Flavonoids as Dds

74 Current Medicinal Chemistry, 2010, 17, 74-95

0929-8673/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Design and Development of Nanovehicle-Based Delivery Systems for Preventive or Therapeutic Supplementation with Flavonoids G. Leonarduzzi, G. Testa, B. Sottero, P. Gamba and G. Poli*

Department of Clinical and Biological Sciences, University of Turin at San Luigi Gonzaga Hospital, 10043 Orbassano (Turin), Italy

Abstract: To date more than 4000 compounds are recognized to belong to the class of flavonoids. These natural phenolic drugs are poorly soluble in water and are rapidly degraded and metabolized in the human body, but nevertheless are very promising for their potential contribution to the prevention and therapy of major chronic diseases, including cardiovascu-lar and neurodegenerative diseases and cancer. In recent years a number of flavanols (e.g. catechins), flavonols (e.g. quer-cetin, myricetin) and isoflavones (e.g. genistein, daidzein) have been confirmed to possess strong antioxidant, anti-inflammatory, anti-proliferative and anti-aging activities. Incorporation into lipidic or polymer-based nanoparticles ap-pears to markedly help the oral delivery of flavonoids, as these particles can protect the drug from degradation in the gas-trointestinal tract and, by virtue of their unique absorption mechanism through the lymphatic system, also from first-pass metabolism in the liver. In addition, both oral and parenteral administration of flavonoids exploits a pharmacologic deliv-ery route that guarantees sustained release of the active principle at the desired site of action. A comprehensive review of studies currently available on the in vitro and in vivo experimental administration of flavonoids by means of nanovectors may be of use as a foundation for the development of advanced delivery systems for these powerful compounds, in view of their adoption in primary and secondary disease prevention.

Keywords: Flavonoids, antioxidants, anti-inflammatory drugs, liposomes, solid lipid nanoparticles, polymeric nanoparticles, nanovehicles.

INTRODUCTION

Flavonoids are a large class of plant secondary metabo-lites belonging to the wider family of natural polyphenols. They are present in plant-derived food and beverages, such as fruits, vegetables, cereals, legumes, cocoa, olive oil, tea, coffee and wine, thus comprising an important constituent of the human diet.

Plant polyphenols were originally considered important in plant pigmentation and flavor [1]; they were later recog-nized to play a crucial role in plant growth and reproduction, also providing resistance to pathogens and predators and protecting crops from diseases [2]. Recently, interest in food polyphenolic compounds has steadily increased owing to their antioxidant capacity, but also to their ability to modu-late a number of molecular pathways primarily involved in the regulation of cell proliferation, cell function and inflam-matory reactions. Considerable attention is thus now concen-trated on their possible beneficial implications on human health, such as in the prevention and treatment of cancer, cardiovascular diseases, neurodegenerative processes and other chronic diseases affecting the liver, gut, kidney and lung [3].

These disease processes are frequently associated with an oxidative imbalance of the tissue redox state and with inflammation. Oxidative and inflammatory reactions generate and amplify each other, and combine to form a powerful mechanism of disease progression. Because of their recognized antioxidant and anti-inflammatory properties, flavonoids could make a sizeable contribution to preventing onset and progression of many human diseases, but their bioavail-

*Address correspondence to this author at the Department of Clinical and Biological Sciences, University of Turin at San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano, Torino, Italy; Tel: +39 011 6705422; Fax: +39 011 6705424; E-mail: [email protected]

ability must be significantly improved and their delivery to the diseased organ or apparatus optimized.

The design of suitable molecular carriers for flavonoids is an area of research that is already in progress, but efficient solutions are still far from being developed and applied to humans. It thus appeared of use to comprehensively review the recent literature concerning chemical and biochemical properties of the flavonoids of interest in human pathophysi-ology, their bioavailability, as well as all suitable attempts made thus far to improve flavonoid bioavailability by means of nanotechnologies.

1. FLAVONOIDS OF NUTRITIONAL AND PHARMA-COLOGICAL INTEREST

1.1. Structure and Subclasses

Flavonoids have a basic chemical structure of diphenyl propanes, consisting of two benzene rings joined by a linear three-carbon chain (C6-C3-C6). In most cases, the central three carbons form a closed pyran ring with one of the ben-zene rings, thus forming a structure of 15 carbon atoms ar-ranged in three rings, labeled A, B and C. The various classes of flavonoids differ in the degree of oxidation and in the pattern of substitution of the C ring, while individual compounds within a class differ in the pattern of substitution of the A and B rings [4]. Depending on the variations in the heterocyclic C-ring, flavonoids may be divided into seven subclasses: flavones (e.g., apigenin, luteolin, diosmetin, chrysin), flavonols (quercetin, kaempferol, myricetin, galangin, fisetin), flavanones (e.g., naringenin, hesperitin, erioclictyol), flavanonols (taxifolin), flavanols or catechins (catechin, epicatechin, epigallocatechin, epigallocatechin gallate), isoflavones (genistein, daidzein, glycitein, puerarin), anthocyanins and anthocyanidins (cyanidin, pelargonidin, delphinidin, malvadin, petunidin).

