1. Nanomedicines for Oral Delivery of AST and BR
1.1. Nanomedicines to Treat Inflammatory Bowel Diseases (IBDs)
IBDs, including Crohn’s disease and ulcerative colitis, are relapsing disorders of the gastrointestinal tract (GIT) with no cure, characterized by chronic inflammation and epithelial injury induced by the uncontrolled activation of the mucosal immune system 
. OS is primarily responsible for IBD pathophysiology 
. The ROS in the intestinal mucosa of IBD patients is 10- to 100-fold higher than that in healthy mucosa 
. Whereas the co-administration of anti-inflammatory drugs and antioxidants has shown clinical benefits in IBD patients 
, no specific antioxidant treatment against IBD is currently available. Successful oral treatments depend on maintaining the drug’s structures along the gastrointestinal transit and on the feasibility of macrophage and dendritic cell targeting. Both AST and BR suffer gastrointestinal degradation. Targeting macrophages with AST or BR loaded nanoparticles aimed to protect them along the gastrointestinal transit could make IBD treatments more effective than conventional therapies.
AST loaded into caseinate microparticles covered with chitosan- triphenylphosphonium bromide (TPP, a well-known mitochondria-targeted moiety due to its high lipophilicity and stable positive charge 
) and sodium alginate (CC-AST) were prepared by the electrostatic self-assembly method 
. The sodium alginate (a natural polysaccharide containing a large number of carboxyl groups) cover provides pH-responsiveness to microparticles, which in the stomach are protonated and then are agglomerated, protecting thus the AST in the core. Upon reaching the intestine under alkaline conditions, the alginate is deprotonated, and the microparticles acquire negative charge and are redispersed. In vitro studies showed that CC-AST are internalized by RAW264.7 macrophages, accumulate in mitochondria, and inhibit ROS and mitochondrial membrane depolarization in LPS-induced macrophages. In a murine model of colitis, the previous and during-induction administration of CC-AST relieves colitis and significantly inhibits the expression of the inflammatory markers IL-1β, IL-6, TNF-α, cyclooxygenase-2, myeloperoxidase (MPO), iNOS, and NO in a more potent way than free AST. In addition, CC-AST protects the integrity of the colon tissue structure, maintaining the expression of the tight junction protein zonula occludens-1. Two dominant phyla, Firmicutes
, represent more than 90% of the intestinal commensal microbes of the human gut microbiota. The balance between Firmicutes
is related to host health: in the murine colitis model the abundance of Bacteroides
is increased with the concomitant decreased ratio of Firmicutes/Bacteroides.
CC-AST increases the relative abundance of Firmicutes; Lactobacillaceae
, on the other hand, improves the intestinal environment, relieving inflammation.
The same authors have recently reported the preparation of ultrasonic assisted self-assembled Np, where AST was loaded into ROS-triggered self-disintegrating mitochondrial-targeted Nps made of poly (propylene sulphide) (PPS) and Rhodamine 123 (RD) covalently modified with sodium alginate 
. PPS is a hydrophobic ROS-responsive functional group that can be converted from hydrophobic sulphide groups to hydrophilic sulfoxide/sulfone groups under ROS stimulation. RD, on the other hand, was used as a targeted ligand to mitochondria. The combination of PPS with sodium alginate at a 3:1 w
ratio was required for the self-association of Nps. It was observed that the protonation of sodium alginate in simulated gastric fluid induced Nps aggregation with further deprotonation in simulated intestinal fluid, swelling, and AST release; the incubation with H2
instead disintegrated the Nps. The Nps accumulate in mitochondria, inhibit ROS, and protect against mitochondrial membrane depolarization in LPS-induced Raw264.7. The previous and during-induction administration of Np relieves the severity of colitis, protects the integrity of colon tissue, and restores the expression of ZO-1 and occludin (while free AST and void Nps do not). The abundance of Lactobacillus
and the Firmicutes/Bacteroides
ratio of gut microbiota are significantly improved.
