Employ of Anthocyanins in Nanocarriers for Nano Delivery: In Vitro and In Vivo Experimental Approaches for Chronic Diseases

Anthocyanins are among the best-known phenolic compounds and possess remarkable biological activities, including antioxidant, anti-inflammatory, anticancer, and antidiabetic effects. Despite their therapeutic benefits, they are not widely used as health-promoting agents due to their instability, low absorption, and, thus, low bioavailability and rapid metabolism in the human body. Recent research suggests that the application of nanotechnology could increase their solubility and/or bioavailability, and thus their biological potential. Therefore, in this review, we have provided, for the first time, a comprehensive overview of in vitro and in vivo studies on nanocarriers used as delivery systems of anthocyanins, and their aglycones, i.e., anthocyanidins alone or combined with conventional drugs in the treatment or management of chronic diseases.


Introduction
With the increasing prevalence of chronic diseases worldwide and the emerging need to find safer, more effective, cheaper, and lower toxic new therapeutic agents, it is not surprising that phenolic compounds are receiving special attention and importance, given their characteristics (low price, low toxicity, and few, if any, side effects) and the common belief that they can be more effective than synthetic drugs [1,2]. These secondary metabolites are produced by plants to promote their normal cell metabolism and protect them from abiotic (e.g., humidity, light, temperature, moisture, soil, water, pH, and salinity) and biotic factors (e.g., microbial infections) [3]. Their daily consumption by individuals is also promoted and considered a useful strategy to promote health status and prevent numerous diseases, with few or no side effects, unlike conventional drugs [3][4][5]. In fact, phenolics have been used in traditional medicines since ancient times due to their remarkable ability to relieve gastrointestinal complaints, pain, headaches, colds, coughs, and other ailments [6,7]. Therefore, old proverbs, such as "an apple a day keeps the doctor away", are more relevant than ever.
Among these natural molecules, special attention has been paid to anthocyanidins, and their glycosides, i.e., anthocyanins [8]. Both are water-soluble and non-toxic natural compounds, have high molecular weights, and are very affordable. They are considered to be the main contributors to the blue-violet and red colors of many fruits, flowers, and vegetables [9]. In addition, there is a great demand and many potential applications to be the main contributors to the blue-violet and red colors of many fruits, flowers, and vegetables [9]. In addition, there is a great demand and many potential applications because they show remarkable effects as antioxidant, anti-inflammatory, antimutagenic, antidiabetic, and anticancer agents, both in vitro and in vivo; provide neurological and cardiovascular protection; improve memory ( Figure 1); and can be used as additives for beverages, foods, and cosmetics [8,[10][11][12][13][14][15][16][17][18][19]. However, anthocyanidins and anthocyanins have low bioavailability because they are poorly absorbed and rapidly metabolized and excreted, which, in turn, compromises their biological and pharmacological benefits [13]. In addition, their high susceptibility to environmental factors, such as chemical factors (e.g., ascorbic acid, light, metal ions, oxygen, pH), processing (e.g., heat), and gastrointestinal digestive processes (enzymes, proteins, and pH), makes them highly sensitive, reactive, unstable, and susceptible to decomposition [9,20]. Additionally, both have the ability to irreversibly bind with some molecules, resulting in structural changes, and, consequently, making them inaccessible for absorption [13]. IGF-1: insulin-like growth factor 1; TG: triglycerides; VLDL: very low-density lipoprotein; HDL: high-density lipoprotein. Adapted from [8,[10][11][12][13][14][15][16][17][18][19].
In view of the above, and in order to overcome these drawbacks and improve their pharmacokinetic, pharmacodynamic, and therapeutic properties, i.e., their bioaccessibility, bioactivity, absorption, stability in the gastrointestinal tract, solubility, and/or better control of their release, the development of novel delivery systems may be an important approach. This is mainly due to their ability to form a physical barrier between anthocyanins and destabilizing factors, protecting them from degradation and enhancing their biological potential [21][22][23][24][25][26]. Several methods exist for the improvement of drug delivery, namely physical (electrospinning/electrospraying, freeze-and spray-drying, lyophilization), chemical (complexation processes), and/or a combination of both (liposomal systems, emulsification, gelation, coacervation) [13,20]. The main biopolymers used for encapsulation are polysaccharides such as cellulose, chitosan, mucilages, natural gums, pectin, starch, and derivatives and proteins such as caseinate, gelatin, soy protein, and whey [9,[27][28][29]. Moreover, the encapsulation of anthocyanins also allows for their use at the industrial level [30]. Comparing the nano and microencapsulation, the former has been preferred due to its higher stability and encapsulation efficiency [28]. Therefore, taking into account all these aspects, the main objective of this work was to provide a ↓: decrease; RONS: reactive oxygen and nitrogen species; NO: nitric oxide; MAO: monoamine oxidase; IGF-1: insulin-like growth factor 1; TG: triglycerides; VLDL: very low-density lipoprotein; HDL: high-density lipoprotein. Adapted from [8,[10][11][12][13][14][15][16][17][18][19].
In view of the above, and in order to overcome these drawbacks and improve their pharmacokinetic, pharmacodynamic, and therapeutic properties, i.e., their bioaccessibility, bioactivity, absorption, stability in the gastrointestinal tract, solubility, and/or better control of their release, the development of novel delivery systems may be an important approach. This is mainly due to their ability to form a physical barrier between anthocyanins and destabilizing factors, protecting them from degradation and enhancing their biological potential [21][22][23][24][25][26]. Several methods exist for the improvement of drug delivery, namely physical (electrospinning/electrospraying, freeze-and spray-drying, lyophilization), chemical (complexation processes), and/or a combination of both (liposomal systems, emulsification, gelation, coacervation) [13,20]. The main biopolymers used for encapsulation are polysaccharides such as cellulose, chitosan, mucilages, natural gums, pectin, starch, and derivatives and proteins such as caseinate, gelatin, soy protein, and whey [9,[27][28][29]. Moreover, the encapsulation of anthocyanins also allows for their use at the industrial level [30]. Comparing the nano and microencapsulation, the former has been preferred due to its higher stability and encapsulation efficiency [28]. Therefore, taking into account all these aspects, the main objective of this work was to provide a comprehensive review of in vitro and in vivo studies on nanocarriers employed to deliver anthocyanins and anthocyanidins alone or in combination with active pharmaceutical ingredients. Moreover, different pH conditions lead to different colors and affect the stability of anthocyanins [41]. As far as we know, anthocyanins are more stable under acidic conditions [31]. Moreover, pH values below 3 contribute to the formation of red and orange anthocyanin compounds, as flavylium cations predominate here, and increase their water solubility, while pH values of 6 to 7 favor the appearance of violet species and pH values above 7 to 8 lead to the appearance of the blue color ( Figure 3) [42][43][44]. In contrast, at basic pH, anthocyanins are oxidized and degraded, losing their color as they kinetically and thermodynamically compete for the hydration of flavylium ions, leading to the formation of carbinol pseudobases, chalcones, and quinonoid anions [31,45]. The chemical degradation of chalcones leads to the formation of phenolic acids [9,46].   Moreover, different pH conditions lead to different colors and affect the stability of anthocyanins [41]. As far as we know, anthocyanins are more stable under acidic conditions [31]. Moreover, pH values below 3 contribute to the formation of red and orange anthocyanin compounds, as flavylium cations predominate here, and increase their water solubility, while pH values of 6 to 7 favor the appearance of violet species and pH values above 7 to 8 lead to the appearance of the blue color ( Figure 3) [42][43][44]. In contrast, at basic pH, anthocyanins are oxidized and degraded, losing their color as they kinetically and thermodynamically compete for the hydration of flavylium ions, leading to the formation of carbinol pseudobases, chalcones, and quinonoid anions [31,45]. The chemical degradation of chalcones leads to the formation of phenolic acids [9,46].  Other factors that increase the sensitivity of anthocyanins include storage and processing conditions, temperature, concentration, and exposure to light, oxygen, other phenolic compounds, proteins (such as enzymes), molecules (such as water), sulfites, and metal ions [21,40,[47][48][49].

