Biomedicines | Free Full-Text | Advances in Nrf2 Signaling Pathway by Targeted Nanostructured-Based Drug Delivery Systems

Biomedicines | Free Full-Text | Advances in Nrf2 Signaling Pathway by Targeted Nanostructured-Based Drug Delivery Systems

3.1. Organic Nanostructures

Sabzichi et al. [56] formulated luteolin in nanophytosomes and tested their efficacy in MDA-MB 231 cells. They observed the suppression of Nrf2 and downstream genes like HO-1 and MDR1. These changes collectively led to significantly high apoptosis of cancer cells compared with luteolin alone. In earlier studies, luteolin proved to be an inhibitor of Nrf2; however, poor solubility and penetration restricted its application, which was overcome in this study by using nano-based phytosomes [57].
Yan et al. [58] reported a nanospray of ligustrazine on postoperative abdominal adhesion in rats, and it was observed that protein expressions of MMP-9, Nrf2, heme-oxygenase-1, and NQO1 were increased. In the same study, MCP-1 protein, TNF-α, and IL-1β were also found to be elevated. Similarly, a nanoformulation of roselle seed oil significantly inhibited the paracetamol-induced mRNA expression of pro-inflammatory cytokines, including TNF-α and IL-6 [59]. The same study also showed increased levels of Nrf2 and glutathione due to nanoformulation treatment. Pandhita et al. [60] reported a nano-based formulation of curcumin against cisplatin-induced renal injury. They observed upregulation of Keap1 and Nrf2 activation due to nanoformulation treatment. In addition, chitosan-coated curcumin nanocrystals were investigated against an LPS-induced sepsis model and HepG2 and J774 cells [61]. The nanocrystals conferred protection via Nrf2 activation with elevated SOD and GST levels. The results also revealed NF-κB downregulation and suppressed cytokine levels. Likewise, another study also showed that the liposomal nanocurcumin alleviated copper sulfate (CuSO4)-induced testicular lipid peroxidation, inflammation, and apoptosis via Nrf2/HO-1 signaling [62]. Here, it was postulated that the nanocurcumin activates Nrf2/HO-1 signaling by promoting the levels of Nrf2 and Bcl-2 with the enhancement of endogenous antioxidants, such as HO-1, GSH, and SOD, thereby protecting from tissue injury.
One of the most potent Nrf2 activators is CDDO-Me, a triterpenoid oleanolic acid analog that reportedly regulates inflammation, oxidative stress, and cell death. CDDO-Me is undergoing a Phase I trial in cancer patients [63]. To this end, in a most recent study, encapsulation of an Nrf2 activator, CDDO-methyl into polymeric nanoparticles, led to the upregulation of the expression of antioxidants and cytoprotective proteins for athero-protection [64]. They showed the accumulation of nanoparticles in atherosclerotic plaque of ApoE−/− and LDLr−/− mice, which were accompanied by the activation of Nrf2. The targeted delivery of nanoparticles to atherosclerotic plaque led to the elevation in expression of the Nrf2-regulated genes GCLC and NQO1. This study supports the paradigm that nanosystem administration of redox-active therapeutics can counteract the inflammatory response via Nrf2 activation. Furthermore, in another study, a molecular probe biosensor based on a multiplexed microfluidic device was designed as a nanoengineered platform to identify potent Nrf2 activators [65]. Similarly, nanoselenium is a potent antioxidant [66] and, therefore, has been studied for its antioxidant activity against cadmium-induced hepatotoxicity. The results revealed that nanoselenium mitigates oxidative stress via the Nrf2 signaling pathway and up-regulates the expressions of Nrf2 and their downstream targets, including HO-1, NQO-1, GST, and SOD [67].
Oridonin nanoparticle was developed using polyethylene glycol and poly-lactic-co-glycolic acid and proved effective against breast cancer by inducing ROS generation and also activating the Nrf2/HO-1 signaling pathway [68]. The polyethylene glycol-capped gold nanoparticles prevented acute kidney injury caused by renal ischemia-reperfusion by lowering lipid peroxidation, IL-1β, and TNF-α; upregulating the AMPK/PI3K/AKT pathway; and increasing Nrf2 expression [69].