In a similar approach, AST was loaded into Np having a core made of TPP-modified whey protein isolate-dextran conjugate (for mitochondrial targeting) and covered with hyaluronic acid (HA) modified with lipoic acid for macrophage targeting and a GSH-stimulated release feature (HL-TW-AST) 
. Whereas HA has a strong affinity by cluster of differentiation protein 44 (CD44), overexpressed on the surface of activated macrophages in colitis tissues, the disulphide bonds of lipoic acid are reduced in the presence of high intracellular GSH in inflamed cells, releasing the Nps core
. The lipoic acid–HA coating protects from early AST release in the stomach; macrophage and mitochondria targeting were shown in vitro on Raw264.7 cells. On LPS-induced macrophages, HL-TW-AST reduces ROS; reconstitutes to normal levels the mitochondrial membrane potential; increases the CAT, SOD, and GSH levels; reduces the iNOS, NO, TNF-α, IL-1β, and IL-6 levels; and increases the anti-inflammatory IL-10 cytokine levels. In murine colitis models, the previous and during-induction administration of HL-TW-AST markedly alleviates clinical symptoms (body weight loss, colon length, and spleen weight) and inflammation (decreases the level of malondialdehyde (MDA, a stable metabolite of lipid peroxidation); increases the levels of CAT and GSH; reduces MPO, iNOS, TNF-α, IL-1β, and IL-6; and increases IL-10 levels) more extensively than free AST. The anti-inflammatory effects of HL-TW-AST are mediated by the modulation of TLR4/MyD88/NF-κB signaling pathway. The HL-TW-AST administration is also shown to improve the composition of gut microbiota and the production of short-chain fatty acid.
A simpler approach was recently launched, where an o/w emulsion of AST in olive oil and soy lecithin was encapsulated in alginate microspheres by high-pressure spraying and ionic gelation with CaCl2 
. In vitro, the protection of AST along the GIT was observed, as well as its release in the colon thanks to the degradation of alginate by the gut microbiota. The microparticles increased the colon length, increased the liver weight, and reduced the spleen weight increase in a murine model of colitis. In the colon tissue, the microparticles are observed to reduce the levels of IL-6 and IL-1β, MPO, and iNOS and increase the levels of IL-10, ZO-1, occluding, SOD, and GPx, accompanied by increased Firmicutes
Scavenger receptors class A (SR-A1) are involved in the innate immune response in intestinal inflammation 
. SR-A1 negatively regulates NF-κB signaling and stimulates the production of reparative cytokines, shifting macrophage phenotypes from pro-inflammatory (M1) to anti-inflammatory (M2) 
. Recently, nanostructured archaeolipid carriers (NACs) were loaded with BR plus dexamethasone (NAC-Dex). The NAC consisted of a compritol and BR core, covered by a shell of polar archaeolipids (PA) extracted from the halophilic archaea Halorubrum tebenquichense
and Tween 80 
. The shell provides macrophage targeting because of its high content of 2,3-di-O-phytanyl-snglycero-1-phospho-(3′-sn-glycerol-1′-methylphosphate) (PGPMe), a ligand for SRA1 
. The shell provides also structural endurance since the PA (mixture of the sn
2,3 ether linked phytanyl saturated archaeolipids) is resistant to hydrolysis, oxidation, and stereospecific phospholipases 
. NAC-Dex was observed to display high anti-inflammatory and antioxidant activities on a gut inflammation model made of Caco-2 cells and LPS-stimulated THP-1-derived macrophages, by reducing TNF-α and IL-8 release and ROS production. NAC-Dex also reverses the morphological changes induced by inflammation (normal microvilli, well-defined tight junctions, desmosomes, interdigitations, and F-actin filaments) and increases the transepithelial electrical resistance, partly reconstituting the barrier function. After in vitro gastrointestinal digestion, NACs retain their size and structure, while important, the anti-inflammatory activity of NAC-Dex remains intact, indicating the high structural resistance of Nps prepared with lipids extracted from halophilic archaebacteria.
1.2. Nanomedicines to Treat Liver Damage
In a model of liver damage, orally administered liposomal AST before an LPS challenge was used to decrease and normalize the serum levels of glutamate-pyruvate transaminase, blood urea nitrogen (BUN), creatinine, and glutamate-oxaloacetate transaminase to levels comparable to those induced by the antioxidant N-acetylcysteine, more efficiently than free AST 
. Liposomal AST also reduced the serum levels of NO, IL-6, and TNF-α more efficiently than N-acetylcysteine and free AST. In addition, the hepatic levels of MDA, SOD, and GPx were restored, and those of CAT were partially alleviated, while N-acetylcysteine and free AST provided moderate relief. The increased levels of iNOS and nuclear NF-κβ induced by LPS, were also decreased more efficiently than N-acetylcysteine and free AST. The authors suggest that liposomes increase the AST bioavailability, although neither the gastrointestinal stability of liposomal AST nor its biodistribution are reported. Moreover, a potential mechanism by which the AST bioavailability is increased is not provided; apparently, the AST bioavailability was increased despite the poor structural stability of liposomes in the GIT.