Nanovehicles for Flavonoids Current Medicinal Chemistry, 2010 Vol. 17, No. 1 75

In all the subclasses, rings B and C are linked at C2, with the sole exception of the isoflavones (linked at C3). Flavones, flavonols and isoflavones show a double bond between C2 and C3, whereas flavanones, flavanonols, and flavanols con-tain a saturated three-carbon chain. These subclasses differ among them for the presence of a carbonylic carbon atom at C4 (flavones, flavonols, flavanones, flavanonols, and isofla-vones) and a hydroxyl group at C3 (flavonols, flavanonols, and flavanols) [5]. Anthocyanins are glycosides, having glu-cose, galactose, rhamnose, xylose or arabinose conjugated to the aglycon nucleus via the C3 hydroxyl group. The de-glycosylated or aglycone forms of the anthocyanins are known as anthocyanidins; these are salt derivatives of the 2-phenylchromenylium cation, also known as the flavylium cation [6].

Flavonoids can undergo various modifications, such as O-glycosylation, C-glycosylation, O-acylation, ring conden-sation, and polymerization, which yield a large number of diverse and highly-complex structures. The combination of flavonoids and lignan-like structures generate flavonolignans [7]. Silymarin is the most important flavonolignan mixture, and is extracted from seeds of the milk thistle (Silybum marianum): its active constituents are silybin and isosilybin (each as a pair of diastereoisomers, A and B), silydianin and silychristin [8]. Silybin, known also as silibinin, is the major and most active component (60-70%): it is obtained through coupling two radical species, derived respectively from the one-electron oxidation of dihydroflavonol taxifolin and of coniferyl alcohol. This reaction leads to an adduct that un-dergoes cyclisation through the nucleophilic attack of the B-ring hydroxyl group on the quinone methide, which is yielded by coniferyl alcohol, with the consequent formation of sylibin diastereoisomers A and B [7].

The chemical structures of the above described com-pounds are depicted in Fig. (1).

1.2. Food Sources

Apart from various vegetables and fruits, flavonoids are found in seeds, nuts, grains, spices, as well as in beverages, like wine (particularly red wine), tea, and (at lower levels) beer [9]. In particular, the flavones apigenin and luteolin are abundant in cereal grains and aromatic herbs (parsley, rose-mary, thyme, celery, sweet red and hot peppers), while their hydrogenated analogues (flavanones) hesperetin and naringin are almost exclusively present in citrus fruits and juices, grapes, and prunes [10]. The flavonols quercetin and kaempferol are present in vegetables (broccoli, red cabbage, yellow onions, fennel, tomato), fruits (apples, cherries, apri-cots, blueberries, black grapes), and green and black tea, whereas the flavanonol taxifolin is present in many citrus fruits and juices (especially oranges) and grapes [11]. Isofla-vones are common in legumes, including soybeans, black and green beans, chick peas [12]. The flavanols catechin, epicatechin (EC), epigallocatechin (EGC), and their gallate esters are widely present in green and black tea, wine, cocoa, apples, grapes and berries. Anthocyanins are abundant in red fruits such as berries, grapes and strawberries, while antho-cyanidins are present in apples, pears, berries, black grapes, persimmons, black currants, sorghum and barley grains, co-coa, and red wine [11].

Flavonoids play different roles in the ecology of plants: flavones, flavonols and anthocyanidins, through their attrac-tive colors, may act as visual signals for pollinating insects, whereas flavanols, thanks to their astringency, are a defense system against insects [2]. They can also function as stress protectors in plant cells by scavenging the reactive oxygen species (ROS) produced by the photosynthetic electron transport system and by UV radiation [13].

2. BIOCHEMICAL AND BIOLOGICAL PROPERTIES

In addition to their physiological roles in plants, many biochemical and biological properties in humans and animals have been attributed to these compounds: they exhibit not only strong antioxidant activity [14] but also the ability to modulate important cellular signaling pathways and enzy-matic activities involved in pathophysiological events such as cell proliferation, inflammation, immune response, plate-let aggregation, cytotoxicity [15-18]. Recent studies suggest that these effects of flavonoids could be related to their abil-ity to bind to DNA and RNA [19, 20].

Numerous reports have highlighted their beneficial ef-fects on human health and their potential utilization as thera-peutic tools [21-25]. In particular, the main research field regarding the biological action of these dietary compounds is that related to inflammation. An excessive inflammatory response is unanimously considered as a critical event in aging and in many human diseases, including cardiovascular diseases, neurodegenerative diseases, cancer, type II diabetes and obesity. In this connection, an increasing number of epi-demiological and experimental studies consistently support the beneficial contribution of flavonoids in combating in-flammatory-related diseases [24, 26-35].

2.1. Antioxidant Properties

Numerous studies have focused on the antioxidant prop-erties of flavonoids, which appear to be due to their efficacy as metal chelators and as free-radical scavengers, as well as to their ability to influence enzymatic and non-enzymatic systems that regulate cellular redox balance [14, 18, 36].