Anthocyanin-Rich Sources
Anthocyanins and their aglycones are the main contributors to the color of many fruits, flowers, and vegetables and are considered the key of the biological potential associated with these products [39]. The recommended daily intake is over 100 mg and ranges up to 1000 mg per day. However, Zamora-Ros et al. [50] reported that the daily intake of anthocyanins in Europe ranges from 19 in women. In contrast, in the United States of America (USA), daily consumption ranges from 180-215 mg [51]. Among anthocyanin-rich sources, in Europe, apples, berries, grapes, and pears (50%), and red wine (21%) are the main contributors to daily anthocyanin intake, while in the USA, berries, wine, grapes, and bananas (20, 16, 11, and 11%, respectively) are the main sources [52].
Indeed, anthocyanins possess remarkable and recognized health-promoting properties, so, it is highly recommended to increase their consumption to promote health status and prevent the occurrence and/or development of chronic diseases [19,29,53]. Thus, strong correlations have already been reported between the total amount of anthocyanins extracted from sweet cherries and the ability to inhibit the enzyme α-glucosidase (r = 0.9929) and to affect the growth of colon adenocarcinoma Caco-2 cells (r = 0.6674) [11]. In addition, recent studies have shown that anthocyanins have a remarkable ability to reduce oxidative stress in hepatocellular HepG2 carcinoma cells [10], as well as impair the growth of AGS gastric cancer cells and reduce inflammatory iNOs and COX-2 expression and resulting nitric oxide radicals on RAW 264.7 macrophages [12].
Of course, it should be noted that anthocyanin content is highly dependent on genotype/variety, climate, ripening stage, local origin, and agronomic and postharvest practices and conditions [89]. For example, Howard et al. [90] reported that blueberry jams stored at 4 • C had 65% more anthocyanins after 6 months of storage than those stored at 25 • C. Similar data were obtained for their juices after an 8-month study [84].