Recently, AST loaded into lactobionic acid (LA, targeting asialoglycoprotein receptors on hepatocytes) modified hydroxypropyl-β-cyclodextrin (AST-LA-CD) was administered for liver-targeting 
. AST-LA-CD released 12% and 25.6% of AST after 2 h in simulated gastric fluids and 4 h in simulated intestinal fluids, respectively. In vitro, AST-LA-CD showed increased cellular uptake and prevented mitochondrial depolarization and ROS induced by H2
, compared with non-targeted CD and free AST. After oral administration of Nile Red labeled-LA-CD (where AST was replaced with the hydrophobic fluorescent dye Nile Red, a molecule used to track its biodistribution) a higher fluorescence in liver was shown compared with non-targeted CD. This result could suggest that the LA-HD nanocarriers had stronger liver-targeted ability as compared with HD nanocarriers. However, free absorption of the dye could not be discarded given that Nile Red was not covalently attached to LA-CD. Therefore, no rigorous evidence showing cyclodextrin absorption to gain blood circulation is provided.
1.3. Nanomedicines to Treat Inherited Retinal Degeneration
Retinal degeneration is a heterogeneous group of retinopathies affecting the outer layers of the retina, damaging the photoreceptor layers and the pigmentary epithelia, and perturbing the visual field, causing night blindness and altering color perception. If the molecular mechanism of the disease is still unknown and no specific treatment is available, the OS is known to be involved in the apoptosis of photoreceptor cells. Orally administered polysorbate 20 micelles loaded with AST (AST micelles) improved the architecture and functionality of the retina in a chemically induced mouse retinal degeneration model 
. The AST micelles preserved photoreceptor responsiveness and inhibited photoreceptor loss in the degenerative retina, corroborated by electroretinography results and behavior tests, more efficiently than lutein (which is known to access the retina because of its ability to cross the blood–retinal barrier). Unfortunately, the work did not test the activity of free AST. The AST micelles significantly reduce the mRNA level of caspase-3 (mediator of photoreceptor apoptosis) and Bax and increase the mRNA level of the anti-apoptotic Bcl-2, showing anti-apoptotic effect. In addition, the MDA and 8-OHdG levels are reduced and the SOD and Mn-SOD levels in the retina are increased to a higher extent than those induced by lutein. Whereas AST PK and BD of micellar AST were not determined, this, together with the oral administration of liposomal AST, is another example of increased AST bioavailability mediated by nanomedicines.
2. Nanomedicines for Topical Delivery of AST and BR
2.1. Nanomedicines to Treat Atopic Dermatitis (AD) and Psoriasis (PS)
OS and inflammation are involved in the pathogenesis and complications of cutaneous inflammatory diseases, such as AD and PS 
. Topical treatments are ideal for direct action on the diseased cell: the first-pass metabolism and further systemic effects are avoided, they can be chronically applied, and the patient’s adhesion is good. However, AST and BR, in addition to being sensitive to oxidation induced by light and heat, do not permeate across the stratum corneum (SC) to enter the viable epidermis.
The AD is a chronic inflammatory affection of the skin, characterized by a massive release of proinflammatory cytokines and increased levels of IgE. In a recent work, despite not reporting skin permeation, liposomal AST was observed to decrease the severity of symptoms in a murine model induced by phthalic anhydride (displaying dermatitis, epidermal thickening, and mast cell infiltration) 
. The topical treatment suppressed inflammatory mediators (iNOS and COX-2) and OS (reduced MDA levels and increased GSH, GPx, and HO-1) to a higher extent than free AST dissolved in acetone:olive oil 4:1 v
ratio. Liposomal AST reduces STAT3 and p65 phosphorylation, inhibiting the activation of STAT3 and NF-kB. Remarkably, STAT3 is critical for regulating IgE levels, IgE-based allergen sensitization, and mast cell degranulation 
PS, on the other hand, is a multifactorial autoimmune skin disease, where OS plays a key role in promoting a vicious cycle between keratinocytes and immune cells. Recently, BR was loaded into NAC plus vitamin D3 (NAC-VD3) 
. The combination of BR and VD3 in the NAC core produced a synergic antioxidant effect (measured by DPPH) and protected VD3 against thermal degradation. NAC-VD3 were extensively captured and displayed high anti-proliferative (65%), anti-inflammatory (IL-8 release), and antioxidant activities (ROS reduction) on a psoriatic model made of CaCl2
differentiated-imiquimod stimulated HaCaT cells and on lipopolysaccharide-induced THP-1 macrophages.
