co- production, the continuous NO-generation after subsequent workers used liposome vesicles as capsule for NO donor molecules.41 Liposomes, phospholipid bilayer vesicles, are also Figure 4: Gas-releasing frameworks prepared by the encapsulation of gas donors into the cavities of mesoporous silica. There exist examples of gas-donor@Mesoporous silica systems for all gasotransmitters (e.g. CO, NO and H 2S), demonstrating the high versatility of mesoporous silica to prepare gas-releasing materials by means of gas-donor encapsulation strategy. a) Representation of Al-MCM-41 mesoporous silica loading the cationic [Mn(PaPy3)(NO)]+ complex. The irradiation with visible light elicits the NO- release response, exerting an antibiotic effect against a skin pathogen (e.g. A. baumannii). Reprinted with permission from Ref. 47. Copyright 2012 American Chemical Society. b) Encapsulation of the cationic photoCORM [Mn(tacn)(CO)3]+ into mesoporous silicas functionalized with anionic sulfonic groups. The photoresponsive properties of the pristine CORM are maintained and the hybrid material release CO under photoactivation with visible light. Adapted with permission from Ref. 50. Copyright 2016 American Chemical Society. c) Schematic representation of the encapsulation of Diallyl trisulfide (DATS) in a mesoporous silica. The interaction of DATS with the bioavailable glutatathione results in the release of H2S. The both slower diffusion of glutathione inside the pores and DATS outside the pores enable a more controlled release of H 2S. Reproduced from Ref. 55 with permission from The Royal Society of Chemistry. widely used as drug nanocarriers because they prevent the leaching of the entrapped molecule.42, 43 S-nitroso-N- MCM-41 in saline solution after 24 h incubation (2.92% and acetylpenicillamine (SNAP), which is known to be a light- 3.44% of adsorbed Mn-NO were leached from Al-MCM-41 and induced NO donor molecule was encapsulated into liposomes, Si-MCM-41, respectively). The mechanism of Mn-NO loading followed by doping them into the synthetic solution of the into Al-MCM-41 was supposed to entail cation exchange mesoporous silica. The sol-gel reaction of this mixture between [Mn(PaPy3)(NO)]+ and Na(I) ions inside the host and containing SNAP, liposomes and tetraethyl orthosilicate (TEOS) passive diffusion. On the other hand, passive diffusion was the provided the liposome/mesoporous silica composite materials. only driving force for Mn-NO loading into Si-MCM-41. Therefore, the NO release from the functional substrate was Congruent with the higher loading capacity of Al-MCM-41, its measured by implementing a self-referencing amperometric releasing amount of NO by light irradiation was also higher than NO microsensor. It was found that the evolution of NO from the Si-MCM-41 (0.43 mmol g-1 for Al-MCM-41 and 0.30 mmol g-1 for mesoporous silica surface was dependent on light irradiation Si-MCM-41). The control on NO release from Al-MCM-41 and was within the range of biological relevance (14 pmol·cm – allowed studying its antibiotic effect on A. baumannii, a skin and 2·sec–1).44 In addition, the mesoporous silica composite could be soft-tissue infections (SSTI) model showing effective clearance used as a functional growth surface for mammalian cells.45, 46 upon light-triggered NO release (Figure 4a). Mascharak and co-workers attempted to limit the leaching Zhao and co-workers took an advantage of the porosity of of NO donor molecules by using host-guest electrostatic mesoporous silica to accommodate a photolytic NO-donor interactions.47 Thus, they compared the uptake amount of the ([NH4][Fe4S3(NO)7], Roussin’s Black Salt) in a core-shell positively charged metal-nitrosyl NO donor complex upconversion nanoparticle@mesoporous silica nanocomposite. ([Mn(PaPy3)(NO)]ClO4, denote as Mn-NO) into a negatively 48 The irradiation of the nanoparticles with low-damage near- charged aluminosilicate mesoporous material (denote as Al- infrared irradiation (λ = 980 nm, within of the therapeutic MCM-41), and neutral mesoporous silica material (Si-MCM-41). window) activated the NO-release, being only possible for the Al-MCM-41 showed a 20% higher uptake when compared to Si- bare NO-donor under higher energetic irradiation. By MCM-41 (Al-MCM-41: 2.39 wt.%, Si-MCM-41: 2.04 wt.%,). In modulating the laser output power, the amount of gas addition, Al-MCM-41 showed less leaching of Mn-NO than Si- generated was controlled, resulting in a dose-controlled NO- release systems. This feature was later demonstrated by in vitro to their inherent luminescence. The good encapsulating cell studies, where different biological responses with NO-dose features of the anionic Al-MCM-41, its stability in physiological dependence were achieved by simply tuning the power media and its large pores were exploited by Barea and co- irradiation of the NIR light used as a trigger. workers to encapsulate both photoCORM [Mn(tacn)(CO)3]+ and Mesoporous silica can also be employed as vehicles to cisplatin within the same carrier, suggesting the possibility of transport guest drugs to the targeted tissue. In addition, the producing CO releasing solid platforms with multiple extent of the leaching of the encapsulated NO-prodrug could therapeutic effects.53 Diallyl trisulfide (DATS) is known as enable a more sustained NO-release. This strategy was glutathione (GSH)-triggered H2S donor, however, its fast employed by Deng et al. toward the efficient treatment of releasing kinetics hinders its therapeutic use as a rapid increase Primary Open-Angle Glaucoma (POAG).49 Nitric oxide has of H2S concentration may cause adverse effects in the proved to reduce elevated intraocular pressure (IOP), a strong risk factor for developing POAG. Likewise, sodium nitroprusside metabolism.54 Wang and co-workers encapsulated DATS inside (SNP), a N-donor, has been proposed as potential therapeutic mesoporous silica nanoparticles in order to control the release agent against this disease. However, its low uptake through the kinetics of H2S (Figure 4c).55 DATS was loaded into mesoporous cornea together with its poor stability limit its practical use. The silica nanoparticles (MSNs) by stirring mixture of DATS, MSNs encapsulation of SNP inside mesoporous silica resulted in a and distilled water for 12 h. Surprisingly, 99% of DATS molecules slower release of the N-donor in PBS, extended during 50 hours. were encapsulated into MSNs (which accounts for a loading of In vivo studies on Cav1 gene knockout mice demonstrated that 2.5 mmolg-1). This very high loading indicates that there is a SNP@mesoporous silica nanoparticles were able to go through strong interaction between DATS and Si-OH of MSNs surface. cornea and reach the target tissues. In addition, the topical eye The low leaching (less than 10%) observed upon 24h incubation drop application of SNP@mesoporous silica (0.004 mg of SNP) in PBS further supported this hypothesis. The H 2S releasing resulted in IOP reduction of 22 % after 3.5 h, followed by a experiments performed in the presence of GSH showed slower sustained IOP reduction during 48 h. On the contrary, the dose of the same amount of SNP solution did not affect IOP, release kinetics from the composite nanoparticles in demonstrating the beneficial effect of NO-donor encapsulation. comparison with free DATS. The biomedical applications of the CO-prodrugs, known as CO-releasing molecules (CORMs), DATS-MSN nanoparticles were demonstrated by injecting them have also been incorporated into the cavities of mesoporous into mouse’s veins. The nanoparticles effectively released H2S silica to generate CO-releasing materials. For instance, Barea into vein without causing any of the adverse effects caused by and co-workers functionalized the pore surface of MCM-41 and high concentration of H2S (such as an increase in blood pressure SBA-15 mesoporous silica with alkanesulfonic groups to confer and heart beat rate). Later, the same group demonstrated that an anionic character to the frameworks. By cation exchange, the hybrid system DATS@MSNs promoted the proliferation and they successfully encapsulated the cationic photoactive CO differentiation of endothelial cells in vitro and alleviated releasing molecule (photoCORM) [Mn(tacn)(CO) 3]+ (where tacn inflammatory response caused by hypoxia–reoxygenation. is 1,4,7-triazacyclononane), which could not be encapsulated Likewise, in vivo studies in mouse models confirming these into the neutral matrices (Figure 4b). Both hybrid materials kept benefits, showing that DATS@MSN was able to protect the the photoactivity of the pristine CORM, but offering a more endothelium of aortic allografts from ischemia–reperfusion sustained CO-delivery kinetics in physiological media when injurie.56 irradiated with white light. Unfortunately, the host-guest 4.2 Chemical attachment of gas donors interactions were not strong enough and led to partial leaching of the cationic CORM when incubated in saline buffer. 