There is considerable evidence for the effectiveness of flavonoids as scavengers of ROS such as peroxyl, alkyl per-oxyl, hydroxyl and superoxide radicals, and reports indicate three chemical features as most likely responsible for this activity, namely an ortho-dihydroxy structure in the B-ring, and the presence, in the C-ring, of a 2,3 double bond and/or of a 4-oxo function [14]. Based on the same mechanisms, flavonoids have also been shown to be effective in regard to reactive nitrogen species (RNS), namely nitric oxide (NO) and peroxynitrite (ONOO-), counteracting the damage in-duced by these molecules [37-41].

Due to the presence of vicinal hydroxyl groups, several flavonoids can function as chelators of redox-active metal ions (e.g. copper, iron), thus preventing metal-catalyzed free radical formation and lipid peroxidation [42, 43]. Moreover, flavonoids and some of their metabolites may inhibit the activity of several enzymes responsible for ROS/RNS pro-duction, among which are xanthine oxidase, myeloperoxi-dase and reduced nicotinamide adenine dinucleotide phos-phate (NADPH) oxidase [17, 44].

76 Current Medicinal Chemistry, 2010 Vol. 17, No. 1 Leonarduzzi et al.

Fig. (1). Structures and main chemical features of the more common flavonoids and flavonolignans.


On the other hand, flavonoids might up-regulate enzymatic and non-enzymatic systems responsible for removal and de-toxification of oxidant species, namely reduced glutathione (GSH), GSH peroxidase, GSH reductase, GSH S-transferase, superoxide dismutase, catalase, as has been demonstrated in animal models for rutin [45], quercetin [46], the isoflavones daidzein and, to a lesser extent, genistein [47], the catechins epigallocatechin gallate (EGCG) [48] and EC [49]. An inter-play between polyphenolic compounds and defense systems has been fully confirmed by in vitro investigations of the antioxidant efficacy of EGCG [50], daidzein [51] and genis-tein [52]. Certain flavonoids, such as the catechins and quer-cetin, have been shown to regenerate ascorbate and α-tocopherol by means of electron transfer reactions [53-55], thus displaying a further antioxidant mechanism.

It is significant that flavonoids might also, under defined conditions, exert a marked pro-oxidant activity, thus becom-ing cytotoxic: they can undergo transition metal- or peroxi-dase-catalyzed reactions leading to the formation of ROS and highly reactive phenoxyl radicals, which can damage biological molecules such as proteins and DNA [56].

2.2. Anti-inflammatory Effects

Flavonoids have been shown, in vitro and in vivo, to down-regulate the expression of a wide spectrum of pro-inflammatory genes such as cyclooxygenase (COX), lipoxy-genase (LOX), inducible nitric oxide synthase (iNOS) and several pivotal cytokines, mainly through the inhibition of mitogen-activated protein kinase (MAPK)- and nuclear fac-tor-kappa B (NF-κB)-mediated signaling and gene transcrip-tion [28, 35, 57]. Notably, NF-κB activation requires in-volvement of MAPK [58].

The ability to inhibit the arachidonic acid pathway is par-ticularly important; this occurs at the level of phospholipase A2, COX and LOX, thus lowering the production of prosta-glandins and leukotrienes, key mediators of the acute in-flammatory cascade [59, 60].

The flavonoids present in red wine, cocoa, green and black tea, in particular quercetin, catechins and kaempferol, modulate several steps of the inflammation cascade in differ-ent human and animal cell types [61-64]. The effect of fla-vonoids on iNOS activity and NO production have also been investigated intensively: quercetin, kaempferol, genistein, myricetin and EGCG have been found to inhibit iNOS ex-pression and consequently iNOS release and activity [59, 65-67] by down-regulating extracellular signal regulated protein kinase 1/2 (ERK 1/2) and p38 MAPK phosphorylation [68] and preventing the binding of NF-κB to iNOS gene promoter [69]. As regards NF-κB, flavonoids have been shown to pre-vent its activation, by inhibiting, in vitro and in vivo, the phosphorylation of inhibitor κB (IκB) protein and by block-ing the transcription factor binding to the specific DNA con-sensus sequences [70-74]. Moreover, several flavonoids in-terfere with the production and/or function of pro-inflammatory cytokines, chemokines and adhesion mole-cules, including tumor necrosis factor α (TNFα), interleukin-1β, -6, -8 (IL-1β, IL-6, IL-8) monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), intercellular adhesion molecule (ICAM), vascular cell adhe-sion molecule (VCAM) and P-selectin [74-78] by inhibiting

the MAPK pathways, blocking NF-κB nuclear translocation and COX-2 synthesis [74, 79, 80].