Absorption, Distribution, Metabolism, and Excretion (ADME) and Bioavailability of Anthocyanins
Although there are existing studies on the ADME processes and the bioavailability of anthocyanins, understanding these issues is still a major challenge [143]. However, they are necessary to fully reveal and explore the biological potential of anthocyanins [9,41]. Nowadays, it is believed that the absorption of anthocyanins is higher than the expectable (<28.00%) once anthocyanins undergo extensive metabolism in the colon [33,[144][145][146]. Nevertheless, their percentages are highly variable, largely depending on the individual's sex, genetic profile, age, pre-systemic metabolism, physiological and pathological conditions, dietary habits, lifestyle, intestinal flora, and the presence of possible intolerances, as well as chemical structure, molecular size, glycosylation pattern, and behavior in the gastrointestinal tract, including solubility, transport, and permeability [9,147]. For example, pelargonidin aglycone and derivatives are more readily absorbed than other anthocyanins when they have lower substituents on the B ring [148]. Comparing the sugar residues, malvidin 3-O-arabinoside shows higher absorption than its 3-O-glucoside derivative [149]. In addition, it is important to consider the maturity degree of the food matrix, while cooking practices can also influence the availability process, as well as their levels and ability to be released from the food matrix, and whether they are administered alone or with other foods and beverages, and/or after a fasting period [41]. Nevertheless, anthocyanins are generally readily absorbed, reaching maximum concentrations ranging from 1.4 to 592 nmol/L (doses of 68-1300 mg) 30 min to 4 h after ingestion [150,151]. The recovery rate in urine is less than 2% 24 h after ingestion [152].
As mentioned earlier, most anthocyanins found in foods are glycosides [9]. After consumption, anthocyanins are partially degraded by the microbiota in the mouth [153]. They are then passed to the stomach, where the low pH promotes the stability of anthocyanins and their persistence in the form of glycosides [22]. Unlike other flavonoids, anthocyanins can be absorbed along with their sugar moieties [143]. They require the action of bilitranslocase because they are unable to cross cell membranes by passive diffusion due to their hydrophilic nature [49,148]. Therefore, anthocyanins are delivered by bilitranslocase to the portal vein of the liver to be distributed by systemic circulation to target tissues and organs (bioavailability) [148].
However, the majority of anthocyanins ingested in the diet are transported by glucose transporters (GLUT) 1 and 3 to the small intestine, where they are degraded, possibly increasing their absorption and thus bioavailability [41,49]. For this purpose, anthocyanins are deglycosylated in a first step by cytosolic β-glucosidase (CGB) in the intestinal lumen and lactase-phlorizin hydrolase (LPH) in the brush border of intestinal epithelial cells, producing more lipophilic aglycones that are more easily absorbed [144]. Therefore, the released aglycone can enter the intestinal epithelial cells by passive diffusion or via sodiumdependent transporters GLUT 1 and GLUT 2, metabolic detoxification through phase I (the reactions of hydrolysis, oxidation, and reduction), and phase II enzymatic metabolism (conjugation reactions such as methylation, glucuronidation, and sulfation mediated by the enzyme catechol-O-methyltransferase, uridine-5 -diphospho-glucuronosyltransferase, and sulfotransferase mediated, respectively) [41,143,154]. Similar reactions may also occur in the liver and kidneys [154]. The absorption of the non-acylated anthocyanins is four times higher than that of the acylated ones [155].
The non-absorbed anthocyanins can be excreted in the urine and bile [156] or enter the colon, where they are metabolized by 300-500 different bacteria, mainly of the genera Bacteroides, Bifidobacterium, Clostridium, and Eubacterium, which convert them to lower molecular-weight compounds, namely phenolic acids, aldehydes, or other phenolics, to increase their absorption [41,144,153]. For example, it has been previously reported that 16 different metabolites derived from the degradation of anthocyanins were identified after the consumption of red raspberries [154]. In addition, 29 different metabolites were detected in the urine of 20 volunteers who had consumed anthocyanin-containing beverages including caffeic, trans-isoferulic, vanillic, trans-ferulic, hippuric, 2,4,6-trihydroxybenzaldehyd, and 4-dihidroxifenilacetic acids derivatives. They peaked 3.5 h after consumption of the beverage [157]. Moreover, this modulation between colonic bacteria and anthocyanins is very interesting and beneficial because it produces short-chain fatty acids, which together with the new phenolic acids formed, create favorable conditions for the proliferation of probiotic bacteria with remarkable health-promoting properties, such as Bifidobacteria, Actinobacteria, and Lactobacilli [158]. Thus, the conjugated forms may be excreted through bile to jejunum to be then recycled by enteropathic circulation [156]. Unabsorbed anthocyanins are excreted in the stool [33].
Given the great potential of anthocyanins as dietary supplements, several efforts have been made to increase the stability of anthocyanins in the gastrointestinal tract, their absorption and bioavailability, and thus their biological potential [9]. One of the most promising strategies is the encapsulation of anthocyanins, which will be discussed below [9,48].