50 The diversity of chemical functionalities that one can use in the As it was done with the NO-donor ([Mn(PaPy3)(NO)]ClO4, sol-gel process (amine, hydroxyl and carboxylate among others) Mascharak et al. also used the cation exchange strategy to enables to functionalize mesoporous silica through post- incorporate a cationic photoCORM into the negatively charged synthetic reactions. One can use this chemistry to covalently Al-MCM-41 nanoparticles (approx. 60 nm).51 For this, the attach donor molecules of gasotransmitter on the surface of manganese carbonyl complex (fac-[Mn(pqa)(CO)3]+, pqa = (2- these materials. This approach has the disadvantage that pyridylmethyl)(2-quinolylmethyl)amine), denote as Mn-CO, was generally requires multi step chemical reaction. On the other exchanged with the Na(I) ions presenting in the initial Al-MCM- hand, it reduces the extent of leaching due to the covalent 41 structure. In this case, the host-guest electrostatic interaction between the donor and the surface of the interactions ensured a small percentage of leaching when the mesoporous silica. composite was incubated in PBS (i. e. 2% and 12% of Mn(I) Surface modification of mesoporous silica with coordination leaching after incubating for 24h and 60h, respectively). The complexes has been researched by a number of groups in the released CO from the [Mn-CO]@Al-MCM-41 nanoparticles late 1990s and early 2000s.57, 58 Since then, surface modified upon light irradiation was used to induce vasorelaxation of rat fumed silica, silica gel and silica nanoparticles have been aorta muscle rings. A similar strategy was employed to extensively developed and applied to the field of gas biology.59- 61 Longhinotti et al. immobilized cis-[Ru(bpy) (L)NO]3+ and incorporate the luminescent photoCORM [Re(CO)3(pbt) (PPh3)], 2 pbt = 2-(2-pyridyl)-benzothiazole) into the pores of Al-MCM- [Fe(CN)4(L)NO]– complexes on surface functionalized 41.52 In this case, not only the intracellular delivery of mesoporous silica.62 The procedure entailed four sequential biologically relevant amounts of CO was demonstrated, but also surface functionalization reactions, including amine the nanoparticles could be easily tracked inside the cells, thanks functionalized mesoporous silica (Si-APTS), amine terminal converted to isonicotinamide (ISN) and Ru(II) and Fe(II) metal means of the Förster resonance energy transfer (FRET) process. complexes immobilization on the surface of Si-APTS-ISN by More interestingly, the visible light irradiation triggered the NO- reacting the functionalized silica with cis-[Ru(bpy)2Cl2] and release, with the concomitant fluorescence restoration of the Na2[Fe(CN)4(dmso)2] respectively. Finally, metal-nitrosyl carbon dots. Therefore, the nanocomposites showed a dual- functionalities were introduced by bubbling of NO gas to the function, because they not only enabled a controlled NO- prepared silica materials in acetone. Light-induced release of release under light irradiation, but also counted with a NO in the PBS from both of the prepared materials was fluorescence reporter, allowing the tracking of the gas delivery estimated to be 1.8 mmol g-1. process by fluorescence spectroscopy. An alternative moiety being able to store and deliver NO are S-nitrosothiols (RSNO), a kind of heat-sensitive NO-donor, the amine groups, which can store NO in the form of nitrite and has been also incorporated in mesoporous silica by surface release it under acidic conditions. To immobilize the amine functionalization. The feasibility of functionalizing mesoporous functionality on the surface of mesoporous silica, γ- silica with thiol-terminated organosilanes was used by Malone- aminopropyltriethoxysilane (APTES) was attached on the Povolny and Schoenfisch for, subsequently, generating the S- surface of MCM-41 and mesoporous MFI zeolite,63 which was nitrosothiol sites.68 The same procedure was performed on non- synthesized from MCM-41 by dry-gel conversion.64 Before the porous silica nanoparticles to assess the influence of porosity on modification of APTES, NO was loaded on MCM-41 and MFI the NO-release kinetics. While the non-porous materials zeolite. which released 0.77 and 0.67 µmol·g–1 of NO in the showed a half-life (t1/2) of NO release of 2.92 h in PBS at 37 ºC, acidic aqueous solution, respectively. In contrast, APTES mesoporous silica particles exhibited a much more sustained modified MCM-41 and MFI zeolite showed a release of 27.43 release (t1/2 of 26.6 h). These differences were explained by the and 17.99 µmol·g–1, respectively. The higher release observed “cage effect” shown by the porous material. The confined in the case of MCM-41 is attributed to its higher surface area, microenvironment in the nanometer-scale pores favored the which implies higher degree of surface functionalization. This recombination of the thiyl and NO radical pair after the high releasing amount in acidic conditions postulates these homolytic cleavage of the S-N bond, extending the duration of functionalized materials as efficient NO delivery vehicles for the NO delivery. They also dispersed the mesoporous nanoparticles gastrointestinal organs. The functionalization of the porous in several polyurethane membranes towards their potential framework of mesoporous silica with aminosilanes was also applications as coating of medical devices. Note that the employed by Schoenfisch et al. to generate novel NO-releasing nanocomposites exhibited a much longer prolonged NO-release materials.65 They prepared mesoporous silica with different (until 30 days), with negligible particle leaching after suspension particle size (ranging from 30 to 1100 nm) and functionalized in PBS for 21 days. covalently the pores with terminal secondary amines. By reacting with NO, they were transformed in N- diazeniumdiolates able to release NO in physiological media. The materials showed different NO-payloads and release kinetics in PBS. However, these differences could not be correlated with the distinct particle size of the carriers. A further analysis by powder small-angle X-ray scattering (SAXS) suggested that the different pore structure found in the functionalized systems determined these features. While more ordered pore system enhanced the diffusion of water through the cavities, resulting in faster NO-release kinetics, more heterogenous pore structures hindered the solvent diffusion and resulted in more sustained NO-release profiles. In another work,66 the amine groups in the outer surface of a NH2- functionalized mesoporous silica were used to covalently attach erythrosine, a singlet oxygen photosensitizer (PS), while the internal surfaces were used to conjugate a nitro aniline- derivative as a photoactive NO-donor. The antitumoral drug doxorubicin (Dox) was further encapsulated into the porous matrix, generating a multidrug carrier. In vitro studies on Figure 5: Core-shell Fe3O4@Mesoporous Silica nanocomposite with melanoma A375 cell line showed that, under light irradiation, photoresponsive antitumoral properties: a) Scheme of the generation of S- the triple-drug nanomaterial exhibited a higher cytotoxicity nitrothiols and S-transferring in the mesoporous silica shell and of the photo- thermal responsive release of nitric oxide under near-infrared irradiation, b) and c) than the dual system Dox-PS@mesoporous silica, suggesting an Antitumoral mechanism of the nanocomposite. The presence of transferring increased activity due to the nitric oxide release. A similar increases the specificity of the system toward tumoral-cells while the NIR strategy was used to confer NO-releasing functionalities to a irradiation activates the NO-release. Likewise, the NO-released exerts a signaling fluorescent carbon dot@mesoporous silica nanocomposites.67 function, favoring the accumulation of doxorubicin in the cells, resulting in a high The conjugation of a nitro