2.3. Vascular Protection

A number of epidemiological studies have associated fla-vonoids with a reduced risk of cardiovascular disease [81-87]. In addition, these polyphenolic compounds have consis-tently been shown to inhibit the development of atheroscle-rotic lesions in animal models [88-92]. They may exert vas-cular protection by improving endothelial function, inhibit-ing oxidative damage and cell-cell interactions within the arterial wall, lowering high blood pressure and angiogenesis [26, 86, 93-99].

Flavonoids have been repeatedly shown to reduce plas-matic concentrations of lipids as well as low density lipopro-tein (LDL) susceptibility to oxidation [100-102]. They can ameliorate endothelial dysfunction and regularize blood pressure by enhancing the production of vasodilating factors, such as NO, and inhibiting the synthesis of vasoconstrictor factors, such as endothelin-1 and angiotensin II [103-109]. They inhibit platelet activation and aggregation [110, 111], leukocyte adhesion to endothelium and their migration into vascular intima [112, 113], proliferation and migration of smooth muscle cells (SMC) [114, 115]. Flavonoids may also exert anti-angiogenic effect by inhibiting the expression of vascular endothelial growth factor (VEGF) in vascular SMC [116, 117].

2.4. Effects on Brain Functions

Several epidemiological studies indicate that consump-tion of flavonoids such as quercetin and catechins is associ-ated with a lower incidence of Parkinson’s disease [118] and Alzheimer’s disease (AD) [119, 120]. Of note, the ability of flavonoids to cross the blood-brain barrier (BBB) is favored by their relatively high lipophilicity [121] and depends on the interaction with specific efflux transporters present in the BBB [122].

Likely mechanisms of protection appear to be quenching of oxidative stress-induced injury and neuroinflammation, restoration of neuronal communications, and improvement of cognitive and motor performance. As regards AD, catechin gallates prevent β-amyloid (Aβ)-induced neurotoxicity in vitro, in part by avoiding accumulation and aggregation of Aβ fibrils [123-125]. Catechins have been shown to modu-late the processing of amyloid precursor protein (APP) through activation of α-disintegrin and matrix metalloprote-ase-10 (MMP-10), contributing to the α-secretase cleavage of APP [126]. Further, EGCG has been demonstrated to promote the non-amyloidogenic α-secretase pathway of APP in neuronal cells via protein kinase C (PKC)-dependent sig-naling [127] and to prevent neuronal loss by activating sev-eral cell survival signals, involving c-Jun-N-terminal kinase (JNK) and p38 MAPK [127, 128].

The neuroprotective effects were originally attributed to the well-known antioxidant activities of this group of com-pounds [129,130], but it is now clear that they are also de-pendent on non-antioxidant mechanisms: a) modulation of the intracellular signaling cascade which regulates neuronal survival, death and differentiation; b) modulation of gene


expression; c) interaction with mitochondria [33, 131, 132]. Several intracellular signaling pathways have been reported to play a central role in flavonoid-induced neuronal survival, namely PKC, ERK, and Akt/phosphatidylinositol 3-kinase (Akt/PI3K) [127, 133-135], or neuronal death, i.e. JNK and NF-κB [136, 137]. EGCG has also been shown to reduce SH-SY-5Y neuroblastoma cell loss by decreasing the expres-sion of pro-apoptotic genes bax, bad, gadd45 and fas-ligand [138, 139].

2.5. Effects on Cancer Development

In vitro and in vivo studies have highlighted the direct and indirect anti-carcinogenic effects of flavonoids, which they exercise by quenching oxidative stress and inflamma-tory response, inducing apoptosis, suppressing MMP secre-tion, inhibiting cell growth, tumor cell invasion and angio-genesis [31, 34, 140-144]. Several different flavonoids have been demonstrated, in human and in animal models, to pre-vent the development of different types of malignant tumors, affecting the lung, breast, prostate, gastrointestinal tract, skin, and including metastatic formations [24, 31]; many mechanisms for their cancer-preventive activity have been proposed [31, 34, 145-147].

Flavonoids might act as antioxidants by preventing the DNA-damage that can be induced by free radicals or car-cinogens, thus blocking the initiation step of cancer [148]. Moreover, since NF-κB and COX-2 are not only involved in inflammation but also in cell proliferation, apoptosis and angiogenesis [149], the flavonoid-activated inhibition of aberrant arachidonic acid metabolism and COX activity might have a beneficial effect on neoplastic growth [150, 151].

Flavonoids have been demonstrated to inhibit cell prolif-eration in many kinds of cancerous cell lines [30, 141] by blocking the cell cycle at the G1/S or G2/M phase through down-regulation of cyclins and cyclin-dependent kinases [152-154]. The anti-tumor effect of flavonoids could also be exerted through the stimulation of apoptotic death, via acti-vation of caspases (caspase-3, -9, and -8) and pro-apoptotic proteins (p53, p27 and Bax) as well as by inhibiting anti-apoptotic proteins (Bcl-2 and Bid) and the release of cyto-chrome c [155-158].