2.2. Nanomedicines to Treat UV-Induced Skin Damage
UV irradiation generates ROS and induces skin damage such as inflammation, oxidative alteration of collagen, production of melanine, DNA alteration, and skin cancer in the long term. A topical pre-treatment with liposomal AST is effective to prevent morphological changes in a UV-induced mouse skin damage model, such as wrinkles and inflammation, better than free AST dissolved in DMSO 
. Liposomal AST inhibits the increase in thickness of the SC
induced by UV irradiation, preventing the decrease in the collagen amount below the SC
layer. In addition, the combination of iontophoresis with cationic liposomal AST as a pre-treatment inhibits the UV-melanin production in the basal layer, indicating than liposomal AST prevents melanocyte damage from UV radiation. More recently, it was shown that the application of liposomal AST reverts the pathological changes induced by skin irradiation, such as the organization of collagen fibers and the dermis thickness, accompanied by decreased expression of Ki-67 (proliferation index related to cancer progression), MMP-13, and 8-OHdG (indicator of oxidative DNA damage) and increased SOD activity 
2.3. Nanomedicines to Treat Dry Eye Disease (DED)
DED is a multifactorial disease, where the OS induced by the decreased volume of tears, their excessive evaporation, and hyperosmolarity play a key role. The daily application of topical drops containing liposomal AST on a murine model of DED prevented the increase in the fluorescein score (as a corneal damage measure) and the upregulation of age-related markers (p53, p21, and p16). The medication, however, did not increase the tears volume 
. Liposomes displaying a small positive charge showed increased affinity by the cornea compared with neutral liposomes, efficiently ameliorating its deterioration, as a clinical symptom of DED, measured by superficial punctate keratopathy.
2.4. Nanomedicines for Otoprotection
A frequent secondary effect of cisplatin chemotherapy is ototoxicity, which is produced by an excess of ROS. Aiming to increase the penetration through round window membranes, AST was loaded into lipid-polymer hybrid Nps made of a Peg-PLA and AST core covered by a shell of lipids (AST-Nps), prepared by an emulsion and evaporation technique 
. The AST-Nps impair mitochondrial membrane potential reduction and avoid apoptosis (by suppressing the release of pro-apoptotic proteins, cleaving caspase 3/9 and cytochrome-c, and increasing the expression level of Bcl-2) induced by cisplatin on HEI-OC1 cells (House ear institute-organ of corti 1). AST-Nps but not free AST penetrate the round window membrane and maintain AST concentrations in the perilymph in the inner ear for 24 h after a single administration on guinea pigs. AST-Nps efficiently provide otoprotection to zebrafish hair cells against cisplatin and to guinea pigs exposed to cisplatin, especially in the higher and the ultrahigh frequencies.
