domain: chim sdv phys

text:
Joana Claudio Pieretti

Milena Trevisan Pelegrino

Ariane Boudier

Amedea Barozzi Seabra
email: amedea.seabra@ufabc.edu.br

Recent progress in the toxicity of nitric oxide-releasing nanomaterials

Keywords: Nitric oxide donors, nanomaterials, cytotoxicity, cell sensitization, multidrug resistance cells, in vivo biocompatibility. Materials Advances

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

rsc.li/materials-advances

Materials Advances

Materials Advances

Introduction: The biological importance of nitric oxide

Nitric oxide (NO) is a small signaling molecule associated with several cellular functions. 1,2,3 NO is relatively a simple molecule, composed only by a covalent bound between nitrogen and oxygen atoms with an unpaired electron on the antibonding 2* y , therefore, a free radical. 4 Until the 1970 decade, NO was majority known as an atmospheric pollutant and a toxic agent. 2,5,6 However, the Nobel Prize of Medicine and Physiology awarded by Louis J. Ignarro, Robert F. Furchgott and Ferid Murad identifying NO as the EDRF (endothelium derived relaxing factor) shed light on the importance of this signaling molecule in several physiological and pathophysiological processes. [7][8][9][10][11] In 1992, NO was elected the 'molecule of the year' by the journal Science due to the increasing number of published articles regarding its impactful cellular effects. 12 NO is endogenously synthesized by the action of cluster catalytic enzymes named nitric oxide synthase (NOS). NOS enzyme family includes three isoforms: (i) endothelial (eNOS), (ii) neuronal (nNOS) and (iii) inducible (iNOS). 1,13,14 Both eNOS and nNOS synthesize NO at lower amounts for a longer time-period in the presence of calcium, while iNOS produces NO only when induced by an external signal at higher concentrations for longer time frame. 1,15,16 All members of NOS enzyme family catalyze the oxidation of Larginine to L-citrulline with formation of one NO molecule. 4,16 In cancer biology, NO serves multiple functions, being considering an anti-and proapoptotic agent, promoting vasodilation, angiogenesis and interaction with macrophages. 4,6,14,15,17 For instance, a member of NOS family, eNOS, in endothelium has been linked to a prognostic factor for patients with stomach adenocarcinoma (STAD). 13 In this sense, NO is well recognized for playing a dual role in cancer cells, having deleterious or beneficial features. 2,6,18 In some studies, NO or NOS levels are correlated with tumor suppression, however, in other studies to tumor progression and metastasis. 1,2,18 NO interaction with cellular targets can vary based on spatial and temporal distribution of NO influx within tumor cells and tumor microenvironment. 17,19 Thus, key factor linked to modulate the dichotomous effect of NO are the amount of NO released, the time-period and the spatial distribution of this signaling molecule in tumor tissue. 20 In addition, tumor microenvironment and heterogeneity also play an important role in the dichotomous effect of NO. 1 Thus, for proposing the use of exogenous NO to cancer cells/tissues, it is of fundamental importance to tailor the desired amounts of NO released to the target site of application. To this end, the combination of NO donors with nanomaterials represents an important approach. 3,9,21,22 Tumor tissue has unique vascular characteristics such as hypervasculature, irregular morphology and enhanced permeability. 23 Tumor tissue recovery of some substances from tumor interstitium into lymphatics is slow; this effect is observed for macromolecules, lipids and nanoparticles. 22,23 Therefore, nanoparticles naturally stay within tumor tissue for a longer time-period, compared with other drugs, this is a well-known phenomenon called enhanced permeability and retention (EPR) effect. 22,23 EPR is one of the reasons to explain why nanomaterials are intensively developed for cancer treatment. 24 Interestingly, NO has been linked to mediate the enhanced permeability. Indeed, NO scavenger and NOS inhibitor suppressed the EPR effect in mice with sarcoma solid tumor. 23,25 NO donors are a class of molecules capable to mimic NO flux conditions and modulate its concentration. 3,17 Due to the NO instability, the use of NO donors has been successful used to exogenously delivery NO to a target site, in different biomedical applications. 19,26 The most common classes of NO donors, are sodium nitroprusside, organic nitrite and nitrate, furoxans, benzofuroxans, diazeniumdiolates (NONOates), S-nitrosothiols (RSNOs), Roussin's black salt (RBS) and metal nitrosyl complexes. 3,17,[27][28][29][30][31] The molecular representations of commonly used NO donors are shown in Figure 1. NONOates and RSNOs are highlighted as the most used NO donors in oncological therapeutic applications due to NO release features and feasibility to be produced. 3,32,33 NONOate releases NO only in the presence of protons and can be classified according to its secondary amine structure, which varies its half-life. 3,21,33 In RSNOs, NO is covalently bound to a sulfur group (RS); and NO is spontaneously released from the homolytic S-N cleavage. 26,27 RSNOs is also involved in Stransnitrosation reactions that has been linked to be a relevant step on the downward signaling route created by NO. 19,26,34,35,36 Another commonly used NO donor is the RBS, which is a metal-nitrosyl complex that releases NO after illumination. Although NO donors have allowed the use of exogenous NO in biomedical applications, these molecules have limitations regarding the NO concentrations spatially and temporally reach. 26,27 In order to overcome this challenge, the combination of nanomaterials and NO has been extremely successful. 3,27,32,38,39,40 Nanomaterials have been applied to cancer diagnosis and treatment with positive results. 23,41,42 Cancer is a leading cause of death worldwide; its treatment usually involves surgical resection, radiation and chemotherapy. Although several treatments are conventionally used with certain degree of success, there are some drawbacks such as collateral effects and multi-drug resistant cancers. 3,18,22,30,40 In this sense, the combination of nanomaterials and NO donors can be used alone or with chemotherapy or radiotherapy agents. 3,27,43 This combination is cooperative; the nanoparticle is a carrier for NO allowing it to reach the target site at suitable concentrations.