aniline-derivative photo NO-donor to antitumoral activity against Multidrug Resistant cancer. Reprinted with permission from Ref. 69. Copyright 2017 John Wiley & Sons, Inc. the pore surface quenched the emission from carbon dots by In another work, the terminate-thiol groups where used for Morris and co-workers were pioneers in exploiting the both generating S-nitrosothiols and conjugating S-transferring OMSs in MOFs to incorporate NO. 75 In the first study, (a cancer-cell targeting group) on a core-shell [Cu3(btc)2]n (btc = 1,3,5-benzentricarboxylic acid, also known as Fe3O4@mesoporous silica nanocomposite (Figure 5a). The HKUST-1), which has Cu(II) OMSs, was used. The first evidence antitumoral drug doxorubicin was encapsulated into the of Cu-NO coordination was provided by the NO sorption cavities of mesoporous shell, generating a multidrug carrier.69 isotherms. These isotherms were characterized by a hysteresis The nanocomposite was able to convert near-infrared in the desorption branch. The number of NO molecules that irradiation into heat, enabling to trigger the NO-release by were not desorbed corresponded to the predicted number of photoactivation. Remarkably, the concomitant release of OMSs in the HKUST-1 structure (i. e. 2.21 mmol·g–1). Further doxorubicin and NO, triggered by NIR photoactivation for 5 min, evidences were provided by infrared spectroscopy performed showed a synergistic effect toward the reduction of the cell- in the NO-loaded HKUST-1, which showed the characteristic viability of multidrug resistant breast cancer cell-line (MDR- peak at 1887 cm–1 corresponding to the stretching vibration MCF-7) (Figures 5b and 5c). In vivo studies on MCF-7/ADR mode of NO coordinating to Cu(II) ions . The release of NO was tumor-bearing BALB/c nude mice confirmed these results. The triggered by treating the sample with water vapor, similar to the treatment with the multifunctional nanocomposite and NIR case of zeolites described in Section 3. However, a moderate showed a significant tumor inhibition, superior than the parallel release of 2 µmol·g–1 corresponding to 0.1% of the initially treatments with the pristine drugs. adsorbed NO was found. The low NO release is rationalized by the strong coordination of the Cu-NO bond. Despite the low 5. Metal-organic frameworks releasing efficiency, the release amount was enough to inhibit platelet aggregation confirming the potential of MOFs in the Metal-organic frameworks (MOFs), also called as porous field of gasotransmitters release. coordination polymers (PCPs), which are constructed via the Suitable coordination strength between metal and NO is a assembly of metal ions with organic linkers, are receiving key factor to obtain a material that performs well over the considerable attentions due to their structural characteristics, whole adsorption-storage-delivery cycle. In order to study the highly ordered microporosity, large surface area, and the wide influence of the transition metal ions on the NO release, the range of potential applications in gas storage, gas separation, same research group employed isostructural MOFs containing catalysis and sensors.70-72 Recently, by taking advantages of different metal ions in the adsorption/release of NO. Namely, MOFs’ porosity, researchers have used these materials in they used [Ni2(dobdc)]n (denote as CPO-27-Ni, dobdc = 2,5- biomedical applications, in particular to store and release drug dioxido-1,4-benzenedicarboxylate) and [Co2(dobdc)]n (denote molecules.73 Compared to other porous materials introduced as CPO-27-Co) for NO storage and delivery (Figure 6a).76 CPO- above, the most characteristic feature of MOFs is their inherent 27-Ni and CPO-27-Co showed the total NO uptake of 7 mmol·g– inorganic-organic hybrid nature, which enables to design and 1 and 6 mmol·g–1, respectively, at 25 °C and 6 mmol·g–1 and 5 synthesize a virtually unlimited number of structures by mmol·g–1, respectively, of hysteresis in the desorption branch, appropriate combination of metal ions and organic linkers. which correspond to the number of OMSs in each structure. Furthermore, the inherent hybrid nature of MOFs provides an These results indicate that most of NO was adsorbed through opportunity to expand the possible strategies towards coordination to the OMSs and approximately 1 mmol·g–1 of NO gasotransmitter delivery either by taking advantage of the was physically adsorbed into frameworks. Crystal structures of inorganic part (i. e. OMSs, catalysis) or the organic part (i. e. both frameworks77 clearly show that NO is coordinated to metal post-synthetic modifications, prodrug ligands) or both parts site with a bent geometry, which corresponds to NO acting as a acting synergistically. Their inherent porosity can be also used one-electron donor. NO releasing from CPO-27-Ni and CPO-27- to host gas-donor molecules. Thus, the release of Co was triggered by wet gas (11% relative humidity). Unlike the gasotransmitters from MOFs has been achieved by four HKUST-1, these two MOFs released practically the same characteristic strategies; (1) the release of coordinating gas amount of NO as previously adsorbed. It is noteworthy that a molecules at open metal sites by exchanging with water NO releasing capacity of 7mmol g-1 is the highest reported value molecules, (2) the catalytic decomposition of gas precursors at for porous materials thus far. In order to test the biomedical open metal sites, (3) encapsulation of gas-donor molecules applications of these materials, the vasodilation of pre- inside their cavities and (4) incorporation of organic ligands contracted pig coronary arteries induced by NO-loaded CPO-27- within the MOF structure with the ability to adsorb and release Ni was tested. The results showed a rapid relaxation of pig gasotransmitters, or being themselves gasotransmitter donor coronary arteries immediately after exposing it to an aqueous moieties that can be activated upon external stimuli. suspension containing pellets of CPO-27-Ni. 5.1 Coordination on open metal sites The possibility to synthesize CPO-27 analogue structures with different divalent metal ions enabled to systematically The presence of OMSs decorating the pores of MOFs have study the effect of the nature of the OMSs on the adsorption shown great potential for selectively adsorb molecules with and release of NO.78 Long and co-workers reported the NO lone pair electron and, as in the case of zeolites, they can be loading and releasing properties of CPO-27-Fe (based on used to store and release gasotransmitters.74 Fe(II)).79 Interestingly, while the loading capacity was similar to the examples shown above (i.e. 6 mmol g-1), the releasing profile was different, showing an initial burst release followed promising strategy to make these materials more by a slow and sustained release of NO that occurs over the biocompatible. The MIL-88 series of MOFs composed by iron(III) course of 10 days. These results further prove the key role of carboxylates combine a non-toxic behaviour and the presence the OMSs in the loading and releasing profiles of NO. of OMSs, which can be used to capture NO. 82 Therefore, they A unique feature of MOFs is that they combine OMSs with are suitable candidates as bio-friendly NO delivery vehicles. tunable microporosity. Therefore, one can take an advantage of Serre and co-workers systematically studied the NO these properties to design multifunctional carriers, delivering loading/release properties of these materials by synthesizing more than one substance. Barea and co-workers explored this them with different functionalities ([Fe3OX(−O2C−R−CO2−)3]n, R strategy using CPO-27-Ni as the host structure.80 First, the = terephtalic acid, fumaric acid, 2-nitroterephtalic acid and metallodrug with antitumor activity RAPTA-C ([Ru(p- dobdc, ; X = F, OH, Cl or −O2C−CH3).83 The uptake in all cases fell cymene)Cl2(pta)], pta = 1,3,5-triaza-7-phosphaadamantane) between 1 and 2.5 mmol·g–1. However, the release amount was was introduce into the pores of the framework by limited in the range of 0.1 mmol·g–1 due to the narrow pores in physisorption. Subsequently, NO was loaded into the composite the structure and the strong binding of Fe-NO. In order to material. Interestingly, the NO loading capacity (6 mmol·g–1) improve the NO releasing capacity of the Fe(III) based remained almost unaltered after the incorporation of RAPTA-c, frameworks, the performance