The anti-proliferative and pro-apoptotic effects of fla-vonoids might imply activation of NF-κB [159], inhibition of Akt/PI3K, ERK and activating protein-1 (AP-1) pathways [156, 160, 161], as well as inhibition of growth factors and relevant receptors, such as platelet-derived growth factor (PDGF), PDGF receptor (PDGFR) and epidermal growth factor receptor (EGFR) [162, 163].

Interestingly, flavonoids including EGCG and other cate-chins, quercetin, genistein, luteolin and the anthocyanins, can counteract angiogenesis, which is a key event in tumor growth, invasion and metastasis, by down-regulating VEGF, VEGF receptor (VEGFR), PDGF, PDGFR, EGFR and MMP [30, 31, 141, 164-166]. Further, dietary flavonoids have been shown to interfere with cancer cell adhesion and movement processes, by inducing cytoskeletal alterations, disrupting the stress fibers and reducing myosin II regulatory light chain phosphorylation, inhibiting cell adhesion to fibronectin, re-

ducing integrins and suppressing urokinase-type plasmino-gen activator (uPA) expression [167-169].

2.6. Other Diseases

The beneficial effects of flavonoids, especially quercetin and EGCG, on diabetes and obesity are attracting increasing attention [170-174]. Relevant mechanisms of protection likely interfere with the modulation of the energy balance, endocrine systems, food intake, lipid and carbohydrate me-tabolism and redox status of cells and tissues [175].

In regard to diabetes mellitus, it has been reported that quercetin [176], EC, EGCG [177] and apigenin [178] protect pancreatic β-cells, and that black tea extracts reduce blood glucose levels during food intake in diabetic mice [179]. Moreover, the few observational and interventional clinical studies carried out so far on the potential contribution of catechins, and especially of EGCG, to treating the metabolic syndrome, consistently report beneficial effects in terms of reducing body fat, improving glucose tolerance and improv-ing overall cardiovascular health [174].

Regarding their beneficial action on obesity, it has been reported that the consumption of flavonoids increases energy expenditure, preventing weight gain in humans [180]. Sig-nificantly lower body weight and plasma lipids were also found in human volunteers [181] and in animal models [182-184] after consumption of tea extracts rich in catechins. Studies with adipocytic cell lines and animal models indicate that flavonoids may exert an anti-obesity effect by inhibiting ERK signaling, activating AMP-activated protein kinase and down-regulating adipocyte marker proteins and lipogenic enzymes [171, 173].

3. BIOAVAILABILITY OF FLAVONOIDS.

3.1. Absorption and Metabolism

Even in the presence of a large daily intake of flavonoids contained in dietary sources, it often appears that the plasma and/or tissue concentrations of these compounds are not suf-ficient to exert the expected pharmacological effects, particu-larly in the case of flavonols (quercetin). Despite the fact that isoflavones (genistein, daidzein) and flavanols (catechins) are relatively much better assimilated than flavonols [185], the bioavailability of flavonoids is generally low, because of a great number of factors, including their chemical structure and molecular weight, relatively low hydrosolubility, absorp-tion and metabolism in the gastrointestinal tract, inter-individual metabolic differences, lack of site specificity in distribution, and rapid elimination.

Most flavonoids enter the gastrointestinal tract in the form of esters, glycosides, or polymers and are not easily absorbed. Better bioavailability is provided by the break-down of these conjugates to the corresponding aglycones, which are easily diffusable through cell membranes [186, 187]. This breakdown may occur in the acidic environment of the stomach or may be operated by microflora in the gut. In the latter case, microbial degradation of tea flavonoids (catechins) has been shown to be an additional factor that can modulate the process of absorption of polyphenols in the small bowel [188]. Once absorbed by the intestinal epithe-


lium, flavonoids undergo extensive biotransformation, with the generation of different conjugated products, predomi-nantly glucuronides, but also sulphates and methylated de-rivatives, first in the intestine and then in the liver, where conjugates are secreted into the bile [11, 189]. Because of the rapidly increasing consideration given to flavonoids in re-gard to new therapeutic approaches to AD and other neu-rodegeneration processes, the observation that their methy-lated derivatives appear to cross the BBB much better than their phenolic counterparts is noteworthy [190].

Alongside the metabolic biotransformation and conse-quent degradation that occurs thanks to both the intestinal microflora and the gut-liver pathways, the actual bioavail-ability and cell/tissue accumulation of the different flavon-oids closely depends on the multidrug-resistance-associated proteins (MRP-1 and MRP-2), i.e. ATP-dependent efflux transporters that are almost ubiquitous. This metabolic path-way, also named phase III metabolism, has been analyzed in particular in connection with the intestinal absorption of catechins. MRP-2, which is located on the apical membrane of cells of the small-bowel epithelium, exports the already intracellular flavonol back to the intestinal lumen, thus modulating the actual intestinal importation of the com-pound. On the contrary, MRP-1, situated on the vascular pole of enterocytes, favors transport of the flavonoid from inside the cells into the blood [11]. Most likely, the actual flux of a given flavonoid from the gut lumen to the blood and the various organs depends on tissue distribution of MRP-1 and MRP-2 as well as on their substrate’s affinity. However, the overall mechanism of MRP-mediated flavon-oid transport appears to be far more complex, based on the confirmed observation of the ability of certain phase II meta-bolic derivatives of flavonoids to act as competitive sub-strates of that system of membrane transporters. Indeed, the potential use of flavonoids to overcome transporter-mediated chemotherapy resistance due to the frequent overexpression of MRP in several cancers is based on this property. At least as regards quercetin, the glucuronidated metabolites appear to be better competitive inhibitors of both MRP-1 and MRP-2 than either the parent compound or its methylated form [191].