Anthocyanins in Nano-Delivery Systems
Given the toxicity of many synthetic drugs, the search for and development of new effective drugs is essential [4]. Anthocyanins are a target of many studies in this regard [2,[159][160][161][162][163].
However, these phytochemicals are very sensitive to external factors (e.g., pH variations, water, temperature) and have a short half-life [164]. Therefore, it is imperative to develop new delivery systems that do not exhibit toxicity and are able to increase the stability of anthocyanins and make them kinetically and thermodynamically stable, as well as improve their solubility and pharmaceutical properties [165].
Among the various alternatives studied, those related to nanotechnology and nanoencapsulation have attracted the greatest interest in many fields, such as medicine, namely in the prevention, diagnosis, and treatment of numerous diseases through active or passive targeting [166][167][168][169][170]. They are preferred over micro-delivery systems because the latter are more unstable in the physiological environment due to their large particle size, low zeta potential, and low encapsulation efficiency [171]. Therefore, various approaches to nanoencapsulation are currently being investigated for the different routes of administration, including intravenous, nasal, oral, parenteral, intraocular, and dermal topical applications [172]. In addition, most of these approaches can also easily cross the blood-brain barrier, which increases the therapeutic potential of the molecule(s) [170,173]. This technique can be applied because anthocyanins are able to generate van der Waals and hydrogen bonds as well as hydrophobic interactions with these nanocarriers, which increases their stability [24,26,165]. The latter has a particle size between 1 and 1000 nm and may consist of organic components (e.g., polymer and lipid-based nanoparticles, such as nanoemulsions, liposomes, and nanoparticles of solid lipid), inorganic coatings (e.g., metallic nanostructures of gold and titanium dioxide, such as nano-quantum dots and nanodiamonds), or a combination of both. Most of them are biodegradable and non-toxic [171,174].
Biopolymers are the most commonly used for nanoencapsulation because they incorporate various types of hydrophobic and hydrophilic molecules (e.g., drugs, proteins, phytochemicals, and other molecules), minimizing their undesirable effects and enhancing their benefits [175,176]. Among them, carbohydrates derived from alginate and chitosan, natural gums, and protein derivatives are the most popular [164,177]. They are very effective in encapsulating anthocyanins and contribute greatly to shifting the flavylium cation structure, protecting them from negative environmental effects, thus promoting their stability in the gastrointestinal tract and increasing their bioavailability [20,160,178]. Thus, several studies have already shown that encapsulation of anthocyanins with chitosan and derivatives can improve their physical and oxidative stability, preserve their antioxidant activity, and promote a slower degradation during simulated gastrointestinal digestion and environmental storage [22,27,164,179,180]. Pectin is also considered a promising coating because of its distinct ability to protect anthocyanins from different pH ranges and temperatures, as well as from damage by ascorbic acid [23,181]. In addition, it promotes a slower release of anthocyanins [182]. Zhao et al. [183] developed a nanoliposome of anthocyanins from blueberries coated with pectin and demonstrated a slower release of anthocyanins in simulated gastric fluid (≤35.9%), but faster release in simulated intestinal fluid, caused by the degradation of vesicles by the enzyme pancreatin. Liposomal micelles were also found to be effective in increasing the stability and resistance of blueberry anthocyanins in the gastrointestinal tract, resulting in a bioavailability greater than 90% [184]. In addition, the combination of pectin and chitosan has also been a subject of numerous studies. So far, they have already shown a promising ability to protect anthocyanins from degradation by milk and white fluorescent light, as well as from different pH and temperature levels, to maintain their antioxidant capacity and to improve retention during simulated ingestion and cellular uptake into human Caco-2 intestinal cells [13,28,185]. Similar results were obtained with gum arabic [186,187], α-lactalbumin [48], β-lactoglobulin [2], β-glucan [21], inulin and oligofructose carbohydrates [5], chondroitin sulfate [24], and casein conjugated with carboxymethyl cellulose [160]. The combination of whey protein with glucose also showed a remarkable ability to protect anthocyanins from high temperatures (80 • C) at acidic pH [188], while the use of ovalbumin conjugated with dextran appears to provide added value for the protection of anthocyanins from hydrogen peroxide-induced oxidative damage, as its conjugation showed a remarkable ability to increase anthocyanins' chemical stability, cellular uptake, and intestinal absorption in human Caco-2 intestinal cells [26]. Peptides are also promising materials for nanoencapsulation. For example, C6M1, a peptide composed of 18 amino acids, was previously reported to maintain the free radical scavenging ability of cyanidin 3-O-glucoside and to be resistant to higher pH values, temperature, and concentrations of metal ions [162]. Apoferritin nanocages exhibit similar capabilities and also show the ability to facilitate the transport of this anthocyanidin through the cell monolayer of Caco-2 cells [189]. Finally, the use of EUDRAGIT ® L100 and polyethylene glycol 2000 can also improve the bioavailability of anthocyanins from açaí berries [190].
Nanoencapsulation involves two primary structures: the first is the nanospheres core structure, in which the polymer matrix is dispersed and/or adsorbed by the bioactive compounds, and the second is the nanocapsule, which may consist of water or oil and a polymer shell [171,191]. When nanoparticles reach the outer cell membrane, they can interact with components of the plasma or extracellular membrane and enter the cell by endocytosis [26,192,193]. In addition, some biopolymers allow controlled release of the molecules that they coated at specific times and cells/organs [9,194].
The most promising in vitro and in vivo studies on the effects of nanoencapsulated anthocyanins on human health are summarized and discussed below ( Figure 4 and Table 2).