The lipid-based nanotechnology for drug delivery systems can be applied in several forms, and solid lipid nanoparticles are the most effective and stable form. Interestingly, solid lipid nanoparticles exhibited several unique advantages, such as tissue targeting, controlled drug release kinetics, minimal immune response, high bioavailability, and loading efficiency [70]. Some reports stated that the encapsulation of oridonin, a natural product with solid lipid nanoparticles, enhanced the antitumor effect in different cancer cell lines, such as breast cancer and hepatocellular and lung cancer cells [71]. Here, nanoparticles induced cell cycle arrest at S and apoptotic rates. In this regard, another investigation demonstrated the importance of the Nrf2–NF-κB cross-link in anticancer activity [72]. When astaxanthin was encapsulated into solid lipid nanoparticles, a protective effect was observed against DMBA-induced breast cancer via the mTOR/Maf-1/PTEN pathway [73]. Moreover, astaxanthin-loaded nanoparticles suppressed p-AKT levels, further suppressing the expression of NF-κB and Keap1 with a simultaneous elevation of HO-1 and Nrf2 expressions. In agreement with these results, the antioxidant, anti-inflammatory, and neuroprotective activity of resveratrol-encapsulated solid lipid nanoparticles significantly improved the redox state and suppressed ROS generation [74]. The nanoparticles also caused a reduction in hypoxia-inducible factor 1α (HIF-1α) levels with Nrf2 activation and promoted the expression of HO-1 in vascular dementia [74]. The nanoparticle treatment caused a significant elevation in Nrf2 mRNA expression in the cortex, hippocampus, striatum, and cerebellum. Moreover, another report identified copolymers and trigonelline-entrapped micelles for colon cancer treatment [75]. These micelles had spherical shapes and significantly inhibited Nrf2 activation and ARE-related gene expressions. Furthermore, trigonelline-entrapped micelles enhanced oxaliplatin-induced apoptosis in an Nrf2-dependent manner. Emerging evidence also reveals that apigenin–solid lipid nanoparticles modulate oxidative stress and inflammation in diabetic nephropathy [76]. The anti-inflammatory effect of the nanosystem was studied in the mRNA expression of cytokines, such as IL-6, TNFα, and IL-1β. Moreover, treatment with nanoparticles promoted Nrf2 and HO-1 expression with simultaneous suppression of NF-κB activity and lipid peroxidation, suggesting the existence of antioxidant and anti-inflammatory properties.
Shahin et al. [77] designed, synthesized, and evaluated the potential of caffeic acid phenethyl ester-loaded nanoliposomes in a rat model of acute pancreatitis. They observed protection against malondialdehyde, NF-κB, TNFα, and caspase-3 activity. Furthermore, nanoliposomes also caused Nrf2 activation and up-regulated gene expressions of antioxidants such as glutathione reductase and HO-1, leading to modulation in the Bcl-2/Bax ratio. Thus, the nanoliposomal system more profoundly counteracts oxidative stress and inflammatory response by targeting Nrf2 activation compared with standard drugs without a nanosystem due to efficient cellular uptake and improved pharmacokinetics. A study found that long non-coding RNA (MT1DP) can effectively sensitize A549 and H1299 cells by down-regulating Nrf2 through stabilizing miR-365a-3p and up-regulating ROS, which in turn causes erastin-induced ferroptosis in non-small cell lung cancer. This was achieved by developing a nano-delivery system using folate-modified liposomes [78]. With a mean particle size of 80 ± 5 nm, quinacrine-loaded liposomes, an Nrf2 inhibitor, demonstrated decreased Nrf2 expression. This was followed by its downstream genes, MRP1, Trx, and bcl2, which ultimately produced A549 lung cancer cells that were more sensitive to cisplatin [79].