On the basis of what is currently known about the bioavailability of flavonoids, it could be concluded that indi-vidual members or subgroups of this large class of com-pounds show at least some peculiarities in their biotransfor-mational and metabolic behavior, which however requires support from further pharmacokinetics data in humans. As far as the main aim of this review is concerned, namely to provide an overview of new tools to improve the delivery of flavonoids in preventive and clinical medicine, the bioavail-ability of isoflavones, flavonols and flavanols will now be examined, these being the most promising compounds in regard to such applications.

Despite some controversial data, the absorption effi-ciency of both the aglycone and the glycoside forms of isoflavones are quite similar. The metabolic processing of this group of flavonoids generates mainly glucuronides, with small proportions of sulfate esters. The elimination half-life of daidzein and genistein is 6-8 hours, i.e. relatively slow compared to other groups of flavonoids; the former is mainly

excreted with the urine, the latter with the bile [185]. Analy-sis of the pharmacokinetics of isoflavones in the plasma of human volunteers after soy bean powder uptake showed ge-nistein to have a better bioavailability than daidzein, but both reached micromolar concentrations with the same ease [192]. With regards to secondary products, the formation of equol, a bacterial metabolite of daidzein with higher estrogenic ef-fect than the parent compound, and whose formation shows great interindividual variability, is particularly interesting [185].

Intestinal absorption of quercetin, the flavonol thus far given most consideration for its therapeutic potential, is fa-vored when in the aglycone form. Its metabolism in the gut and liver appears relatively high, so that less than 2% of in-gested quercetin is recovered in the plasma [193]. Human plasma concentration of quercetin is in the low nanomolar range but can be enhanced to the low micromolar concentra-tion upon supplementation [194].

Bioavailability in humans differs markedly among the different catechins, but in general these compounds show a peak level in the sub-micromolar range following oral ad-ministration of two-three cups of green tea [195], and an elimination half-life of 1-3 hours, with highest plasma recov-ery for EGC followed by EC, then EGCG [185, 196]. Plasma EGC and EC were mainly glucuronidated or in some cases sulfated, while most of EGCG was recovered in the free form [196].

3.2. Interactions with the Metabolism of Xenobiotics

Because of the rapidly increasing demand for food sup-plements containing flavonoids, and for appropriate thera-peutic interventions employing these compounds, a safety assessment of flavonoid intake appears mandatory when quantities are ingested that greatly exceed normal dietary levels. Potential toxicity of flavonoids has recently been re-viewed [56]. In general, only megadoses of specific com-pounds have been shown to exert toxic effects. For instance, with regard to phenol-ring-containing flavonoids, generation of cytotoxic phenoxyl radicals following oxidation by per-oxidases can occur, leading to excessive ROS production and mitochondrial toxicity [56]. However, adverse effects might also be induced by moderate amounts of supplemented fla-vonoids due to their possible interference with the metabo-lism of other drugs, as has been summarized in recent review articles [189, 197]. Several flavonoids, for example the isoflavones and flavonols, are known to undergo phase I metabolism, being good substrates for cytochrome P450 (CYP). Even if modulation of this metabolic pathway in some cases has been found to be useful, as in the case of daidzein, whose CYP1A1-dependent metabolites display tumour growth inhibitory effects stronger than the parent compound [198], in many other cases it may be harmful, altering the pharmacokinetics of therapeutic drugs. For in-stance, with regard to the cytochrome CYP3A4, which is the major CYP isoenzyme in the intestine and is responsible for the metabolism of approximately 50% of all prescribed drugs, quercetin, ECG, EGCG and sylibin have been shown to exert strong down-regulation, with consequent risk of in-creased toxicity especially of drugs with limited therapeutic windows [197]. The relatively rapid metabolic disposal of


flavonoid compounds of nutritional and therapeutic interest, together with the sustained evidence of their interference with the metabolic fate of other compounds and drugs, clearly amount to a compelling indication for developing advanced methods to improve oral delivery of flavonoids. Nanoparticles may protect flavonoids from degradation in the gastrointestinal tract and, by virtue of their unique ab-sorption mechanism through lymphatic system, also provide protection from first pass metabolism in the liver. Metabolic interaction with other drugs would thus be minimized. In addition, oral but also parenteral administration of flavonoids would take advantage of a pharmacological delivery route that guarantees sustained release of the active principle at the intended site of action.