of Fe-MIL-100 which indicates that the coordination of NO at the OMSs of the (Fe3OX[btc]2.xH2O), which contains big mesoporous cavities framework was not influenced by incorporated RAPTA-C within its structure, was evaluated. As expected, the releasing molecules. Importantly, the release of both bioactive molecules efficiency was improved when compared to the MIL-88 was very similar as in the case in which only one of the structure releasing up to 15 % of the initially chemisorbed NO molecules was encapsulated, indicating the potential use of (0.35 mmol g-1) over the course of 7h.84 In this regard, the MOF MOFs as multi drug delivery vehicles. bioMIL-3 [Ca2(azbz-TC)(H2O)(DMF), azbc-TC= 3,3’,5,5’- One of the key aspects when considering MOFs for azobenzenetetracarboxylate], counting with OMSs, was also biomedical applications is their toxicity both in vivo and in assessed as a NO-releasing materials due to the high vitro.81 Therefore, the synthesis of MOFs with gasotransmitter biocompatibility of calcium cations.85 It showed a NO payload of releasing properties with non-toxic building blocks is a 0.8 mmol·g–1 and a sustained release during 10 hours under wet nitrogen gas. Recently, Serre and co-workers have proved the feasibility of coordinating NO to the OMSs of a titanium-based MOF, namely Ti12O15(mdip)3(OH)6(H2O)6 (mdip: 3,3′,5,5′- tetracarboxydiphenylmethane) by the in situ formation of complexed nitrites (Figure 6 b).86_ENREF_84 After a thermal treatment, bridged Ti-OH-Ti groups were generated in the secondary-building units, which after reacting with NO formed chemisorbed nitrites (NO payload of 1.3 mmol·g–1). The material showed a slow NO release when suspended in PBS, not previously observed in NO-releasing MOFs due to the lability of the metal-NO bonds and/or the low stability of the frameworks in physiological media. In this case, given the high stability of the MOF, the sustained release was associated to the water- exchange of the chemisorbed nitrites. The material inhibited mitochondrial respiration in a concentration-dependent manner, demonstrating the control of the NO-releasing amount. The treatment of HeLa cells with the NO-releasing material favored the migration of the cells compared to the unloaded one, postulating its employment in wound-healing applications. As mentioned above, all three gasotransmitters have lone- paired electrons. Therefore, it is possible to use OMSs in MOFs to store and deliver not only NO but also CO and H2S. N. Metzler-Nolte and co-workers used the MIL-88 (Fe) analogues for CO adsorption and release.87 In-situ ultrahigh vacuum-FTIR Figure 6: Examples of NO-releasing metal-organic frameworks showing open metal and Mössbauer spectroscopy studies of MIL-88B and MIL-88B- sites. a) Crystal structure of [Co(C8H2O6)(NO)2] MOF prepared by the NH2 confirmed the presence of Fe(II)–CO and Fe(III)–CO chemisorption of nitric oxide to the unsaturated- cobalt sites. In detail, the view of NO bound to the cobalt atoms in the MOF structure Reprinted with permission coordinations after the desorption cycle. In addition, the effect from Ref. 76. Copyright 2008 American Chemical Society, b) Schematic of the ligand functionality was also studied. It was found that representation of the of the formation of chemisorbed nitrates on the titanium- the binding strength decreased in the case of MIL-88B-NH2 due based MOF Ti12O15(mdip)3(OH)6(H2O)6. Reprinted with permission from Ref. 86. to the electron donating effect of the amino group. CO releasing Copyright 2020 John Wiley & Sons, Inc. quantification was carried out through the myoglobin assay under physiological conditions in phosphate buffer at pH = biomedical applicability of this MOF was further optimized by 7.4.88 The CO release for bare MIL-88B and the amino hybridizing it with biocompatible polymers such as functionalized version was 0.36 mmol·g–1 and 0.69 mmol·g–1 polyurethane, poly(vinyl alcohol) and chitosan. Remarkably, the respectively. These values are consistent with the higher performance of the MOF was not diminished by its coordination strength in the Fe-CO bond in the case of the incorporation into polymeric supports, which enhances its unfunctionalized MIL-88B. practical implementation.94-96 Morris and co-workers, further widen the scope of MOFs in the delivery of gasotransmitters by incorporating H 2S in the 5.3 Encapsulation of gas-donor molecules structure of CPO-27-M (M = Ni, Zn).89 In close analogy to the The almost infinite possible combinations of metal ions- case of NO, H2S was coordinated to the OMSs of both /clusters and organic linkers to prepare metal-organic frameworks showing a maximum uptake close to 6.2 mmol·g–1. frameworks have resulted in a huge library of structures, some The differences between the Ni and Co analogue appeared in of them showing mesoporosity. Thanks to the larger size of the their releasing performance. While the H 2S releasing capacity of cavities, the framework can host bigger molecules than the gas CPO-27-Ni was 1.8 mmol·g–1, in the case of CPO-27-Zn was 0.5 itself (i.e. gas-donor molecules). The resulting gas-donor@MOF mmol·g–1. This difference is attributed to the difference in systems can modify the gas-release kinetics of the bare gas- coordination strength between the OMSs and H2S. The capacity donor (i.e. slowing down the gas release) and/or reduce the of CPO-27-Zn to store H2S and release it under physiological toxicity risks associated to the guest molecule by trapping it conditions enabled to test its vasodilation-induced properties in inside the pores and, therefore, limiting its leaching to the pre-contracted pig arteries. The immersion of the pre- physiological media. contracted arteries in organ bath containing H2S-loaded CPO- Barea et al. were pioneers in using the guest-encapsulation 27-Zn resulted in an artery relaxation of 39%. strategy to prepare a novel CO-releasing material based on a MOF.50 The cationic photoCORM [Mn(tacn)(CO)3]Br (tacn = 5.2 Catalysis 1,4,7-triazacyclononane) was incorporated in the anionic metal- OMSs located inside MOFs can be used as catalytic sites, which organic framework bio-MOF-1 (NH2(CH3)2)2[Zn8(adeninate)4- are easily accessible by the substrates owing to the intrinsic (BPDC)6]·8DMF·11H2O (BPDC = 4,4′-biphenyldicarboxylate) by porosity of MOFs. Thanks to these complementary properties, means of a cationic exchange approach (Figure 7a). Although MOFs have shown outstanding potential as catalysts. 90 On the other hand, several catalytic mechanisms of NO releasing by decomposition of RSNO are widely known including copper- mediated pathway.91 Reynolds and co-workers used the Cu(II) OMSs in the HKUST-1 structure to catalyze the decomposition reaction of a S-nitrosothiols (RSNO) into NO. Thus, HKUST-1 and S- nitrosocysteine (CysNO) were suspended in an ethanolic solution and the NO generation was monitored. It was found that when the MOF was present the NO generation was sustained over the course of 12h. On the contrary, when only Cu(II) salts such as CuCl2 and Cu(CH3COO)2 were employed only a burst release was found. These results suggest that the combination of OMSs and narrow pore architecture are responsible for the sustained NO release. 