3.2. Inorganic Nanostructures

Graphene oxide (GO) is a two-dimensional nanomaterial, and its particle-like properties make them suitable for studies with inflammatory responses. The effects of different GO sheets (small, ultrasmall, and large) were investigated on inflammatory responses in macrophages [80]. They found that GO small sheets inhibit IL-1β, IL-6, and TNF-α secretion. Specifically, the authors suggested that the GO small sheets can not influence NLRP3 inflammasome activation; however, they reduced the expression of pro-IL-1β. It was suggested that the GO small sheets are efficiently internalized and inhibit the expression of IL-1β and IL-6 mRNA in LPS-stimulated macrophages. To this end, the authors then investigated the mechanism of this inflammatory gene regulation, and they found that Nrf2 activation up-regulates the expression of anti-inflammatory genes and suppresses IκBζ−NF-κB-mediated pro-inflammatory mediators, including IL-1β and IL-6.
Among several nanoparticles, silica nanoparticles have been shown to induce an inflammatory response even after short-term exposure, owing to their large surface area and optical transparency. Nano-SiO2 has been reported to induce carcinogenesis via suppressed DNA methylation [81]. In cancers, Nrf2 plays a contrary role, and its loss leads to malignant cellular transformation [82]. In contrast, accumulating evidence also suggests that Nrf2 activation promotes cancer cell growth and metastasis in different tumor types [83,84,85]. In this context, nano-SiO2 exposure leads to carcinogenesis in human bronchial epithelial cells via DNA hypomethylation and altered methylCpG binding protein expression. They also observed Nrf2 activation by CpG island demethylation within the promoter region of Nrf2. All these changes lead to the Nrf2 upregulation, which might be the reason for alleviating the nano-SiO2-induced carcinogenesis [86]. Furthermore, they reported an enhancement in antioxidants, such as HO-1, SOD1, and GST levels, due to nano-SiO2 exposure. In an earlier study, it was reported that nano-SiO2 (10–20 nm) exposure up-regulated the levels of PKR-like endoplasmic reticulum (ER)-regulated kinase (PERK), Nrf2, and HO-1 in A549 cells [84]. Next, they developed A549-shNrf2 cells and observed Nrf2 activation due to nano-SiO2 exposure; however, the changes observed were less profound than the A549 cells. It was also reported that the Nrf2 expression level was reduced significantly in Nrf2−/− ICR mice exposed to nano-SiO2 along with ROS elevation compared to the wild type. Thus, these results suggest that Nrf2 activation protects against nano-SiO2-induced toxicity. In a recent study, Argenziano and co-authors [87] designed a chitosan-shelled nanobubble (siNrf2-NBs) for siRNA against Nrf2, demonstrating the downregulation of target genes in M14 cells. The treatment was administered in combination with ultrasound to treat melanoma cancer cells.
Nowadays, gold nanoparticles (AuNPs) and their derivatives have also been a focus for their different applications [88]. However, AuNPs also reportedly show toxicity signs [89,90,91]. In this context, Bajak and co-workers studied the plasmonic excitation effects of AuNPs (5 and 30 nm) on Caco-2 cancer cells [92]. Interestingly, small AuNPs (5 nm) induced the expression of Nrf2-responsive genes, such as HMOX, G6PD, oxidative stress-induced growth inhibitor 1 (OSGIN1), and glutathione peroxidase 2 (GPX2). Furthermore, these results revealed oxidative stress and a disturbed redox state due to the AuNPs, indicating their anticancer property via the Nrf2 pathway. However, low concentrations of AuNPs affect GSH levels only slightly and, thus, activate Nrf2 in a ROS-dependent manner. Here, it is worth mentioning that γ-glutamyl cysteine ligase and glutathione synthetase, which are associated with GSH synthesis, are regulated by the Nrf2 pathway [93]. Thus, the interaction of AuNPs to GSH can be related to Nrf2 activation. Nrf2 activation by AuNPs and their plasmonic excitation can be a new platform for treating several pathologies. In a murine model of streptozotocin-induced diabetic nephropathy, pomegranate peel extract was tested in conjunction with stabilized gold nanoparticles. It reduced inflammatory mediators by modulating the MAPK/NF-κB/STAT3/cytokine axis and also activated PI3K/AKT/Nrf2. Eventually, this led to an increase in antioxidant enzymes, a decrease in blood glucose, a decrease in triglycerides, an increase in insulin, and a return of pancreatic β-cell dysfunction [94].