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In addition, as a vasodilator, NO can increase tumor permeability allowing nanoparticle accumulation on tumor site. 23 All these features together can provide an alternative strategy to overcome setbacks of traditional cancer treatment.

In this scenario, the scope of this review is to point out the recent advances in the design of nanomaterials capable of releasing NO and their activity against cancer cell lines, highlighting their efficiency against multi-drug resistant tumor, in vivo results, and selective toxicity. We aimed to overview recent publications available on reliable platforms such as Web of Science and PubMed from the last five years (2016-2021), focusing on NO-releasing polymeric nanomaterials and/or polymer-coated nanomaterials.

Cytotoxicity of NO-releasing nanoparticles against cancer and non-cancerous cells

Since few years after NO was defined as the endothelium-derived relaxing factor (EDRF), 7,[START_REF] Ignarro | Nitric Oxide Biology and Pathobiology[END_REF] NO was described as an important signaling molecule, involved in cytotoxic mechanisms, demonstrating relevant cytoprotective properties. 5,[START_REF] Kröncke | [END_REF] Regarding its cell cytotoxicity, NO has been reported as a possible modulator of apoptosis, angiogenesis, invasion, metastasis, and also as a regulator of the cell cycle. 46 The cytotoxic effect of NO on tumor cells relays, mainly, in the suppression of DNA synthesis and cellular respiration, shifting iron metabolism, modulation of apoptosis by activating caspase family proteases, upregulation of p53 protein and other apoptosis-related proteins. 46 More recently, NO has also

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Open been mentioned as a sensitizing agent, demonstrating promising cytotoxic effects against tumoral cells when allied to traditional therapeutic approaches (chemotherapy and radiotherapy). 3,47 As previously mentioned, NO is a relatively unstable molecule, therefore NO donors have been employed as a strategy to promote an effective delivery. More recently, the combination of NO donors with nanoparticles has improved the use of NO as an antitumoral therapeutic strategy. 3 In this scenario, NO has been allied to different kind of nanoparticles, ranging from metallic nanoparticles, as gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), and quantum dots (QDs); 48 metal oxide nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPIONs); zeolites and other metal-organic frameworks; 49 porous nanomaterials, as silica nanoparticles (SiO 2 ); 50 to polymeric nanoparticles. [51][52][53][54] Depending on the nature of the nanoparticles, they can have a direct cytotoxicity, or they can act as nanocarriers of active drugs, enhancing the intratumoral penetration, thus promoting a more efficient drug delivery. 55 Generally, the empty nanocarrier itself does not present toxic or cytotoxic effect, being only a nanovehicle for improving drug delivery. When compared to control cells or in vivo models, empty nanoparticles should demonstrate similar pattern, as no antitumoral effect should be promoted. For instance, poly(lactic-co-glycolic acid)-based nanoparticles (PLGA NPs), approved by the US Food and Drug Administration (FDA) for medical use, demonstrate promising potential to be combined with different drugs, other nanoparticles or molecules, since these nanoparticles do not demonstrate significant cytotoxic effects without the mentioned combinations. 56 On the opposite, there are some types of nanoparticles that present intrinsic properties that may lead significant cytotoxic potential, as well as contributing to the diagnosis, such as the theragnostic nanoparticles. 57 AuNPs, for example, exhibit unique optical and electronic properties that make them suitable for cell and/or tumor imaging, besides presenting size and shape-dependent toxicity against cancerous and non-cancerous cells. 58,59 AgNPs demonstrate higher cytotoxicity against cancer cells when compared to AuNPs, drastically reducing cell viability, as demonstrated for colon cancer cells, regulating redox processes and the cell cycle, mediating cell apoptosis and DNA disfunction. 60 Moreover, AgNPs may demonstrate a synergic toxic effect in combination with NO. 54 For instance, it is well-known that AgNPs may cause toxicity due to the ability to generate reactive nitrogen and oxygen species, leading to oxidative damage in mammalian cells and/or microorganisms. 61 Similar to AgNPs, NO sub products, such as peroxynitrite (ONOO -) are also key players for increasing the oxidative stress and causing cellular damage. 62 NO fast reacts with superoxide (O 2 •-) leading to the formation of the harmful peroxynitrite, described as following:

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NO • +O 2 •- ONOO - (Eq. 1)
In this sense, combining functional nanoparticles, such as AgNPs, with NO releasing molecules consists in a promising strategy for increasing the generation of NOx species, such as peroxynitrite, increasing oxidative damage to cancer cell lines.

In this section, we aim to overview the recent progress in the cytotoxicity promoted by NO-releasing polymeric nanoparticles and/or polymer-coated nanoparticles and their interference in cellular mechanisms, which might be directly related to the NO delivered, or to the combination of the NO donor with the nanoparticle and/or other chemotherapeutic drugs.

Cytotoxicity of NO-releasing polymeric nanoparticles against tumoral and non-tumoral cell lines

Polymer-based nanoparticles have stand out in nanomedicine and drug delivery, mostly due to the synthesis facility, high biocompatibility, and the possibility of achieving different designs, such as polymeric nanocapsules and/or nanospheres, micelles, and liposomes. 60 The last one stands out for being approved by FDA, being used for delivering the chemotherapeutic doxorubicin (DOX), improving its efficacy. 63 In recent years, NO has been combined to different polymeric nanoparticles in order to promote or potentiate cytotoxic effects against tumoral cells. 42 PLGA NPs-containing the NO donor dinitrosyl iron complex were developed to promote a sustained NO release, regarding the concentration, localization, and duration. 64 Nanoparticles with 120 nm in size were responsible for promoting EPR effect concentration of 50 mmol L -1 , in which NO should demonstrate significant cytotoxic effects. 51 The potential cytotoxic effect of RSNO encapsulated in CS NPs was confirmed against different tumoral cell lines: hepatocellular carcinoma (HepG2), skin melanoma (B16-F10), chronic myeloid leukemia (K562), and vincristine resistant chronic myeloid leukemia (Lucena Similar results were reported for RSNO-loaded CS NPs functionalized with hyaluronic acid (HA). 66 Cytotoxic results were verified for all tested concentrations (5-200 μg mL -1 ), achieving 0% of cell viability at the highest evaluated concentration. Interestingly, the HA did not contribute to the cytotoxicity, although it might promote a higher selectivity for cells that overexpresses CDD4 protein. 66 Another targeted nanomaterial was reported by Liu and coworkers, in which molecular imprinted nanoparticles (MIPs) were designed to target tumor that overexpresses sialic acid. 30 The cytotoxicity was evaluated against different tumoral cell lines: HepG2 cells, mammary cancer cells (MCF-7), and compared to non-tumoral

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Open hepatocellular cells. The NO-releasing nanoparticles demonstrated selectivity for sialic acid overexpressing cells (HepG2 and MCF-7), evidenced by a higher cellular uptake and cytotoxicity against these cells when compared to non-tumoral hepatocellular cells, with low sialic acid expression. Interestingly, HepG2 cells that survived de NO-releasing MIPs were re-cultured and treated with the nanoparticles, and no acquired resistance was observed. This is an important result for future studies with NO-releasing nanoparticles, as it is directly related to the NO chemical biology. NO and its sub products are known to cause oxidative and nitrosative stress, DNA deamination, besides interacting with different proteins, comprehending a complex mechanism that may hinder the development of resistance mechanisms in tumoral cells. 30,68 Despite focusing on NO, other gasotransmitters also present important biomedical roles, including cytotoxicity against tumoral cells. (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST). 71 Biological pathways occur through sulfhydration, a post-translational modification of specific protein cysteines. 69 Regarding antitumoral properties, H 2 S suppresses cellular bioenergetics, reduces the intracellular levels of antioxidant glutathione, and modulates p53 and nuclear factor-κB (NF-κB) pathways. 72 When cell were treated with NO/H 2 S-releasing nanoparticles, an enhanced angiogenesis was observed, resultant from the NO/H 2 S crosstalk. 70 applications, and further studies are welcome. The crosstalk between NO and H 2 S, and its biological responses, is under intensive investigation. 73