3. Nanomedicines for Intra-Articular Delivery of AST
Nanomedicines to Treat Osteoarthritis (OA)
OA is a degradative disease of the cartilage caused by the mechanical wear and tear of the joints that mainly affects the hips and knees, and its incidence increases with age. OA involves the progressive loss of cartilage, remodeling of bone, inflammation, and deformation of the joint. Regardless of the causes, joint destruction is associated with the presence of proinflammatory cytokines including TNF-α, IL-1β, IL-6, immune cell subsets including macrophages, neutrophils, and activated synoviocytes 
and expression of MMP that degrades the joint. In addition to inflammatory mediators, ROS and RNS play a key role in joint damage 
. Macrophages and synoviocytes are the most abundant cells in the inflamed synovial and are fundamental for the progression of chronic inflammation and tissue destruction 
. A ROS-responsive triblock copolymer (poly (ethylene glycol)-polythioketal-poly (ethylene glycol) (PEG-PTK-PEG)) through a simple and direct reaction between polythioketal (PTK) and m-PEG-acrylate, for AST delivery into the articulation was recently synthesized 
. The polymer contains the lipophilic PTK segments in the middle of two hydrophilic PEG segments with a hydrophilic/lipophilic ratio of 31%, so that above the critical micellar concentration are formed micelles with a hydrophobic core where AST was encapsulated. In LPS-induced bone marrow-derived macrophages, PEG-PTK-PEG@AST micelles showed intracellular and extracellular ROS-scavenging effects higher than PEG-PTK-PEG and free AST, indicating a synergistic effect of PEG-PTK-PEG and loaded AST. Likewise, PEGPTK-PEG@AST micelles induce the transformation of the pro-inflammatory M1 phenotype provoked by LPS, to the anti-inflammatory M2 phenotype, and reverse the effect of LPS on IL-1β and TNF-α expression. In the OA rat model, PEG-PTK-PEG@AST micelles are gradually decomposed over time, remaining at day 7 in the OA tissues, after intra-articular injection. PEG-PTK-PEG@AST micelles show the best ROS-responsive scavenging ability and in vivo M1 transformation into the M2 phenotype. PEG-PTK-PEG@AST micelles demonstrate the most significant inhibitory effect on the expression of pro-inflammatory factors (MMP-2, IL-1β, TNF-α, and PGE-2) and on promoting the expression of marker proteins of cartilage anabolism (Col-2, aggrecan, and Sox-9), inhibiting the expression of cartilage catabolism (MMP-9 and MMP-13). The cartilage structure in the PEG-PTK-PEG@AST micelles group is better preserved.
4. Nanomedicines for Endovenous Delivery of AST
4.1. Nanomedicines to Treat Liver Injury
The 3R,3′R isomer of AST resulted in the best isomer to be loaded together with capsaicin (CAP) (1:2 molar ratio) in liposomes bilayers to exert a synergic antioxidant effect 
. Liposomal AST-CAP showed a protective effect in a murine model of liver injury upon intravenous administration, better than liposomal drugs loaded alone or combined treatment 
. Liposomal AST-CAP significantly decreased the aspartate transaminase and alanine aminotransferase levels.
4.2. Nanomedicines to Treat Diabetic Nephropathy
Diabetic nephropathy is a severe long-term complication from type 1 and 2 diabetes, which is presented as a decreased glomerular filtration rate, glomerulosclerosis, tubulointerstitial fibrosis, and renal tubular epithelial cell damage. The lesions are irreversible and lead to renal failure. OS is an important pathological mechanism of diabetic nephropathy, where the hyperglycemia promotes the generation of abundant ROS. The increased accumulation of proteins from the extracellular matrix (fibronectin and collagen IV) caused by ROS causes glomerulosclerosis and tubulointerstitial fibrosis 
. Excessive ROS are aberrantly generated by renal-infiltrating or endogenous cells, then react with biomolecules to trigger kidney injury and renal dysfunction 
. Aiming to reduce renal OS, AST was loaded in glucose-modified pegylated liposomes (AST-Glu-lipos) targeting glomerular mesangial cells that overexpressed glucose transporter 1 (GLUT1) on cell membrane 
. AST-Glu-lipos retained AST in media with serum and pH 7.4, while a faster release at pH 5.5 was observed in a simulated lysosomal acidic environment. In vitro AST-Glu-lipos were more internalized, reducing ROS and apoptosis in Human Renal Mesangial Cells (HRMCs), especially high-glucose-induced HRMCs, to a higher extent than glucose lacking liposomes (AST-lipos) and free AST. In a diabetic rat model, while AST is filtered and excreted in urine, intravenously administered AST-lipos and AST-Glu-lipos were observed to accumulate in the renal cortex, including glomeruli and renal tubules, mainly mesangial cells, the latter being accumulated to a higher extent. AST-Glu-lipos increased the levels of SOD and Gpx and improved renal functionalism in terms of urine protein, serum creatinine, and BUN, as well as significantly improving renal pathological morphology.
5. Nanomedicines for Nose to Brain Delivery of AST
The OS is one of the key factors in the pathogenesis of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The neuroprotective activity of AST is reported by many studies. Aiming to increase its brain delivery, AST loaded into solid lipid nanoparticles (SLNs) prepared by the solvent displacement method was nasally administered 
. The AST-SLNs were observed to protect from GSH consumption and from lipid peroxidation in PC-12 cells (from rat pheochromocytoma) upon H2
-induced cellular injury, either as pre-treatment or as a post-treatment. After intranasally administering 99m
Tc labeled AST-SLN to healthy animals, the amount of brain radioactivity compared with intravenous administration was increased.