92 A further step towards the integration of the catalytic NO releasing property into functional materials was provided by the same authors. To this end, HKUST-1 was immobilized in cotton fabrics to produce NO on surfaces.93 The composite material showed to be active in the catalytic decomposition of S- nitrosocysteamine (CysamNO) to produce NO. In fact, a NO release of 7.1 mmol·g–1 was found over a period of 4h in ethanol. However, as in earlier cases, the poor hydrolytic Figure 7: Schematic representations of the encapsulation of gas-donors into the stability of HKUST-1 hindered its application in the biomedical cavities of metal-organic frameworks. a) Encapsulation of the photoCORM field. In order to overcome this shortcoming, the water stable [Mn(tacn)(CO)3]Br into the porous framework bio-MOF-1 MOF [(Cu4Cl)3(BTTri)8(H2O)12]·72H2O (where BTTri is 1,3,5- ((NH2(CH3)2)2[Zn8(adeninate)4(BPDC)6]·8DMF·11H2O) by cationic exchange b) Representation of the light triggered CO-release from the hybrid system tris(1H-1,2,3-triazol-5-yl)benzene) was synthesized and photoCORM@MOF [Mo(CNCMe2CO2H)3(CO)3]@[Al(OH)(stilbenedicarboxylate)]n. evaluated. The high chemical stability of the MOF allowed The MOF-based system releases CO under light irradiation while keeps trapped measuring its catalytic performance and NO release in PBS most of the metal-fragments encapsulated when suspended in physiological buffered solution. Thus, CuBTTri was able to release NO with buffer. Adapted with permission from Ref. 98. Copyright 2018 American Chemical the amount of 2.7 mmol·g–1 in the course of 4 hours. The Society the material showed a high payload (0.25 mmol of CORM per a two-step post synthetic modification of HKUST-1 was gram of material), the scarce stability of the framework in implemented. First, 4-(methylamino)-pyridine (denote as 4- physiological media led to almost full leaching of the CORM map) was coordinated to the OMSs. Subsequently, the after 6 hours of incubation in PBS. secondary amines were converted to NONOate functionality by A CO-releasing material was prepared by the encapsulation reacting them with NO gas at high pressure. This second step of the photoCORM [Mo(CNCMe2CO2H)3(CO)3] in a hierarchical was conditioned by the first step. The reaction did not occur structure of [Zn2(dobdc)]n.97 The microporous nature of the Zn- when the loading of 4-map was close to 100% due to the pore MOF with a pore aperture of 1.0 x 1.4 nm may hamper the blocking effect. On the other hand, the reaction proceeds when diffusion of the bulky Mo-CORM (ca. 1.3 nm). To overcome the loading was lower than 60%. As a proof of concept, the NO these problems, the authors used a hierarchical structure of delivery was confirmed in deionized water. However, the NO [Zn2(dobdc)]n showing textural mesopores. The CORM was release was accompanied by significant 4-map leaching. Cohen successfully encapsulated by one-pot synthesis-encapsulation and co-workers simplified the strategy by implementing it to (0.3 mmol of CORM per gram of material). The hybrid system two different MOFs, which contain the 2-aminoterephthalate exhibited a more sustained CO-release than the pristine CORM moiety in their structure.102 Therefore, the NONOate when irradiated with UV light. In addition, the material was able functionality was introduced into [Zn4O(NH2-bdc)]n (denote as to keep trapped the 87 % of the Mo-fragment incorporated IRMOF-3)103 and [Zn4O(NH2-bdc)(btb)4/3]n (denote as UMCM-1- after incubation in physiological media, limiting its potential NH2, btb = benzene-1,3,5-tribenzoate).104 The yield of the toxicity. The same CORM was also encapsulated in the cavities reaction (NONOate formation) for IRMOF-3 and UMCM-NH2 of the mesoporous MOF CYCU-3 [Al(OH)(SDC)]n, (H2SDC: 4,4- was 44% and 83% respectively, which is consistent with the stilbenedicarboxylic acid) (Figure 7b).98 The coordination- higher porosity of the later. The releasing amount of NO in modulation method99 was used to prepare several materials water was moderate in both cases (less than 10%) due to the with different particle features, ranging from pseudosphericals very rapid degradation of both structures. nanoparticles to micrometric needles. Noteworthy, the use of The poor hydrolytic stability of the NONOate functionalized higher concentration of modulator (50 times the linker MOFs limits their scope as NO releasing vehicles in water (or concentration) led to a denser framework (CYCU-3’), whose physiological media). However, they can be employed in the structure was elucidated by continuous rotation electron sustained release of NO in solid state upon exposure to humid diffraction (CRED). Although CYCU-3’ showed a lower porosity gas. To this end, a ligand bearing three amine functionalities than pristine CYCU-3, there channels were big enough to was employed in the synthesis of the MOF [Cu3(TDPAT)(H2O)3]n accommodate the CORM molecules, showing both systems framework (denote as Cu-TDPAT, TDPAT = 2,4,6-tris(3,5- similar payloads. Remarkably, the lower porosity in CYCU-3’ dicarboxylphenylamino)-1,3,5-triazine).105 The amine resulted in higher stability, enabling to retain the 75 % of Mo- functionalities were converted to NONOate functionalities by fragments encapsulated after being suspended in PBS during 72 reaction with NO gas, likewise previously introduced IRMOF-3 hours. In addition, the hybrid CYCU-3’ system enabled a more and UMCM-1-NH2. The NO releasing experiments in the solid sustained release of CO, both in solution and solid state, state revealed that the Cu-TDPAT NONOate released NO when increasing its potential toward different applications. contacted to humid gas (RH = 85%) over the course of 7 days and with a maximum release of 0.175 mmol·g–1. 5.4 Chemical attachment of gas-donors Most of the NO-releasing MOFs discussed so far used The versatility of MOFs enables to use functional ligands with humidity (water molecule) to trigger the release. However, ability to store and deliver gasotransmitters in their synthesis or humidity is a not an easy to control parameter in real incorporate them by post-synthetic modifications. This strategy physiological conditions, which implies that the degree of offers the advantage of ensuring a very high payload of the control in the delivery of the bio gas is limited. On the other donor moiety because it is incorporated in the structure. It also hand, light is a more suitable stimulus because it is non-invasive limits the extent of leaching, as in most of the cases the gas and can be manipulated in terms of intensity, wavelength and molecules are strongly attached to the organic moiety. location. Therefore, the use of photodonor ligands as part of the Diazeniumdiolate (NONOate) is probably the most famous framework is a promising strategy to develop new NO-releasing functionality. It is easily synthesized from photoresponsive gasotransmitters releasing materials. secondary amines under the high pressure of NO gas and Furukawa, Kamei and co-workers first implemented this release two molecules of NO by acid-catalyzed spontaneous strategy by using imidazole ligands bearing the nitro decomposition with the first-order kinetics, thus the releasing functionality in the synthesis of zeolitic imidazole frameworks rate of NO can be precisely predicted. Moreover, its (ZIF) (Figure 8a). Aromatic molecules with nitro groups are well decomposition rate can be tuned by subtle changes in its known for their NO photogeneration.106, 107 Thus, the reaction structure.100 Therefore, many kinds of NONOate derivatives, of 2-nitroimidazole (2-nIm) or 5-methyl-4-nitroimidazole which have various half-lives ranging from few seconds to (mnIm) with Zn(II) ions yielded [Zn(2nIm)2]n (NOF-1) and several hours, have been tested in experimental models of [Zn(mnIm)2]n (NOF-2).108 Interestingly, both frameworks cardiovascular diseases. showed light triggered NO release in the solid-state with a Rosseinsky and co-workers were the first to synthesize the maximum release of 3 mmol·g–1. The light used for these NONOate moiety inside a framework structure.101 In this study, experiments was set at 365 nm. Interestingly, The NO release duration and rate could be controlled by light on/off and strong coordination of high valence metal ions, such as Ti(IV) intensity. It is noteworthy that the light triggered NO releasing and Al(III) with carboxylates,110 and the BNN moiety decorating efficiency of the ligands themselves was significantly lower (less the pores, which hinders water and phosphate inclusion than 0.5 mmol·g–1) than the respective frameworks. The preventing hydrolytic degradation. difference in photoreactivity between the frameworks and the Even though the most common strategy consists in ligands is attributed to the structuring effect of the frameworks. introducing in the MOF synthesis ligands with the potential to The spatial segregation of the photoreactive moieties within the release gasotransmitter molecules under certain stimuli, framework avoids the aggregation induced quenching observed behaving as pro-drugs, it is also possible to exploit the affinity in the ligands in the solid-state, which paves the way to new of these gases towards certain organic groups. This was recently solid-state NO releasing materials. The solid-state delivery of demonstrated by Alkordi and co-workers. They showed that the NO of NOF-1 crystals enabled to integrate them into substrates NH2 functionality decorating the pores of the UiO-66-NH2 can that can then be used as functional extracellular scaffolds. Using trap up to 6.2 mmol·g–1 by chemisorption. The loaded this cell culture