Cytotoxicity of NO-releasing polymer-functionalized nanoparticles against tumoral and non-tumoral cell lines

Polymer-coated metal or metal-oxide nanoparticles have also been reported as promising templates for delivering NO. 22,48,74 The surface functionalization enables the delivery of therapeutics, such as NO or traditional chemotherapeutics, and also promotes a targeted delivery to cancerous cells. 74,75 Among different nanoparticles, AuNPs have been one

of the most employed in drug delivery. 50 Similarly to what was demonstrated for CS NPs, AuNPs without hyaluronic acid and NO coating, suggesting an important role in drug internalization combined with NO cytotoxic effects. 50 The apoptotic and necrotic effect of NO was also evident on osteosarcoma cells (MG63) when combined with a hybrid nanoparticle based on SPIONs and AgNPs. 74

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Open Apoptotic and necrotic cells comprehended 25% of the cell population after 24 h treatment with hybrid nanoparticles based on SPIONs and AgNPs, coated with NO-releasing chitosan, at a concentration of 100 μg mL -1 , which corroborated with a pronounced cell viability reduction. Moreover, it was possible to observe the interference in the cell cycle, inducing a cycle arrest at S phase. Thus, it is evident that NO plays an important role against tumoral cells, combined or not with other therapeutics. Still, it is important to better comprehend the cytotoxic mechanism of NO against different cell lines, as well as the innovative proposed combinations with active nanomaterials and/or drugs.

Cytotoxicity of NO-releasing nanoparticles against multidrug resistant cancer cells

As reported in the previous section, when evaluating the potential cytotoxicity of NOreleasing nanoparticles against different cell lines, resistant cells demonstrated higher IC 50 when treated with RSNO-CS NPs, 51 compared with no resistant cells, confirming the necessity to employ higher dosages against these cell lines. We have recently published a review focused on the overview of recent innovative combinations of NO and nanomaterials, allied or not to chemotherapeutics, that represent potential approach to overcome resistance mechanisms. 3 An illustrative scheme shown in Figure 2 describes possible resistance mechanisms that can be reverted by nanoparticles combined with NO donors.

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Open Some mechanisms have correlated NO release to the downregulation of P-gp protein through the inhibition in MDR-1 mRNA levels. 77 A similar design was reported by Wu and coworkers, combining an important RSNO, S-nitrosoglutathione (GSNO), with a vastly used chemotherapeutic, DOX, in polymeric block nanoparticles based on poly(ethylene glycol) and poly(propylene sulfide) (PEG-PPS NPs). 80 Even at the lowest evaluated concentration (1 μg mL -1 of DOX), a statistically significant increase in the cytotoxicity against HepG2 cells was

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Open observed for the encapsulated drug combined with GSNO. When comparing tumoral to nontumoral cells, the cytotoxicity of the nanomaterial was 37-fold higher, evidencing a high selectivity, this is of great importance when it comes to chemotherapeutic drugs. Through apoptosis analysis, it was evident that GSNO encapsulated in PEG-PPS NPs at a concentration of 25 μmol L -1 already interferes in cell mechanisms and leads to a small percentage of apoptotic cells. Although, when combined with DOX and the PEG-PPS NPs, a synergistic effect was observed by reaching almost 100% of late apoptotic cells, in contrast with 23.6% for DOX encapsulated and 2.41% for GSNO encapsulated nanoparticles. With these data, it is clear that NO donor can sensitize the resistant cancerous cells, promoting a pronounced effect of the chemotherapeutic.

For a micelle nanoparticle with a similar design, combined with DOX, it was observed a pattern similar to the previously discussed when evaluated against MCF-7 cells. 78 Therefore, we expect to encourage researchers to further investigate the mechanism of NO in the MDR reversal/sensitization, as it consists in a promising advance in the area of chemotherapy.

Combination of NO-releasing nanomaterials with traditional anti-cancer therapies

Recently, in cancer treatment, the combination of chemotherapy or radiotherapy with NO donors is a promising strategy that might overcome drug resistance. Furthermore, the addition of nanomaterials allied to NO donors may enhance this positive effect.

Nanomaterials are able to load either NO donors/generators or chemotherapeutic drugs.

Interesting, the in clinical applications, administration of NO donors at subtoxic doses along with suboptimal doses of chemotherapeutics can lead to additive or synergistic effects, which are suitable for overcoming chemoresistance. 47 Such combinations (NO donors, nanomaterials and chemo/radio therapies) can also activate the anti-tumor immune response.