In another study, both silver (AgNPs) and AuNPs reportedly showed uptake into the cytoplasm; however, no uptake was observed within the nucleus of Caco-2 cells [95]. Also, the AgNPs reduced the total glutathione and upregulated the expression of Nrf2 and HO-1. However, these AgNP-induced changes were significantly reversed by siRNA silencing of Nrf2 transcripts [95]. A study in Nrf2-knockout HK-2 renal epithelial cells treated with AgNPs showed G2/M growth arrest, high phospho-CDC25C, phospho-CDC2, and GSH depletion [96]. In addition, it was observed that the pre-treatment with N-acetylcystein caused Nrf2 pathway upregulation of GCLC and GCLM expression, thereby enhancing the GSH levels. Therefore, taken together, all these results show that the Nrf2–GSH pathway can be involved in the AgNP-mediated toxicity in epithelial cells.
8-Oxoguanine DNA glycosylase 1 (OGG1) is a DNA repair protein marker for ROS generation. When human Chang hepatocytes were treated with AgNPs, OGG1 protein expression was significantly suppressed [97]. They revealed that AgNPs downregulated OGG1 expression with concealed Nrf2 binding to the promoter region of OGG1 and, thereby, decreased nuclear Nrf2 levels. It has also been reported that an Nrf2 transcription factor binding site is localized in the promoter region of the human OGG1 region, which correlates the levels of OGG1 with the redox state of the cells [98]. ERK and AKT reportedly phosphorylate Nrf2, leading to the Nrf2 release from Keap1, followed by its translocation into the nucleus [99,100]. The results revealed that AgNPs downregulate ERK and AKT and, further, OGG1, intensifying oxidized DNA bases. Nrf2 and stimulation protein-1 (SP-1) have binding sites in the OGG1 promoter region [98]. On the other hand, the AKT pathway plays a crucial role in regulating Nrf2 and SP-1 [101,102]. Similarly, Nrf2 activation is also regulated by the ERK pathway and the upregulation of OGG1 [103,104]. Therefore, the AKT and ERK pathways are responsible for the upregulation of OGG1.
It is also now established that iron oxide nanoparticles loaded with curcumin (FeNPs) exhibit ROS-scavenging ability in chondrocytes and reduce the elevated levels of IL-1β [105]. These results were further corroborated by the observation that Nrf2 activation occurred along with the suppression of the nod-like receptor protein-3 inflammasome (NLRP3) activation. Also, all these changes lead to the inhibition of matrix-degrading proteases and other inflammatory factors. Quite recent data report that bovine serum albumin-encapsulated magnetite nanoparticles entrapped with epigallocatechin 3-gallate induced ROS generation and apoptosis in lung adenocarcinoma cells [75]. The nanoparticles exhibited elevated Nrf2 and Keap1 expressions, elevating cell apoptosis.
Similarly, studies on cobalt nanoparticles (CoNPs, 30 nm), mostly used in artificial joint replacements, showed ROS generation and activation of the Nrf2 signaling pathway [106]. On the other hand, selenium nanoparticles (SeNPs) suppress oxidative stress in cells [107,108]. Multiple studies studied the potential mechanisms underlying the antioxidant activity of SeNPs in different cell lines. In IPEC-J2 cells, SeNP treatment activated Nrf2, mitogen-activated protein kinase (MAPK), and the AKT pathway and simultaneously raised the levels of HO-1 and NQO-1 [109]. Moreover, SeNP treatment also promoted phosphorylated-Nrf2 levels without affecting Keap1. Importantly, this phosphorylation was reportedly mediated via p38, ERK1/2, c-Jun N-terminal kinase, and the AKT pathway. Similarly, biogenic SeNPs synthesized using bacteria showed maintenance of intestinal redox homeostasis via an Nrf2-mediated pathway [110]. Biogenic SeNPs (Nrf2) activated Nrf2 and upregulated the expressions of thioredoxin reductase (TXNRD)-1, NQO-1, HO-1, and thioredoxin (Trx) in concentration-dependent manners. These results were more significant as compared with chemically synthesized SeNPs.