4. NANOVEHICLES: ADVANTAGES, LIMITATIONS, CLASSIFICATION AND PHARMACOKINETICS

Conventional oral administration of flavonoids appears to be inefficient, based on various findings such as low solubil-ity and permeability, instability and extensive first pass me-tabolism before reaching systemic circulation [199]. On the contrary, the development of novel drug delivery systems such as nanovehicles has recently markedly improved the efficacy and safety of drugs with unfavorable physicochemi-cal or pharmacokinetic parameters, including flavonoids, as well as enhancing patient compliance. It was thus decided to focus on recent studies dealing with the design and develop-ment of innovative submicron particulate carrier systems, initially in general terms and subsequently in relation to the delivery of flavonoids.

4.1. Drug Delivery Systems

A drug delivery system is a device to deliver a therapeu-tic agent into the human body [200]. Its efficacy depends on its action on target macromolecules located either within or on the surface of particular cells types, such action ideally being as persistent and specific as possible [201]. Designing drug delivery systems is challenging in terms of targeting the drug to specific sites. Several chemical therapeutic agents that are successful in vitro fail to produce the same effect in the human body, and consequently high concentrations would need to be administered, with the probable occurrence or intensification of side effects. These drugs require special formulation technologies to overcome problems such as poor solubility, instability in the biological milieu, and poor bioavailability [202]. For this purpose, sophisticated micro-fabricated (1-1000 µm) and nanofabricated (1-1000 nm) de-vices for drug delivery have recently been developed.

Microdevices act as reservoirs of a given therapeutic agent, which is released into the tissue interstitial space. Due to their size, they cannot cross most of the biological barriers and thus must be delivered directly to the site of interest (lo-cal delivery), minimizing toxicity and prolonging release. On the contrary, nanodevices are delivery systems for intracellu-lar delivery, which are taken in by cells and sorted into dif-ferent cytoplasmic compartments [203].

4.2. Nanovehicle-Based Drug Delivery Systems

Nanometer-sized particles are in the same size-range as antibodies, membrane receptors and proteins: this

biomimetic feature, together with their high surface area/mass ratio, quantum properties and ability to adsorb and carry other compounds, make nanoparticles powerful tools for the medicinal approach (nanomedicine) as well as for imaging and diagnostic purposes [204]. The use of nanoma-terials enables key properties of drugs to be modulated, such as solubility, diffusivity, blood circulation half-life, immu-nogenicity, which potentially can improve treatment of vari-ous diseases including cancer, diabetes, infections, asthma, and allergies [205].

There are several prerequisites for the successful devel-opment and manufacturing of targeted drug delivery vehi-cles. For instance, the biophysicochemical properties of the vehicle, such as size, charge, and surface hydrophilicity, can all impact on the circulating half-life of the particles as well as on their biodistribution. Small molecules, proteins, pep-tides, and nucleic acid loaded nanoparticles are not recog-nized by the immune system and are efficiently targeted to particular tissue types [202]. Moreover, the presence of tar-geting ligands can increase the interaction of drug delivery systems with cells, and potentially enhance cellular uptake by receptor-mediated endocytosis [206].

There are many advantages of employing nanoparticles for therapeutic purposes: 1) specific delivery to a cell or a tissue, optimizing efficacy and minimizing systemic side effect; 2) half-life prolongation in systemic circulation; 3) drug release at a sustained rate or in an environmentally re-sponsive manner; 4) better patient compliance; 5) improved solubility of poorly water-soluble drugs; 6) greater protection against enzymatic degradation and other destructive factors thanks to the complete isolation of drug molecules from the environment; 7) simultaneous delivery of two or more drugs to generate a synergistic effect; 8) the BBB, the branching pathways of the pulmonary system, and the tight epithelial junction of the skin can be bypassed; 9) good biocompatibil-ity and significant reduction of health-care costs [202, 206-208].

There are also some limitations on the use of nanoparti-cles: 1) difficulty of handling, storage and administration, because of their relatively high susceptibility to aggregation; 2) possible access to unintended environments, e.g. the cellu-lar nucleus, with potential harmful consequences like DNA damage and cell cycle changes; 3) generation of pro-oxidant chemical species (under exposure to visible or ultraviolet light, or to transition metals) destabilizing the balance be-tween the production of ROS and the biological system’s ability to detoxify; 4) unexpected pro-inflammatory effects through up-regulation of redox-sensitive transcription factors including NF-κB and AP-1; 5) induction of immune re-sponse; 6) protein denaturation/degradation [202, 209-211].

4.3. Types of Nanovehicles

There are numerous nano-based drug delivery systems: dendrimers [212], micelles [213], nanoemulsion [214], self-emulsifying systems [215], cubic phase gels [216], inorganic nanoparticles [217], nanocrystals [218], nanotubes [219], and quantum dots [220], but this review will chiefly concentrate on lipid based (liposomes and lipid nanoparticles with a solid matrix), and polymeric based (polymeric nanoparticles)


nanocarriers, which are considered to be among the most suitable systems for flavonoid delivery (Fig. 2). 4.3.1. Liposomes

Liposomes are the first system employed for drug deliv-ery: they are concentric vesicles consisting of an aqueous core surrounded by a membranous lipid bilayer and may be small (≤ 100 nm), medium (100-500 nm) or large (≥ 1 µm)

[221]. Thanks to their structure, they can entrap and deliver hydrophilic (in the aqueous compartment), hydrophobic (in the lipid bilayers), and amphipathic (in the vesicular bound-ary zone) drugs.