Chemotherapy remains restricted by poor drug delivery efficacy due to the heterogenous nature of tumor and to the development of tumor resistance. 81 To overcome these issues, considering combined approaches, versatile nanomaterials have been prepared

for cancer treatment. In particular, chemotherapeutic drugs can be allied to NO donors and nanomaterials. DOX and NO donor (RSNO) were loaded in mesoporous silica nanoparticles. 22 The obtained nanomaterial enhanced tumor penetration allowing a localized released of DOX and NO. NO released reacts with superoxide anion yielding the powerful oxidant peroxynitrite, under pathological conditions. This process promotes protein nitration and oxidation, lipid peroxidation, DNA damage. Moreover, peroxynitrite activates pro-matrix metalloproteinases into active matrix metalloproteinases, which degrade collagen in the tumor

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Open extracellular matrix. In other words, NO contributes to disintegrate tumor matrix collagen improving nanoparticle penetration. 22 A nanocarrier containing NO donor (N-NO moiety of a polymer) and paclitaxel was prepared. 82 The polymeric micelles (hydrodynamic diameter of 137 nm) were able to spontaneous release NO, under physiological condition, and demonstrated toxic effects against MDR ovarian cancer cell line OVCAR-8/ADR. Higher cytotoxicity was found for micelles containing both NO donor and paclitaxel, compared with micelles containing only individual drugs. Moreover, these nanoparticles were found in tumor tissue for more than 48 h. 82 Hyaluronan and keratin nanogels containing NO donor and DOX (average size of 60 nm) demonstrated significant and improved toxicity towards breast cancer 4T1cell line, which expresses high levels of CD44 receptor. 83 The natural polysaccharide hyaluronic acid specifically binds to CD44 receptors, which are over-expresses on the cytomembrane of tumor cells. The nanomaterials increased intracellular NO levels, which in turn sensitizes cancer cells enhancing the efficacy of DOX. Thus, in addition of EPR effects, the presence of hyaluronic acid in nanomaterials enhances the tumor targetability.

In addition to chemotherapeutics allied to NO in nanomaterials, irradiation can also be employed. High energy ionizing radiation in radiotherapy treatment damages DNA and other biomolecules causing cell death through photon interaction-biomolecules interactions or by the generation of oxygen and nitrogen radical species, including hydroxyl radicals and nitrogen dioxide. However, as solid tumors are often oxygen deficient, tumor resistance to radiation can be observed. In this tumor hypoxic microenvironment, NO has been reported to act as radiosensitizer, enhancing radical damage of biomolecules. 84 Recently, NO-releasing (nitrosylated maytansinoid containing poly(lactide-co-glycolic)block-poly(ethylene glycol) nanoparticles were used to sensitize non-small cell lung carcinoma to radiotherapy. Nanoparticles were efficiently delivered to tumors thorough the

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Open EPR effect, where upon irradiation generated oxidative stress and NO release. 85 In addition, soft X-ray (45 kVp, 0.18-0.85 mGy) was used in combination with NaYF4:Gd/Tb lanthanide scintillator nanorod as a light transducer for on-demand NO release (from Roussin's black salt) gas-sensitized cancer therapy. 86 This combined therapy can reach up to 3 cm in tumor tissue, making it suitable for solid tumors.

AuNPs were functionalized with nitroimidazole, a cell penetrating peptide (CPP) and poly(ethylene glycol) (PEG), for a combined cancer radiosensitization approach. 47 Nitroimidazole releases nitrite ions, NOx species, while CPP induce nucleus internalization of the nanomaterial. Nitrite ions are reported to easily reduced to NO, especially in a hypoxic and/or acidic tumor microenvironment. 87 The nanomaterial acted in the sensitization of cancer cells upon radiation with clinically used X-ray (6 MeV X-rays). Interestingly, nitrite ion release is trigged by X-ray radiation, and no release was observed without radiation. In vitro studies with epidermoid carcinoma cell line A-431 demonstrated that such nanoparticles combined with radiation have enhanced toxicity. The improved radiotherapy effect can be associated with the action of both nitrite ion/NO in the oxidative damage and the actions of AuNPs themselves in the production of ROS. 42 Recently, soft X-ray luminescence nanotransducer (down to 0.9 mGy) integrating These nanomaterials act as smart materials particular designed to target tumor cells/tissues.

Thus, the tumor microenvironment is of fundamental importance for the design of these nanomaterials. In this sense, near infrared laser was used in combination with hyaluronidasetriggered sized shrinkable hyaluronic acid shells, containing NO donor and DOX. 81 Upon systemic administration, larger size nanoparticles preferably accumulate in tumor site due to the EPR effect and CD44-targeting. 90 At the tumor site, small size dendrimeric produg of DOX and NO are formed via enzymatic degradation enhancing deep tumor penetration of the active drugs, increasing targetable-toxic effects (Figure 3). 1.