platform, the spatiotemporal stimulation of compound released up to 1.1 mmol·g–1 in acidified PBS cells by NO was demonstrated, which subsequently caused an media.111 increase of intracellular Ca2+ ion concentration in the genetically The presence of free moieties with coordination capabilities modified HEK293 cells with TRPC5 channels. decorating the pores of the MOF can be exploited to Besides the nitro aromatic groups, the bis-N-nitroso (BNN) incorporate metal complexes to the structure through post moiety is also known to release NO upon light irradiation. synthetic modifications. A paradigmatic example of this However, its low thermal stability prevents to use it in the direct approach is provided by the reactivity of ligands bearing free synthesis of the MOF. Thus, it must be incorporated in the 2,2-bipyridine moieties towards transition metal ions.112 This framework structure by post-synthetic modification through reactivity was exploited to incorporate the CO photoreleasing nitrosation of secondary amines. Therefore, two MOFs with bis- moiety MnBr(bpydc)(CO)3 (bpydc is 5,5-dicarboxylate-2,2- amino moieties in their structure were synthesized; bipyridine) within the porous structure of the Zr(IV) based UiO- [Ti8O8(OH)4(MeNH-bdc)6]n (preNOF-11) and the 67 [Zr6O4(OH)4(bpydc)6] through post synthetic metalation of [Al8(OMe)8(OH)4(MeNH-bdc)6]n (preNOF-12), where MeNH-bdc the free 2,2-bipyridine centers with MnBr(CO)5 (Figure 8b).113 is 2,5-bis(methylamino)-1,4-benzenedicarboxylic acid. The MnBr(bpydc)(CO)3@UiO-67 (named as CORF-1) was able to Subsequently, the BNN moiety was introduced by post- efficiently release CO in the solid-state upon visible light synthetic nitrosation.109 NOF-11 and NOF-12 released 1.60 irradiation (465 nm) with a maximum release of 4.7 mmol·g–1, mmol·g–1 and 2.78 mmol·g–1, respectively, upon white light which was easily controlled by turning on and off the light (300-600 nm) irradiation in the solid-state. Contrariwise to the source. These features enabled to prepare photoresponsive CO above-mentioned examples, both frameworks showed a releasing cell-growth substrates by immobilizing CORF-1 remarkable stability not only in water but also in phosphate crystals within the biocompatible PDMS polymer. These functional cellular scaffolds were used to observe the cellular uptake of the on-demand generated CO from the CORF-1 crystals. 6. Protein assemblies Protein crystals, which are the solid-state assembly of proteins, engaged interest as porous materials. Typical protein crystals show from 30 to 65% of void volume that are filled by solvent molecules. 114 The pore surface is composed of residual amino acid chains, which have a potential as metal coordination site and, therefore, can be used for anchoring gas-donor to the biomaterial. 115, 116 The low yield in the synthesis of protein crystals and their low stability are the main disadvantages of this strategy. On the other hand, their inherent lack of toxicity is a great advantage. In fact, protein assemblies are the more biocompatible gasotransmitter carriers discussed in this review. Figure 8: Examples of photoresponsive gas-releasing metal-organic frameworks prepared by the chemical attachment of gas donors. a) Chemical structures of 6.1 Chemical functionalization with gas-donor [Zn(2nIm)2]n (NOF-1) and [Zn(mnIm)2]n (NOF-2). Reprinted with permission from Romao and co-workers studied, for the first time, the Ref. 108, b) Post-functionalization of the linker of the MOF [Zr 6O4(OH)4(bpydc)6] with a manganese-carbonyl group to generate a photoresponsive CO-releasing interaction of CO-releasing molecules, namely [fac- material. Reprinted from Ref. 113 with permission from the Royal Society of Ru(CO)3Cl(glycinate)], (CORM-3), and different proteins. They Chemistry. observed the coordination of the carbonyl-metal to side chains of amino acids, forming Ru(CO)2-proteins adducts with the buffered solutions. This high stability stems from two facts; the concomitant loss of one CO and glycinate ligands. However, materials. Thus, researchers can tune the stimuli to deliver gases from porous materials as well as to control its release kinetics to meet the criteria for selected biomedical applications, such as wound healing, vasodilation and anti- inflammation among others. As we have summarized in the review, zeolites offer a robust and non-toxic framework with well-ordered OMS being able to store NO that can be spontaneously released through water exchange or produce it in situ through catalytic reactions. Therefore, while zeolites excel in the prolonged and spontaneous release of NO, it is challenging to modulate their performance toward NO release on demand because humidity and concentration of reactants cannot be easily modulated in vivo. Alternatively, the large pores of mesoporous silica allow encapsulating gas releasing molecules that are sensitive to different stimuli such as light and heat. This strategy offers the Figure 9: Schematic representation of subunit of ferritin with an inner diameter advantage of easily incorporating the advances achieved in the of 8 nm), formation of Ru(CO)2-protein adduct and cellular uptake of Ferritin- Ru(CO)2 composite and intracellular CO-release. Adapted with permission from field of gasotransmitter-releasing molecules into the porous reference 119. Copyright 2014 American Chemical Society. material field. Thus, mesoporous silica have proven to be versatile materials for releasing all known gasotransmitters through different stimuli, which made it possible to attain they did not study the potential CO-releasing properties of important goals in the field of gas biology such as on-demand these new materials.117, 118 Later, Ueno and co-workers proved gasotransmitter release. MOFs provided their versatility in the feasibility of using ferritin as a CO-donor carrier.119 This terms of structures, composition and reactivity to the field of protein includes 24 subunits with 8 nm or inner diameter gas biology, which enabled the development of novel routes (Figure 9). By reacting with [Ru(CO)3Cl2]2, protein-Ru(CO)2 were toward the controlled release of gasotransmitters. For example, formed. With the aim to modulate the CO-releasing properties the incorporation of gas releasing moieties in the organic linker of the system, they used three different ferritin mutants, of the MOF did not only achieve high payloads of gases but also counting with a distinct proportion of side chain aminoamides, their light triggered release in the solid-state paving the way for resulting in 3 novel CO-releasing materials. All composites its implementation in novel devices. All these materials share slowed down the CO-release rate in comparison to the pristine the fact that they are exogenous, which might rise some CORM when suspended in physiological conditions. In addition, concerns on their biocompatibility. In this regard, protein the cellular uptake of the composites was superior to the bare assemblies offer inherently biocompatible scaffolds where to CORM, proving the utility of protein cages as a carrier of CO- localize gasotransmitters at the cost of providing low payloads releasing molecules. In another work, they extended this and stability. approach to attach ruthenium carbonyls to a cross-linked hen However, as highlighted in several examples across this egg with lysozyme crystals. As in the case of the ferritin, the review, the successful implementation of porous materials for novel nanocomposites showed a slower CO-release than the the therapeutic release of gasotransmitters in the real world pristine CORM when suspended in physiological media. application requires an appropriate strategy for their Furthermore, they elicited biological response when incubated materialization/formulation and administration. Thus, in living cells. 