Nano-Polymeric Coatings
The nanoencapsulation of anthocyanins with polymeric coatings has been commo performed and investigated in in vitro conditions [193,196,197]. Among the vario options, the use of the synthetic polymer poly(lactic-co-glycolic acid)-acid is very popu because it has tremendous potential to form stable complexes, is non-toxic a

Nano-Polymeric Coatings
The nanoencapsulation of anthocyanins with polymeric coatings has been commonly performed and investigated in in vitro conditions [193,196,197]. Among the various options, the use of the synthetic polymer poly(lactic-co-glycolic acid)-acid is very popular because it has tremendous potential to form stable complexes, is non-toxic and biodegradable, and the associated results are highly reproducible worldwide [198]. In addition, they display good tissue penetration and are easily manipulated [193]. As far as we know, this synthetic polymer showed great efficiency in coating pelargonidin, at concentrations of 10, 20, and 30 µM, increasing its ability to inhibit the production of reactive oxygen species in L6 muscle cells exposed to the pesticide cypermethrin after 12 and 24 h of exposure [197], as well as protecting these cells from nuclear damage by activating poly(ADP-ribose) polymerase and the p53 pathway, at a concentration of 9 µg/mL [198]. In addition, this coating can also enhance the ability of pelargonidin, at a concentration of 9 µM, to reduce the expression of GLUT 4, insulin receptor substrate 1, peptidase inhibitor 3, glycerol kinase, and protein kinase, and to induce cytochrome complex-mediated apoptosis in alloxan-induced hyperglycemic L6 skeletal muscle cells [196]. Moreover, anthocyanins encapsulated with this polymer also showed a higher capacity to protect human neuroblastoma cells SH-SY5Y from neurodegeneration against Aβ 1-42 -induced toxicity than those not encapsulated. Indeed, encapsulated anthocyanins at concentrations between 50 and 200 µM can remarkably reduce Alzheimer's disease markers amyloid precursor protein, beta-site amyloid precursor protein cleaving enzyme, neuroinflammatory markers phospho-nuclear factor kappa B, tumor necrosis factor alpha, and inducible nitric oxide synthase after 12 h of exposure, and the neuroapoptotic markers B-cell lymphoma 2 and associated X-protein (Bax) and caspase-3 protein expressions, and showed notable scavenging properties and ability to abrogate reactive oxygen species production via the p38-MAPK/JNK pathway, as well as increase the expression of endogenous nuclear factor erythroid 2-related factor 2 and heme oxygenase [199].
Nevertheless, it is important to underline that some polymeric coatings can induce allergic reactions and that the complete degradation of some polymeric coatings, such as poly(lactic-co-glycolic acid) in lactic and glycolic acids, takes months; therefore, their accumulation can lead to disturbances in the microenvironmental pH [199]. Given this knowledge, it is urgent to determine the polymer degradation rate and long-term safety of polymeric drug delivery systems intended for clinical use.

Exosome Coatings
Milk exosomes are another promising strategy once they can take on a nanosize and can be easily customized depending on what they encapsulate [200]. Moreover, they are non-toxic, present a reduced immune response, and can confer protection on circulating genetic material; like other nanoencapsulation coatings, they feature remarkable biocompatibility and tumor targeting [201]. In addition, due to their high stability at lower pH values, milk exosomes can be used as carriers for oral drug delivery, enabling a wide range of preventive and therapeutic applications [174]. Recently, Barkallah et al. [200] demonstrated that exosomes loaded with 10 µg/mL delphinidin are non-toxic to human aortic endothelial cells, and can correct their nitric oxide levels and reduce angiogenesis, after 1 day of treatment. Moreover, Aqil et al. [201] have previously shown that exosomes in combination with anthocyanins at a concentration of 75 µM increase the potential of these phenolics to arrest the growth of A2780, A2780/CP70, OVCA432, and OVCA433 ovarian cancer cells, decrease the P-glycoprotein expression, and reduce the effective cisplatin concentration required to inhibit cisplatin-resistant ovarian cancer cells, after 3 days of treatment. In addition, exosomes composed of anthocyanins from blueberries also showed higher antiproliferative effects than the unencapsulated anthocyanins on colon cancer cell lines HCT-116 and HT-29 at concentrations between 25 and 200 µM, after 24 h of treatment [202], and on human lung cells A549 and H1299, breast cells MDA-MB-231 and MCF7, pancreatic cells PANC1 and Mia PaCa2, prostate cells PC3 and DU145, colon cells HCT-116, and ovarian cells OVCA432, at concentrations ranging from 20 to 100 µM, after 72 h of treatment [174].
However, it is important to take into account that exosomes present a limited transfection efficiency.