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Open liver, and kidney Hemolysis study: no hemolysis induced General toxicity includes measure of body weight a , or general signs of toxicity (behavior, dizziness, respiratory distress) b , or embryonic mortality, morphology modifications or behavior modifications c , or general signs of toxicity on the cardiovascular function (heart rate and mean arterial blood pressure) d , or hemolysis e . Histopathological studies were performed on major organs (heart, liver, spleen, lung, kidney) f and biochemistry analyses on blood g , on the coagulation system h , on the hepatic function i , on the renal function j .

The main animal species used to study NO in vivo effects are mainly mammals and especially mice, except one study in which Zebrafish embryos were used. 101 The mice usually bear tumors which are implanted before testing the NP. Tests on Zebrafishes are often used in the literature to assess the toxicity or the safety of compounds, drugs or even nanoparticles. 109 Moreover, the regulatory agencies, FDA and European Medicines Agency (EMA) accept these data for the approval of investigative new drug. Globally, the articles concerning the biocompatibility of NO releasing/producing NP reported a general good tolerance of the treatment by the animals.

Usually, besides the general signs of toxicity, an exploration of the possible disturbance of the biochemical parameters is explored. After the animal sacrifice, a histopathological study is often performed on main organs. The NO-releasing/producing NP are generally well-tolerated, only two articles reported a toxicity. 21,63 The toxicity can be induced by the NP and/or by the release or the production of NO. NO, the gaseous transmitter with pleiotropic activities in the organisms, at uncontrolled concentration may induce a great variety of toxic side effects (inflammation, respiratory problems, modification in heart rate, in blood pressure). 110 This can be counterbalanced by the very short half-life of NO and consequently, its limited action zone. 104 In parallel, after their parenteral administration, which is the most studied administration route, NP rapidly interact with immune cells and are distributed to reticuloendothelial organs (liver, spleen, lungs). 111 Combined, the properties of NO and NP explain why, besides general indicators of toxicity (body weight, general behavior of animals), other physiological and biochemical parameters must be explored to assess the potential systemic toxicity of the nanoarchitectures.

The interaction with immune cells, with or without an observable inflammation, were observed with some particles loaded with a RSNO. 21,63 In their work, 21 Liu and colleagues explained their observation by high release of high concentrations of NO in an immoderate manner, but the inflammatory effect could be also exacerbated by the nanoparticulate form.

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All these in vivo works can be considered as very preliminary toxicological studies and lay the bases for a more complete work. Indeed, to go further into the development of a drug candidate for a human use in medicine, the regulatory agencies impose many constraints to ensure the final safety of the drug. 112 For example, the toxicology tests must be performed on rodent and non-rodent species according to the Good Laboratory Practices (GLP). The toxicity must be evaluated for a long term: The International Conference on Harmonization (ICH) guidance recommends 9-month chronic toxicity studies in nonrodents. 112 The driving force to deeper evaluates the effects of NO-releasing nanomedicines in combination with traditional anti-cancer therapies is mainly based on the systemic toxicity of these traditional drugs and the increasing problem of MDR. NO-releasing nanomedicine is a promising candidate to mitigate the side effects of chemo/radio therapies. Much effort has been concentrated in the design of smart NO donors able to sensitively generate NO or NO-related species upon exogenous/endogenous stimuli at the desired site of application (tumor tissue), with minimum side effects to normal tissues. 3,74 Undoubtedly, nanotechnology has significantly contributed to the preparation of a variety of smart and efficient multifunctional NO-containing nanostructures in several biomedical applications, including cancer biology. Although several advances have been achieved in this topic, as presented in this work, some issues still need to be

Open

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Fig. 1 .

 1 Fig.1. Chemical structure of selected commonly employed NO donors: (a) Sodium

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  in tumor cells/tissues. When comparing free NO and nanoencapsulated NO at a final concentration of 20 µmol L -1 , a more pronounced cytotoxic effect was observed for the free NO donor. Inferior cytotoxic of encapsulated NO was evidenced by 2-fold increase in the IC 50 in comparison to free NO IC 50 , resultant from a slow NO release in the cell media. The mentioned in vitro results are important to critically analyze the importance of evaluating different parameters (i.e. blood compatibility, NO life-time in blood circulation, targeting efficiency and tissue accumulation) when it comes to NO delivery. Regarding the cytotoxic effect of nanoencapsulated NO, it is usually expected an improved cytotoxicity against tumoral cell lines. Although, despite observing a higher IC 50 for NO after encapsulation, a prolonged circulation of the encapsulated NO donor after intravenous administration was verified, with an outstanding 25-fold increase of the NO donor half-life and a higher accumulation in liver tumoral tissue, when compared to free NO donor. Similar pattern was observed for RSNO (1-10 mmol L -1 ) encapsulated in poly(propylene sulfide) nanoparticles, in which no significant cytotoxicity was observed for the encapsulated molecule, but a 2-fold increase in the accumulation in the targeted tissue was verified in vivo, with no apparent signs of inflammation.65 In contrast, other combinations have demonstrated potential cytotoxic effect against tumoral cell lines.30,51,66 RSNO encapsulated in chitosan nanoparticles (CS NPs), average hydrodynamic size of 108 nm, have demonstrated a sustained NO release for 10 h, at a Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online DOI: 10.1039/D1MA00532D