120 The same authors further gained control on the processing of gasotransmitter releasing porous materials into delivery of CO from protein assemblies by introducing a Mn(I) stable colloids, gels, ointments or shape them into macroscopic based photoCORM.121 Thus, a mutant version of ferritin rich in object through hybridization with other materials such as cysteines was used to coordinate Mn(I) carbonyl species. polymers is a promising strategy toward their practical use. All Interestingly the composite protein assembly only release CO these strategies entail specific challenges that still need to be upon light irradiation at 456 nm. Thanks to the biocompatible overcome. For instance, the intravenous administration of the nature of the CO carrier, it could be used as an intracellular CO porous carriers implies the use of nanoscopic carriers with delivery vehicle. The high degree of control achieved in the CO narrow size distribution and structural and colloidal stability in delivery together with the biocompatibility of the carrier makes physiological media. In this regard, coating the porous carriers this material highly promising to further understand the role of with biocompatible polymers and lipids have shown to be a CO at cellular level. promising strategy to prevent degradation and increase colloidal stability. Alternatively, the use of non-toxic building 7. Conclusions and outlook blocks held by strong interactions in the synthesis of nanoscopic After nearly fifteen years of a multidisciplinary research that porous carriers has shown to increase the combined gas biology and porous materials, the resulting stability/biocompatibility of the porous carrier as exemplified knowledge has led to a versatile toolbox of materials and by titanosilicates and MOFs made with high valence metal ions strategies to load and release gasotransmitters from porous (i. e. Fe(III), Zr(IV) and Ti(IV)). The synthesis of stable nano-sized porous carriers for gasotransmitters together with further 24. P. S. Wheatley, A. R. Butler, M. S. Crane, S. Fox, B. Xiao, A. G. Rossi, I. L. Megson and R. E. Morris, J. Am. Chem. Soc., 2006, studies on their biodistribution will pave the way for their 128, 502-509. efficient intravenous administration. On the other hand, the use 25. H. A. Liu and K. J. Balkus, Chem. Mater., 2009, 21, 5032-5041. of macroscopic devices to exogenously deliver gasotransmitters 26. S. Fox, T. S. Wilkinson, P. S. Wheatley, B. Xiao, R. E. Morris, A. Sutherland, A. J. Simpson, P. G. Barlow, A. R. Butler, I. L. Megson to targeted organs must ensure a robust and reproducible and A. G. Rossi, Acta Biomater., 2010, 6, 1515-1521. performance over several cycles. In this regard, the possibility 27. M. Neidrauer, U. K. Ercan, A. Bhattacharyya, J. Samuels, J. Sedlak, to shape porous materials into gels, 122 liquids,123 melted R. Trikha, K. A. Barbee, M. S. Weingarten and S. G. Joshi, J. Med. liquids124 or monoliths125 seem a promising strategy to process Microbiol., 2014, 63, 203-209. 28. M. L. Pinto, J. Rocha, J. R. B. Gomes and J. Pires, J. Am. Chem. these materials into macroscopic objects without the need of Soc., 2011, 133, 6396-6402. an external matrix that dilute them. 29. M. L. Pinto, A. C. Fernandes, J. Rocha, A. Ferreira, F. Antunes and J. Pires, J. Mat. Chem. B, 2014, 2, 224-230. 30. M. L. Pinto, A. C. Fernandes, F. Antunes, J. Pires and J. Rocha, Microporous Mesoporous Mater., 2016, 229, 83-89. Conflicts of interest 31. R. V. Pinto, A. C. Fernandes, F. Antunes, Z. Lin, J. Rocha, J. Pires There are no conflicts to declare. and M. L. Pinto, Nitric Oxide, 2019, 90, 29-36. 32. E. I. Tocheva, F. I. Rosell, A. G. Mauk and M. E. P. Murphy, Science, 2004, 304, 867. 33. A.-K. Boes, P. S. Wheatley, B. Xiao, I. L. Megson and R. E. Morris, Acknowledgements Chem. Commun., 2008, 6146-6148. 34. G. Turnes Palomino, P. Fisicaro, S. Bordiga, A. Zecchina, E. The authors acknowledge iCeMS for supporting the gas biology Giamello and C. Lamberti, J. Phys. Chem. B, 2000, 104, 4064- materials project. This research was supported by AMED under 4073. 35. W. J. Roth, P. Nachtigall, R. E. Morris, P. S. Wheatley, V. R. Grant Number 20lm0203013j0002. Seymour, S. E. Ashbrook, P. Chlubná, L. Grajciar, M. Položij, A. Zukal, O. Shvets and J. Čejka, Nat. Chem., 2013, 5, 628-633. 36. R. A. Doyle, S. E. Russell and R. E. Morris, Microporous Notes and references 37. Mesoporous Mater., 2019, 280, 367-371. S. E. Russell, J. M. González Carballo, C. Orellana-Tavra, D. Fairen-Jimenez and R. E. Morris, Dalton. Trans., 2017, 46, 3915- 1. A. K. Mustafa, M. M. Gadalla, N. Sen, S. Kim, W. Mu, S. K. Gazi, 3920. R. K. Barrow, G. Yang, R. Wang and S. H. Snyder, Sci. Signal, 38. J. L. Vivero-Escoto, I. I. Slowing, B. G. Trewyn and V. S. Y. Lin, 2009, 2, ra72-ra72. Small, 2010, 6, 1952-1967. 2. R. U. I. Wang, FASEB J., 2002, 16, 1792-1798. 39. Z. Li, J. C. Barnes, A. Bosoy, J. F. Stoddart and J. I. Zink, Chem. 3. R. Wang, Trends Biochem. Sci, 2014, 39, 227-232. Soc. Rev., 2012, 41, 2590-2605. 4. R. M. J. Palmer, A. G. Ferrige and S. Moncada, Nature, 1987, 327, 40. F. Hoffmann, M. Cornelius, J. Morell and M. Fröba, Angew. Chem. 524. Int. Ed., 2006, 45, 3216-3251. 5. D. J. Stuehr, Biochim. Biophys. Acta Bioenerg., 1999, 1411, 217- 41. J. J. Koehler, J. Zhao, S. S. Jedlicka, D. M. Porterfield and J. L. 230. Rickus, J. Phys. Chem. B, 2008, 112, 15086-15093. 6. R. Tenhunen, H. S. Marver and R. Schmid, Proc. Natl. Acad. Sci. 42. V. P. Torchilin, Adv. Drug Deliv. Rev., 2006, 58, 1532-1555. U. S. A., 1968, 61, 748-755. 43. D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. 7. L. Wu and R. Wang, Pharmacol. Rev., 2005, 57, 585-630. Langer, Nat. Nanotechnol., 2007, 2, 751. 8. R. Motterlini and L. E. Otterbein, Nat. Rev. Drug Discov., 2010, 9, 44. D. M. Porterfield, J. D. Laskin, S.-K. Jung, R. P. Malchow, B. 728-743. Billack, P. J. S. Smith and D. E. Heck, Am. J. Physiol. Lung Cell 9. M. S. Vandiver and S. H. Snyder, J. Mol. Med. (Berl.), 2012, 90, Mol. Physiol., 2001, 281, L904-L912. 255-263. 45. C. Zolkov, D. Avnir and R. Armon, J. Mater. Chem., 2004, 14, 10. R. Wang, Physiol. Rev., 2012, 92, 791-896. 2200-2205. 11. D. Babu, R. Motterlini and R. A. Lefebvre, Br. J. Pharmacol., 46. S. S. Jedlicka, J. L. McKenzie, S. J. Leavesley, K. M. Little, T. J. 2015, 172, 1557-1573. Webster, J. P. Robinson, D. E. Nivens and J. L. Rickus, J. Mater. 12. C. C. Romão, W. A. Blättler, J. D. Seixas and G. J. L. Bernardes, Chem., 2006, 16, 3221-3230. Chem. Soc. Rev., 2012, 41, 3571-3583. 47. B. J. Heilman, J. St. John, S. R. J. Oliver and P. K. Mascharak, J. 13. U. Schatzschneider, Br. J. Pharmacol., 2015, 172, 1638-1650. Am. Chem. Soc., 2012, 134, 11573-11582. 14. P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and A. J. 48. X. Zhang, G. Tian, W. Yin, L. Wang, X. Zheng, L. Yan, J. Li, H. Janczuk, Chem. Rev., 2002, 102, 1091-1134. Su, C. Chen, Z. Gu and Y. Zhao, Adv. Funct. Mater., 2015, 25, 15. N. Naghavi, A. de Mel, O. S. Alavijeh, B. G. Cousins and A. M. 3049-3056. Seifalian, Small, 2013, 9, 22-35. 49. C. Hu, J. Sun, Y. Zhang, J. Chen, Y. Lei, X. Sun and Y. Deng, Adv. 16. Y. Zhao, T. D. Biggs and M. Xian, Chem. Commun., 2014, 50, Healthc. Mater., 2018, 7, 1801047. 11788-11805. 50. F. J. Carmona, S. Rojas, P. Sánchez, H. Jeremias, A. R. Marques, 17. S. M. Fix, M. A. Borden and P. A. Dayton, J. Control. Release, C. C. Romão, D. Choquesillo-Lazarte, J. A. R. Navarro, C. R. 2015, 209, 139-149. Maldonado and E. Barea, Inorg. Chem., 2016, 55, 6525-6531. 18. T. Yang, A. N. Zelikin and R. Chandrawati, Adv. Sci., 2018, 5, 51. M. A. Gonzales, H. Han, A. Moyes, A. Radinos, A. J. Hobbs, N. 1701043. Coombs, S. R. J. Oliver and P. K. Mascharak, J. Mat. Chem. B, 19. K. Ling, F. Men, W.-C. Wang, Y.-Q. Zhou, H.-W. Zhang and D.- 2014, 2, 2107-2113. W. Ye, J. Med. Chem., 2018, 61, 2611-2635. 52. I. Chakraborty, S. J. Carrington, J. Hauser, S. R. J. Oliver and P. K. 20. M. E. Davis, Nature, 2002, 417, 813. Mascharak, Chem. Mater., 2015, 27, 8387-8397. 21. S. Wang, Microporous Mesoporous Mater., 2009, 117, 1-9. 53. F. J. Carmona, I. Jiménez-Amezcua, S. Rojas, C. C. Romão, J. A. 22. K. Hadjiivanov, E. Ivanova and H. Knözinger, Microporous R. Navarro, C. R. Maldonado and E. Barea, Inorg. Chem., 2017, Mesoporous Mater., 2003, 58, 225-236. 56, 10474-10480. 23. M. Ozekmekci, G. Salkic and M. F. Fellah, Fuel Process. Technol., 54. G. Caliendo, G. Cirino, V. Santagada and J. L. Wallace, J. Med. 2015, 139, 49-60. Chem., 2010, 53, 6275-6286. 55. X. Sun, B. Kong, W. Wang, P. Chandran, C. Selomulya, H. Zhang, 84. J. F. Eubank, P. S. Wheatley, G. Lebars, A. C. McKinlay, H. K. Zhu, Y. Liu, W. Yang, C. Guo, D. Zhao and C. Wang, J. Mat. Leclerc, P. Horcajada, M. Daturi, A. Vimont, R. E. Morris and C. Chem. B, 2015, 3, 4451-4457. Serre, APL Mater., 2014, 2, 124112. 