Nanolipid Coatings
The nanoencapsulation of anthocyanins with lipids has also been extensively studied owing to their great biocompatibility, small size, high surface-to-volume ratio, and low fusibility [177]. In addition, their synthesis does not require the use of many solvents or aggregation with other nano delivery systems and can be manufactured on a large scale with high reproducibility and provide remarkable protection against enzymatic degradation, allowing the creation of a complex with unique physicochemical properties that can deliver the therapeutic molecule directly to target tissues or cells, minimizing its concentration and frequency of treatment [192]. The lipid chosen to coat the active molecules depends on many factors, such as the type of bioactive molecules, cosurfactants and surfactants, lipids, and cryoprotectants, as well as the target of the loaded bioactive molecule(s). Nowadays, many studies focus on the nasal delivery of drugs and phytochemicals, e.g., for the treatment of Alzheimer's disease. As far as we know, nanoencapsulation of anthocyanins from elderberries, at a concentration of 0.5 mg/mL, with lipids showed a remarkable ability to modulate mitochondrial functionality, by enhancing complexes I and II of the mitochondrial respiratory chain and preserving the mitochondrial membrane potential in the presence of rotenone thanks to their ability to target mitochondria and protect these cells from rotenone and glutamate-induced toxicity [192].
Recently, lipid-derived nano-niosomes systems have also attracted considerable attention because they increase chemical stability and are economical and practical [5]. Anthocyanins encapsulated in niosomes, from black carrots, at concentrations of 6.25 and 100 µg/mL, showed the ability to be released after 10 h and decreased the viability of neuroblastoma cells Neuro 2A, after 2 days of treatment [203], while anthocyanins extracted from Zea mays and Clitoria ternatea (2-2000 µg/mL) showed a remarkable ability to increase collagen production in human gingival fibroblasts and permeate through the esophageal mucosa [163]. In addition, anthocyanin-added nano-niosomes (1%, 2%, 5%, and 10%, v/v) were reported to be non-toxic to normal HT22 hippocampal neuronal cells after 1 day of exposure, and fully internalized in BV2 microglial cells [204].
However, one of the major concerns of using lipids as nanocarriers is their cytotoxic potential due to their ability to create non-specific uptake [192].

Nano-Polysaccharide Coatings
Certain types of polysaccharides are also used to encapsulate anthocyanins, as some of them have a charged residue and can therefore be easily linked to the opposite charge of the phospholipid residue, thus preventing phospholipid hydrolysis in the presence of enzymes and under acidic pH conditions [13,205]. Among them, chitosan and pectin are the most commonly used because both are non-toxic, environmentally friendly, biodegradable, and have high biocompatibility [28]. In addition to their potential advantages, they appear to be a promising, effective drug delivery system to the colon. In in vitro studies, nanoencapsulation of anthocyanins at concentrations of 5, 10, 20, and 40 µg/mL with pectin and chitosan showed greater potential to protect normal rat kidney (NRK) cells from acrylamide-induced damage than non-encapsulated anthocyanins and allowed their controlled release via the gastrointestinal tract [9]. They decrease reactive oxygen species, matrix metalloproteinases, and glutathione levels and provide protection to normal human hepatocyte L02 cells from palmitic acid-induced damage at concentrations ranging from 1.6 to 8.0 µM after 24 h of exposure [176]. Nevertheless, it is important to emphasize that chitosan dissolves only in certain dilute acidic solutions. Chondroitin sulfate is another promising alternative, as it is able to enhance the ability of black soybean anthocyanins at concentrations of 4, 7.5, and 15 µM to scavenge reactive oxygen species in human colon cells HCT-116, after 2 days of treatment [206] and to suppress human cervical cancer HeLa cells more effectively compared to anthocyanins alone, both at 100 µM after 1 day [20]. The combination of chitosan and chondroitin sulfate-loaded black rice anthocyanins at concentrations of 10 and 50 µg/mL also showed remarkable effects on human colon cancer cells HCT-116 by promoting negative changes in mitochondria and, consequently, apoptosis after four hours of exposure [207]. Finally, hyaluronic acid, a natural polysaccharide, together with 20 µg/mL cornflower anthocyanins, can enhance CD44 + apoptosis in colon cancer cells HT29, after 1 h of treatment [208].
Despite this, the employment of polysaccharide coatings shows some drawbacks, such as their cost, undesirable aggregation, and burst release promoted by their small particle size and large surface [209].

Liposome Coatings
Nanoencapsulation of anthocyanins with liposomes has also been investigated, with most studies having shown that they can be used as nanocarriers for drug delivery due to their self-assembling and amphiphilic properties, flexibility, biocompatibility, biodegradability, longer duration of circulation, and low immunogenicity [183]. In addition, since they are composed of an aqueous core and a spherical hydrophobic shell, they can transport hydrophilic, hydrophobic, and amphiphilic molecules, as well as easily cross the blood-brain barrier [173]. Regarding their encapsulation with anthocyanins, Homayoonfal et al. [178] showed that they can enhance the antioxidant capacity of anthocyanins in a very remarkable and effective manner, as well as increase the metabolite activity and replication of human mesenchymal MSC and fibroblast FBL stem cells, at concentrations of 0.5 mg/mL and 10.4 µg/mL, respectively, after 1 week of treatment. Moreover, it has been also reported that there is a positive correlation between the size of nanoliposome particles and the encapsulation efficiency [161].
Nevertheless, it is important to note that liposomes can trigger undesirable immune responses, present a moderate loading capacity, and possibly crystallize after prolonged storage times [209].