1 )

 1 , compared with non-tumoral melanocyte (Melan-A) cells. A cell viability reduction was observed in all evaluated concentrations (5-40 μg mL -1 ), although the resultant IC 50 demonstrated a higher toxicity against B16-F10 and HepG2 cell lines, when compared to nontumoral Melan-A cells. Leukemic cells demonstrated to be less sensible to NO-releasing CS NPs. Lucena 1 exhibited the highest IC 50 , which was expected as it is characterized as a resistant cell lineage derived from K562. When comparing the pair B16-F10 and Melan-A (both skin cells), a 2-fold selectivity for the tumoral cell-line reinforces the potential of NOreleasing CS NPs in the treatment of tumors. The reported cytotoxicity involved different mechanisms, such as caspase-dependent apoptosis, which was associated with oxidative stress promoted by NO, along with an increased superoxide production in the mitochondria and the oxidation of thiol containing proteins.67 Interestingly, it was also observed tyrosine nitration and cysteine S-nitrosation in cellular proteins, demonstrating the involvement of the delivered NO in different cell mechanisms.

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  69 Indeed, a more recent strategy has demonstrated promising results by combining two different gasotransmitters: NO and hydrogen sulfide (H 2 S), co-delivered by a polymeric nanoparticle based on carboxylfunctionalized mPEG-PLGH-thiobenzamide [(methoxy poly (ethylene glycol-b-lactic-coglycolic-co-hydroxymethyl propionic acid)-thiobenzamide)], PTA copolymer. 70 H 2 S is produced in mammalian cells mainly by three different enzymes: cystathionine-β-synthase

  AuNPs were functionalized with hyaluronic acid for targeting CDD4 protein expressed by breast tumoral cells and the consequent lung metastasis. Furthermore, AuNPs were functionalized to deliver the chemotherapeutic paclitaxel (PTX), indocyanine green and NO, from a nitrate ester modified hyaluronic acid. The combined nanomaterial (AuNPs, PTX, indocyanine green and nitrate ester modified hyaluronic acid) exhibited the lowest IC 50 against breast cancer cell line (4T1) when compared to controls (nanoparticles without indocyanine green, and free PTX), evidencing a synergistic effect of PTX, indocyanine green, and NO delivered by AuNPs. The NO role was even more evident in apoptosis analysis, in which the necrotic and apoptotic 4T1 cells reached 52% when treated with AuNPs, PTX, indocyanine green and nitrate ester modified hyaluronic acid, in comparison to 40.9% for

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Fig. 2 .

 2 Fig.2. Schematic representation of NO interference in resistance mechanisms. NO has

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  Micellespresenting 130 nm were obtained from a biodegradable polycarbonate-based copolymer, tailored for NO release from a cyclic nitrate trimethylene carbonate monomer. Once more, NO was correlated to the decrease of P-gp protein expression in a dose-dependent manner, enhancing the chemotherapeutic efficiency. Inorganic nanoparticles, as silica nanoparticles (230 nm), loaded with NO and combined with DOX, reinforced the mechanism of P-gp expression decrease when employed against MCF-7 cells.58 It is already clear that NO has an important involvement in the decrease in the expression of P-gp. Different designs of nanoparticles, drug combinations, and component concentrations may lead to different cytotoxic effects and biochemical mechanisms. Some issues are not as clear as the decrease of P-gp expression and should be further investigated. Do these combinations initiate inflammatory processes? Are there any DNA damages? What is the ideal proportion for promoting apoptosis and/or necrosis? What is the redox balance in MDR cells treated with NO-releasing nanoparticles? Is there any influence in enzymatic activities? Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

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ZnGa 2 O 4 :