In cancer treatment, photothermal therapy and photodynamic therapy proved highly beneficial due to their target selectivity, non-invasive nature, and negligible drug resistance [111,112]. However, the intracellular antioxidant system will partly hinder phototherapy for anticancer treatments. In some instances, the Nrf2 signaling pathway has been shown to promote the resistance of tumor cells to phototherapy [113]. Thus, Nrf2-specific siRNA can be a potential photosensitization strategy to promote phototherapy efficiency via the Nrf2 pathway [114]. However, the targeted delivery of Nrf2–siRNA in tumor cells is also necessary [115,116]. In this regard, a potential nanosystem, including poly(β-amino ester)/poly lactic-co-glycolic acid-based nanoparticles encapsulated with indocyanine green and Nrf2–siRNA, was designed and developed [117]. This nanosystem was then further loaded inside the vesicles of cell membranes derived from oral tongue squamous cell carcinoma cells. These nanoparticles suppressed the Nrf2 signaling pathway and promoted anticancer phototherapy by enhancing ROS accumulation. All these changes led to the apoptosis of cancer cells and reduced tumor growth in animal models. In another report, lung cancer cell death occurred via ferroptosis as a result of zero-valent-iron nanoparticle (ZVI-NP)-induced mitochondrial malfunction, intracellular oxidative stress, and lipid peroxidation. Such cancer-specific ferroptosis increased the degradation of Nrf2 by GSK3/-TrCP via activation of AMPK/mTOR [118]. The authors also revealed the tuning of macrophage polarization from the immunosuppressive M2 phenotype to the antitumor M1 phenotype and suppression of PD-1 and CTLA4 in CD8+ T cells, thereby inducing ferroptosis.
Titanium dioxide nanoparticles enhanced Nrf2 expression in liver cancer cells along with its associated target genes NQO1, HO-1, and GCLC, while Nrf2 loss in Nrf2(−/−) cells significantly increased susceptibility to DNA damage [119]. After conjugating with spironolactone, zinc oxide nanoparticles were created, which ultimately reduced kidney injury by increasing Nrf2 and HO-1 expressions and decreasing inflammatory mediators like TGF-β1, Wnt7a, β-catenin, fibronectin, and collagen [120]. In streptozotocin-induced diabetic rats, cerium oxide nanoparticles (30 mg/kg bodyweight/day for 4 weeks) enhanced sperm fertility by promoting Nrf2 expression, which inhibited sperm DNA fragmentation [121]. Similarly, in streptozotocin-induced type 1 diabetic Swiss mice, nanoceria effectively decreased glucose levels, lowered IL-6 and TNF-α, increased Nrf2 expression, increased superoxide dismutase, and decreased apoptosis [122].
Zinc oxide nanoparticles disrupted the ubiquitin–proteasome system, effectively stimulating the Nrf2 signaling pathway and reducing endothelial damage [123]. Zinc oxide nanoparticles improved the ability of Nrf2–DNA binding, reduced AGE, inhibited NLRP3-mediated inflammasome activation, reduced interleukins, and triggered Nrf2/TXNIP/NLRP3 inflammasome signaling [124]. In diabetic rats, Cyperus rotundus-loaded zinc oxide nanoparticles significantly enhanced antioxidant enzymes and downregulated procaspase-1, caspase-1, IL-18, and IL-1β [123]. In rats exposed to adenine-induced nephrotoxicity, TGF-β1, Wnt7a, β-catenin, fibronectin, collagen IV, α-SMA, TNF-α, and IL-6 were reduced by zinc oxide nanoparticles and the spironolactone-upregulated Nrf2/HO-1 pathway [120]. Similarly, by improving Nrf2 activation and interfering with ubiquitin–proteasome-dependent Nrf2 degradation, copper oxide nanoparticles decreased vascular injury and disease [125].

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