Halloysite Nanotubes Coatings
The use of halloysite nanotubes is also increasing worldwide, due to their unique properties that include particle size, high surface area, and surface-to-volume ratio, and very high dispersion rate [193]. Regarding anthocyanins, they have shown to be effective in suppressing the growth of human breast cancer MCF-7 and human colon cancer HT-29 at a concentration of 500 µg/mL compared to non-encapsulated anthocyanins, after one and 2 days of exposure [8]. Nonetheless, it is important to consider that halloysite nanotubes are inorganic and present toxicity and poor biocompatibility [193].

Dendrimers Coatings
Dendrimers are another promising alternative, as they can reduce toxicity and improve water solubility, as well as possessing pharmacokinetic properties and biodistribution [209]. In addition, dendrimers can also be adjusted in size and shape, in order to favor surface area, loading, and delivery efficiency. They are usually composed of poly(amidoamine), poly(L-lysine), polyamides, polyethers, polyesters, poly(2,2-bis(hydroxyl methyl) propionic acid, and polypropylenimine. Together with anthocyanins, dendrimers can increase, dose-dependently, the cytotoxicity of neuro-2A brain neuroblastoma cell, after 24 h of treatment [194].
However, it is important to note that dendrimers present a complex synthetic route and can only carry low amounts of active molecules.

Nanoemulsions Coatings
Nanoemulsions are another interesting drug delivery system, since their production is economic and they show high loading capacity and efficiency to carrier hydrophobic molecules, as well as a notable capacity to protect bioactive molecules against degradation. In addition, they can be easier manipulated to ameliorate drug release. Nonetheless, their use usually requires conjugation with nonionic surfactants to provide better stability [172]. Nonionic surfactants are recommended because they are safer than their ionic equivalents and are usually accepted for oral administration [172]. Recently, Nazareth et al. [165] reported that nanoemulsions of anthocyanins (400-1050 µg/mL) could be a promising alternative to conventional antibiotics. They demonstrated a remarkable ability to inhibit biofilm formation in Pseudomonas aeruginosa and Yersinia enterocolitica, and anti-quorum sensing activity against Chromobacterium violaceum, in a dose-dependent manner.
Nevertheless, it is important not to forget that some nanoemulsions present some non-compatibility, low viscosity, and can irritate skin.

In Vivo Studies
In vivo studies focusing on the employment of anthocyanins in nanocarriers for nano delivery are still scarce. Nonetheless, some up-to-date reports show promising data involving anthocyanins coated with nano-polymeric, dendrimers, carbohydrates, polysaccharides, and nano-niosomes with notable antioxidant, neurological and cardiovascular properties, and anticancer effects with no apparent adverse effects and, thus, with the potential to be marketed, as mentioned below.

Nano-Polymeric Coatings
However, the use of polymer-based nanocarriers loaded with anthocyanins, at doses of 0.5 and 1 mg/g body weight, has already been reported. In these conditions, anthocyanins had shown the ability to protect fish models exposed to the pesticide cypermethrin from DNA degradation by increasing the penetrability of anthocyanins in liver, kidney, muscle, brain, and ovarian/testicular tissue, and decreasing cytotoxicity by modulating the antioxidant enzymes superoxide dismutase, catalase, and lipid peroxidase, after 7 days of treatment [197]. Moreover, complete tumor ablation was verified in MCF-7-bearing nude mice treated with anthocyanins at 250 µg/mL loaded into polymeric nanoparticles, at doses of 0.5 and 1 mg/g body weight, after 26 days of treatment [166].

Dendrimers Coatings
Anthocyanins encapsulated in exosomes (5 mg anthocyanins and 50 mg exosome protein/kg body weight administered orally by gavage five times a week for a period of 3 weeks) also showed a remarkable ability to reduce tumor size in mice transplanted with A549 human lung cancer cells, with no apparent adverse effects [174]. In addition, they can also reduce the proliferation of colorectal cancer in Apc Min/+ mice inoculated with enterotoxigenic Bacteroides fragilis bacteria and treated for 4 weeks, 3 days per week, with a dose of 8.6 mg/kg/day, by increasing the activity phase II enzymes glutathione S-transferase Mu 1 and uridine 5 -diphospho-glucuronosyltransferase family 1 member A6 and decreasing the expression of aryl hydrocarbon receptor nuclear translocator 1, aryl hydrocarbon receptor, and cytochrome P450 family 1 subfamily A member 1 [202].