56. W. Wang, X. Sun, H. Zhang, C. Yang, Y. Liu, W. Yang, C. Guo 85. S. R. Miller, E. Alvarez, L. Fradcourt, T. Devic, S. Wuttke, P. S. and C. Wang, Int. J. Nanomedicine, 2016, 11, 3255-3263. Wheatley, N. Steunou, C. Bonhomme, C. Gervais, D. Laurencin, 57. S. M. C. Neiva, J. A. V. Santos, J. C. Moreira, Y. Gushikem, H. R. E. Morris, A. Vimont, M. Daturi, P. Horcajada and C. Serre, Vargas and D. W. Franco, Langmuir, 1993, 9, 2982-2985. Chem. Commun., 2013, 49, 7773-7775. 58. B. J. Nablo, T.-Y. Chen and M. H. Schoenfisch, J. Am. Chem. Soc., 86. R. V. Pinto, S. Wang, S. R. Tavares, J. Pires, F. Antunes, A. 2001, 123, 9712-9713. Vimont, G. Clet, M. Daturi, G. Maurin, C. Serre and M. L. Pinto, 59. P. G. Zanichelli, R. L. Sernaglia and D. W. Franco, Langmuir, Angew. Chem. Int. Ed., 2020, 59, 5135-5143. 2006, 22, 203-208. 87. M. Ma, H. Noei, B. Mienert, J. Niesel, E. Bill, M. Muhler, R. A. 60. F. G. Doro, U. P. Rodrigues-Filho and E. Tfouni, J. Colloid, Interf. Fischer, Y. Wang, U. Schatzschneider and N. Metzler-Nolte, Sci., 2007, 307, 405-417. Chem. Eur. J., 2013, 19, 6785-6790. 61. J. H. Shin, S. K. Metzger and M. H. Schoenfisch, J. Am. Chem. 88. A. J. Atkin, J. M. Lynam, B. E. Moulton, P. Sawle, R. Motterlini, Soc., 2007, 129, 4612-4619. N. M. Boyle, M. T. Pryce and I. J. S. Fairlamb, Dalton. Trans., 62. F. O. N. da Silva, E. C. C. Gomes, T. d. S. Francisco, A. K. M. 2011, 40, 5755-5761. Holanda, I. C. N. Diógenes, E. H. S. de Sousa, L. G. F. Lopes and 89. P. K. Allan, P. S. Wheatley, D. Aldous, M. I. Mohideen, C. Tang, E. Longhinotti, Polyhedron, 2010, 29, 3349-3354. J. A. Hriljac, I. L. Megson, K. W. Chapman, G. De Weireld, S. 63. F. Wei, Q. Hou, J. Y. Yang and J. H. Zhu, J. Colloid, Interf. Sci., Vaesen and R. E. Morris, Dalton. Trans., 2012, 41, 4060-4066. 2011, 356, 526-535. 90. J. Lee, O. K. Farha, J. Roberts, K. A. Scheidt, S. T. Nguyen and J. 64. M. B. Yue, L. B. Sun, T. T. Zhuang, X. Dong, Y. Chun and J. H. T. Hupp, Chem. Soc. Rev., 2009, 38, 1450-1459. Zhu, J. Mater. Chem., 2008, 18, 2044-2050. 91. D. L. H. Williams, Acc. Chem. Res., 1999, 32, 869-876. 65. R. J. Soto, L. Yang and M. H. Schoenfisch, ACS Appl. Mater. Int., 92. J. L. Harding and M. M. Reynolds, J. Am. Chem. Soc., 2012, 134, 2016, 8, 2220-2231. 3330-3333. 66. A. L. Tessaro, A. Fraix, A. C. Pedrozo da Silva, E. Gazzano, C. 93. M. J. Neufeld, J. L. Harding and M. M. Reynolds, ACS Appl. Mater. Riganti and S. Sortino, Nanomaterials, 2019, 9, 823. Int., 2015, 7, 26742-26750. 67. D. Afonso, S. Valetti, A. Fraix, C. Bascetta, S. Petralia, S. Conoci, 94. J. L. Harding, J. M. Metz and M. M. Reynolds, Adv. Funct. Mater., A. Feiler and S. Sortino, Nanoscale, 2017, 9, 13404-13408. 2014, 24, 7503-7509. 68. M. J. Malone-Povolny and M. H. Schoenfisch, ACS Appl. Mater. 95. M. J. Neufeld, A. Lutzke, J. B. Tapia and M. M. Reynolds, ACS Int., 2019, 11, 12216-12223. Appl. Mater. Int., 2017, 9, 5139-5148. 69. R. Guo, Y. Tian, Y. Wang and W. Yang, Adv. Funct. Mater., 2017, 96. M. J. Neufeld, A. Lutzke, W. M. Jones and M. M. Reynolds, ACS 27, 1606398. Appl. Mater. Int., 2017, 9, 35628-35641. 70. O. M. Yaghi, M. O'Keeffe, N. W. Ockwig, H. K. Chae, M. 97. F. J. Carmona, S. Rojas, C. C. Romão, J. A. R. Navarro, E. Barea Eddaoudi and J. Kim, Nature, 2003, 423, 705. and C. R. Maldonado, Chem. Commun., 2017, 53, 6581-6584. 71. S. Kitagawa, R. Kitaura and S.-i. Noro, Angew. Chem. Int. Ed., 98. F. J. Carmona, C. R. Maldonado, S. Ikemura, C. C. Romão, Z. 2004, 43, 2334-2375. Huang, H. Xu, X. Zou, S. Kitagawa, S. Furukawa and E. Barea, 72. G. Férey, C. Mellot-Draznieks, C. Serre and F. Millange, Acc. ACS Appl. Mater. Int., 2018, 10, 31158-31167. Chem. Res., 2005, 38, 217-225. 99. T. Tsuruoka, S. Furukawa, Y. Takashima, K. Yoshida, S. Isoda and 73. P. Horcajada, R. Gref, T. Baati, P. K. Allan, G. Maurin, P. S. Kitagawa, Angew. Chem. Int. Ed., 2009, 48, 4739-4743. Couvreur, G. Férey, R. E. Morris and C. Serre, Chem. Rev., 2012, 100. J. A. Hrabie, J. R. Klose, D. A. Wink and L. K. Keefer, J. Org. 112, 1232-1268. Chem., 1993, 58, 1472-1476. 74. X. Kong, E. Scott, W. Ding, J. A. Mason, J. R. Long and J. A. 101. M. J. Ingleson, R. Heck, J. A. Gould and M. J. Rosseinsky, Inorg. Reimer, J. Am. Chem. Soc., 2012, 134, 14341-14344. Chem., 2009, 48, 9986-9988. 75. B. Xiao, P. S. Wheatley, X. Zhao, A. J. Fletcher, S. Fox, A. G. 102. J. G. Nguyen, K. K. Tanabe and S. M. Cohen, CrystEngComm, Rossi, I. L. Megson, S. Bordiga, L. Regli, K. M. Thomas and R. E. 2010, 12, 2335-2338. Morris, J. Am. Chem. Soc., 2007, 129, 1203-1209. 103. M. Eddaoudi, J. Kim, N. Rosi, D. Vodak, J. Wachter, M. O'Keeffe 76. A. C. McKinlay, B. Xiao, D. S. Wragg, P. S. Wheatley, I. L. and O. M. Yaghi, Science, 2002, 295, 469-472. Megson and R. E. Morris, J. Am. Chem. Soc., 2008, 130, 10440- 104. Z. Wang, K. K. Tanabe and S. M. Cohen, Inorg. Chem., 2009, 48, 10444. 296-306. 77. F. Bonino, S. Chavan, J. G. Vitillo, E. Groppo, G. Agostini, C. 105. A. Lowe, P. Chittajallu, Q. Gong, J. Li and K. J. Balkus, Lamberti, P. D. C. Dietzel, C. Prestipino and S. Bordiga, Chem. Microporous Mesoporous Mater., 2013, 181, 17-22. Mater., 2008, 20, 4957-4968. 106. K. Hishikawa, H. Nakagawa, T. Furuta, K. Fukuhara, H. Tsumoto, 78. D. Cattaneo, S. J. Warrender, M. J. Duncan, C. J. Kelsall, M. K. T. Suzuki and N. Miyata, J. Am. Chem. Soc., 2009, 131, 7488-7489. Doherty, P. D. Whitfield, I. L. Megson and R. E. Morris, RSC Adv., 107. S. Sortino, Chem. Soc. Rev., 2010, 39, 2903-2913. 2016, 6, 14059-14067. 108. S. Diring, D. O. Wang, C. Kim, M. Kondo, Y. Chen, S. Kitagawa, 79. E. D. Bloch, W. L. Queen, S. Chavan, P. S. Wheatley, J. M. K.-i. Kamei and S. Furukawa, Nat. Commun., 2013, 4, 2684. Zadrozny, R. Morris, C. M. Brown, C. Lamberti, S. Bordiga and J. 109. C. Kim, S. Diring, S. Furukawa and S. Kitagawa, Dalton. Trans., R. Long, J. Am. Chem. Soc., 2015, 137, 3466-3469. 2015, 44, 15324-15333. 80. S. Rojas, P. S. Wheatley, E. Quartapelle-Procopio, B. Gil, B. 110. T. Devic and C. Serre, Chem. Soc. Rev., 2014, 43, 6097-6115. Marszalek, R. E. Morris and E. Barea, CrystEngComm, 2013, 15, 111. R. R. Haikal, C. Hua, J. J. Perry, D. O’Nolan, I. Syed, A. Kumar, 9364-9367. A. H. Chester, M. J. Zaworotko, M. H. Yacoub and M. H. Alkordi, 81. À. Ruyra, A. Yazdi, J. Espín, A. Carné-Sánchez, N. Roher, J. ACS Appl. Mater. Int., 2017, 9, 43520-43528. Lorenzo, I. Imaz and D. Maspoch, Chem. Eur. J., 2015, 21, 2508- 112. E. D. Bloch, D. Britt, C. Lee, C. J. Doonan, F. J. Uribe-Romo, H. 2518. Furukawa, J. R. Long and O. M. Yaghi, J. Am. Chem. Soc., 2010, 82. P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, T. Baati, J. 132, 14382-14384. F. Eubank, D. Heurtaux, P. Clayette, C. Kreuz, J.-S. Chang, Y. K. 113. S. Diring, A. Carne-Sanchez, J. Zhang, S. Ikemura, C. Kim, H. Hwang, V. Marsaud, P.-N. Bories, L. Cynober, S. Gil, G. Férey, P. Inaba, S. Kitagawa and S. Furukawa, Chem. Sci., 2017, 8, 2381- Couvreur and R. Gref, Nat. Mater., 2009, 9, 172. 2386. 83. A. C. McKinlay, J. F. Eubank, S. Wuttke, B. Xiao, P. S. Wheatley, 114. T. Ueno, Chem. Eur. J., 2013, 19, 9096-9102. P. Bazin, J. C. Lavalley, M. Daturi, A. Vimont, G. De Weireld, P. 115. T. O. Yeates and J. E. Padilla, Curr. Opin. Struct. Biol., 2002, 12, Horcajada, C. Serre and R. E. Morris, Chem. Mater., 2013, 25, 464-470. 1592-1599. 116. Y.-T. Lai, E. Reading, G. L. Hura, K.-L. Tsai, A. Laganowsky, F. J. Asturias, J. A. Tainer, C. V. Robinson and T. O. Yeates, Nat. Chem., 2014, 6, 1065. 117. T. Santos-Silva, A. Mukhopadhyay, J. D. Seixas, G. J. L. Bernardes, C. C. Romão and M. J. Romão, J. Am. Chem. Soc., 2011, 133, 1192-1195. 118. M. F. A. Santos, J. D. Seixas, A. C. Coelho, A. Mukhopadhyay, P. M. Reis, M. J. Romão, C. C. Romão and T. Santos-Silva, J. Inorg. Biochem., 2012, 117, 285-291. 119. K. Fujita, Y. Tanaka, T. Sho, S. Ozeki, S. Abe, T. Hikage, T. Kuchimaru, S. Kizaka-Kondoh and T. Ueno, J. Am. Chem. Soc., 2014, 136, 16902-16908. 120. H. Tabe, K. Fujita, S. Abe, M. Tsujimoto, T. Kuchimaru, S. Kizaka- Kondoh, M. Takano, S. Kitagawa and T. Ueno, Inorg. Chem., 2015, 54, 215-220. 121. K. Fujita, Y. Tanaka, S. Abe and T. Ueno, Angew. Chem. Int. Ed., 2016, 55, 1056-1060. 122. A. Carné-Sánchez, G. A. Craig, P. Larpent, T. Hirose, M. Higuchi, S. Kitagawa, K. Matsuda, K. Urayama and S. Furukawa, Nat. Commun., 2018, 9, 2506. 123. N. Giri, M. G. Del Pópolo, G. Melaugh, R. L. Greenaway, K. Rätzke, T. Koschine, L. Pison, M. F. C. Gomes, A. I. Cooper and S. L. James, Nature, 2015, 527, 216-220. 124. R. Gaillac, P. Pullumbi, K. A. Beyer, K. W. Chapman, D. A. Keen, T. D. Bennett and F.-X. Coudert, Nat. Mater., 2017, 16, 1149-1154. 125. Y. Hara, K. Kanamori and K. Nakanishi, Angew. Chem. Int. Ed., 2019, 58, 19047-19053.