 24 Mn nanoparticles with Roussin's black salt (as light trigged NO donor) was prepared. The authors reported an efficient NO release even after 40 min after stopping the radiation with a deep tumor tissue penetration. Similarly, NaYbF 4 :Tm@NaYF 4 :Yb/Er upconversion nanoparticles containing Roussin's black salt able to release NO upon nearinfrared radiation (980 nm) was reported.88 This combined therapy is able to either release high amounts of NO (for direct toxic effects) or release low concentrations of NO for modulation of P-gp to overcome multi-drug resistance. Bismuth sulfide nanoparticle/bis-Nnitroso nanocomposites were prepared to release NO upon near-infrared radiation (808 nm).89 Materials Advances Accepted ManuscriptOpen Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online DOI: 10.1039/D1MA00532D Other versatile strategies have been developing in recent years to overcome drug resistance in combined therapies involving NO donors, nanomaterials and chemo/radio therapeutic agents. Overall, the combination of light irradiation with nanomaterials and NO donors facilitates the spatiotemporal trigger of drug release and allows a targeted therapy.

81

 81

Fig. 3 .

 3 Fig.3. Schematic representation of the combination with hyaluronidase-triggered sized

Table 1 . 21 Page

 121 Phototriggered NO nanogenerators loaded with doxorubicin

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5 .

 5 Final remarks: Drawbacks, challenges and perspectives As represented and discussed in the work, the recent progress in the design of various promising nanoplatforms of release controlled and therapeutic levels of NO at the desirable site of application have opened new avenues in this interesting research field. Intensive and fruitful research has made several advances in the preparation of smart and versatile nanomedicines able to release NO-on demand in biomedical applications highlighting NO-sensitized synergistic cancer therapy. NO-releasing nanomaterials can have a direct cytotoxic effect (for high NO concentrations) or can sensitize cancer cells (for low NO concentrations, combined with other therapy, such as chemotherapy or radiotherapy). The combined therapy significantly improvedthe response over monotherapy in solid tumors.47 For instance, in radiotherapy, NO enhances DNA damage and inhibits DNA repair by acting as an oxygen-like radiosensitizer in hypoxic tumors.73 Materials Advances Accepted ManuscriptOpen Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online DOI: 10.1039/D1MA00532D

  further explored: (i) in-depth mechanism of NO inducing cell death, and more importantly, cancer cell sensitization should be investigated by using genetic and molecular approaches, (ii) development of nanomedicines stable at room temperature and able to release NO in a controlled manner upon and external or internal stimuli, (iii) further studies (in vitro, in vivo and clinical) of multitherapy by combining NO donors and/or traditional chemotherapeutic drugs, and/or radiotherapy and/or X-ray are still strongly encouraged to be developed, (iv) strategies to enhance deep penetration of NO and/or chemotherapeutic drugs still need to the further developed, by considering the tumor microenvironment. Moreover, a great challenge in the biomedical applications of NO donors is still the ability to accurate measure and real-time monitor the NO generation from the nanomedicines at the target site. To this end, several important and different experimental techniques should be considered. The exactly controlled of the NO release profile from the nanomaterial is of fundamental importance to avoid a burst in the NO release and poisoning. Further in vivo studies Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. are required and the fate of each component in the nanomaterial should be monitored in the biological system, along with the studies of biocompatibility, biosafety and biodistribution prior the clinical applications. Finally, we expect that this work brings new avenues in the development of integrated nanostructures form combined therapeutic modalities allowing a successful and safely use of NO-sensitized synergist effects. Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Article Online DOI: 10.1039/D1MA00532D Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online DOI: 10.1039/D1MA00532D Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Materials Advances Accepted Manuscript Open Access Article. Published on 04 October 2021. Downloaded on 10/7/2021 2:25:30 PM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

  31,37

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Acknowledgements

We have appreciated the support from CNPq (404815/2018-9, 313117/2019-5), FAPESP (2018/08194-2, 2019/07766-5, 2018/03646-2).

illustration of the synergistic effects upon laser irradiation for deep tumor penetration and therapy effects. Reproduced from reference 81 with permission from Elsevier. NaYF4:Yb,Er upconversion nanoparticles (75 nm) conjugated with the natural polymer chitosan and load with the photo NO donor Roussin's black salt and DOX were prepared. 91 Under acid conditions, usually found in solid tumor microenvironment, the entrapped DOX is released from swollen nanomaterials. The enhanced toxic effects demonstrated on colorectal carcinoma cell line (CT26 cells) was attributed to the combined therapy of NO and DOX released from the nanomaterial. As expected the simultaneous release of NO and DOX promoted a synergistic antitumor effect. The concentration range of NO release was found to be up to 2 µmol/L. These effects were attributed to the fact that low doses of NO mediates Pgp increasing DOX uptake, while high doses of NO have a direct toxic effect in coordination with DOX. 91 A similar approach was designed by combing NO-releasing platinum prodrugs and micelles in a photoresponsive therapy. 92

Conflict of interest

The authors declare no conflicts of interest.