Carbohydrate Coatings
Carbohydrate-based derivatives, especially chitosan, have been intensively studied, mainly due to their high loading efficiency, water solubility, and biocompatibility [29]. For example, Chatterjee et al. [175] reported that the use of anthocyanins loaded with chitosan (at a dose of 600 mg/kg body weight/day) from black carrot resulted in the remarkable ability to increase the activity of superoxide dismutase and catalase enzymes in rats. In addition, they can also enhance the potential of cyanidin 3-O-glucoside (5 mL/20 g body weight) to attenuate the oxidative stress induced by selenite sodium in rats treated four times daily for 1 week, enhance the transepithelial transport of liposomes to a depth of 40 mm in the cornea of rats, prolong the residence time on the cornea, and improve permeability in the corneal epithelium, thus increasing superoxide dismutase and catalase activity and decreasing glutathione activity and lipid peroxidation in the lens [205]. Moreover, they can also confer to this anthocyanin a higher ability to balance the B-cell lymphoma-2/leukemia-2 ratio and reduce UVB-induced epidermal damage, by decreasing lipid peroxidation, malondialdehyde, and 8-hydroxy-2 -deoxyguanosine and increasing visual and histological appearance and skin hydration, after 1 day of treatment [210]. In addition, Zhao et al. [9] reported that the use of chitosan combined with pectin can provide the ability of blueberry anthocyanins, at a concentration of 320 µg/mL, to increase the lifespan and reproductive capacity and improve the flexible locomotion behavior of Caenorhabditis elegans from damage induced by acrylamide, heat shock, oxidative stress, and ultraviolet light, and reduce ageing effects.
However, one of the major drawbacks of using carbohydrates as nanocarriers is related to their toxicity.

Polysaccharide Coatings
On the other hand, the use of polysaccharides nanocarried with anthocyanins from black soybean for 5 days showed efficacy in reducing the tumor size of mice previously injected with human cancer cells HCT-116, increasing the number of apoptotic cells, and also contributing to the release of drugs at target sites [206]. In particular, cornflower anthocyanins loaded into the hyaluronic acid were found to be highly effective in increasing myelosuppression in mice transplanted with human colon cancer cells HT29 and treated for 13 days [208]. In addition, Hanafy [14] reported that encapsulation of these phytochemicals with bovine serum albumin can ameliorate cardiomyopathy, completely remove glycogen from tissues, and reduce malondialdehyde and collagen fibers in a fibrotic mouse model treated twice a week for 4 weeks.

Nano-Niosomes Coatings
Nano-niosomes are another promising strategy, mainly thanks to their non-toxicity, greater stability, biocompatibility, capacity of penetration, and potential to control and target bioactive molecule(s) delivery [203]. So far, their encapsulation with anthocyanins has already shown remarkable healing activity in oral cavity wounds of rats [163]; the ability to improve insulin resistance and glucose intolerance, by increasing the antihyperglycemic/anti-diabetic ratios with no apparent toxicity; and lowering animal weight and plasma levels of insulin, glucose, leptin, and total cholesterol in obese mice at 300 mg/kg of body weight [4]. Moreover, nanoencapsulation of anthocyanins with nano-niosomes showed pronounced neuroprotection. For example, nano-niosomes can give anthocyanins a remarkable ability to cross the blood-brain barrier, reach it, and accumulate in mice with Alzheimer's disease treated for 14 days, thus preventing tau hyperphosphorylation and reducing the expression of amyloid beta, beta-site amyloid precursor protein cleaving enzyme 1, and advanced glycation end products receptor, and neuroinflammatory and oxidative stress markers via the GSK-3β/CDK5 pathway [204]. Moreover, in the Aβ 1-42 mouse model of Alzheimer's disease, they may enhance the ability of anthocyanins (12 µg/g) to ameliorate memory impairment, as well as protect their pre-and postsynaptic proteins from Aβ 1-42 -induced synaptic dysfunction, regulate the p-PI3K/p-Akt/p-GSK3β pathway, prevent the hyperphosphorylation of tau protein at serines 413 and 404, and inhibit apoptosis during 14 days of treatment [211]. Although they seem to be a hopeful strategy, it is still difficult to find an appropriate concentration able to balance the relationship between the entrapment efficiency and the affinity of the cell membrane.

Conclusions
Given the worldwide increase in chronic diseases and the urgent need to search for new, effective, safe, and low-toxicity drugs, it is not surprising that many phytochemicals, such as anthocyanins, are considered good candidates. In fact, their pyrogallol, methoxy, and catechol groups confer remarkable health-promoting properties to anthocyanins. Therefore, and considering their limitations (e.g., high sensitivity to pH, rapid absorption, and metabolization), encapsulation seems to be a promising approach. In addition, the combination with other bioactive molecules/pharmaceuticals also appears to be a promising strategy in order to improve the therapeutic effects. Nevertheless, it is imperative to know their safe dosage in humans, since they may act as pro-oxidants under certain conditions, and because in many situations the use of higher dosages may not help to increase their biological potential. Furthermore, it is also important to note that nanoparticles manufacturing needs to be more scalable and robust, and performed under stringent, specific guidelines. Furthermore, clinical studies need to be accurately designed, detailed measured, and controlled. Finally, statistical analyses need to be adequately applied.

Conflicts of Interest:
The authors declare no conflict of interest.