A significant disadvantage of liposomes is their relatively fast elimination from the blood by the cells of reticuloendo-thelial system (RES), primarily in the liver and spleen [222].

Fig. (2). Principal types of lipid-based (liposome and solid lipid nanoparticle) and polymeric-based (polymeric nanoparticles: nanosphere and nanocapsule) nanovehicles.


To reduce this problem, the liposomal structure was modi-fied, developing the so-called long-circulating liposomes by coating their surface with inert and biocompatible polymers, such us polyethylene glycol (PEG) [223]. Further, liposomes may include pH-sensitive components to achieve a pH-sensitive release of their content into the cytoplasm [224]. 4.3.2. Lipid Nanoparticles with a Solid Matrix

Solid lipid nanoparticles (SLN or lipospheres or nano-spheres) are drug carrier systems of 50-1000 nm, with a solid hydrophobic core surrounded by a phospholipid monolayer, also containing surfactants as stabilizers.

SLN have a high specific surface due to their small di-ameters, a spherical shape and favourable electric potential. The main advantages of SLN are excellent physical stability, protection of incorporated drugs from degradation, con-trolled drug release, site-specific targeting and good toler-ability. However, potential limitations have also been ob-served, such as low loading capacity and expulsion of drug during storage. To increase the payload and prevent drug expulsion, a liquid lipid (oil) was blended with the solid lipid component of conventional SLN, thus achieving a lipid ma-trix as irregular as possible. These nanostructured lipid carri-ers increase drug loading capacity and minimize drug expul-sion during storage.

Due to their physical-chemical characteristics, SLN can be administered by the parenteral, oral, transdermal, dermatological, duodenal, and ocular routes [225]. They are also more stable than liposomes in biological systems and, due to their easy biodegradability, they are less toxic than polymeric nanoparticles; these characteristics make them a highly versatile delivery system [226]. 4.3.3. Polymeric Nanoparticles

These are solid colloidal structures composed of natural or synthetic polymers with a size range from 10 nm to 1000 nm [227]. Depending on the shape of the particle, there are two different types: nanocapsules and nanospheres, for which the preparation methods also differ. Nanocapsules are vesicular systems with the drug in a core surrounded by a polymeric membrane, whereas nanospheres are porous ma-trix systems in which the drug is uniformly dispersed [228].

The polymeric materials employed possess high biocom-patibility, biodegradability, stability and surface modification potential; these features enable them to provide the con-trolled release of pharmacologically-active substances to the specific site of action at the therapeutically optimal rate and dose [229]. The most widely used polymers are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), their co-polymer poly(lactide-co-glycolide) (PLGA), poly(methyl methacry-late) (PMMA), poly(vinyl alcohol) (PVA), and poly(alkyl-cyanoacrylate) (PACA) [230-234]. Natural polymers, such as alginate, gelatin, chitosan, and albumin, have also been utilized to avoid the toxicological risk often associated with the use of synthetic polymers [235-238]. Polymeric nanopar-ticles are used to deliver both hydrophobic and hydrophilic compounds, proteins, peptides, genes, DNA, antisense oli-gonucleotides or vaccines [235, 239-241].

To counter the many advantages offered by polymeric nanoparticles, they also possess limitations in the form of

relatively high cytotoxicity [242] and rapid removal from blood. In order to avoid the latter drawback, they can be cov-ered with a layer of non-ionic surfactant, such as poloxamers or poloxamines, reducing opsonization [243], or coated with hydrophilic substances like the non-ionic polymer PEG [244], or with carbohydrates (cyclodextrins) [245].

Interestingly, because conventional polymeric nanoparti-cles cannot permeate the BBB but require receptor-mediated transport, poly(butyl-cyanoacrylate) (PBCA) nanoparticles can be covered with polysorbate 80, thus achieving signifi-cant transport across the barrier [246, 247].

The two principal routes of administration of polymeric nanoparticles are the oral and the intravenous routes. Once they have reached the target tissue, drug release depends on the physicochemical characteristics of both carrier and drug: it may occur by desorption from the colloidal surface, by diffusion through the polymeric wall (capsules) or through the polymeric matrix (spheres), or by nanoparticle erosion [248].

4.4. Pharmacokinetics of Nanovehicles

4.4.1. Particle Size and Surface Properties Size and size distribution influence nanoparticle stability,

drug loading, targeting ability and drug release, and also determine in vivo distribution, biological fate and toxicity. The nanoparticles used in a drug delivery system should be large enough to prevent their rapid leakage into blood capil-laries but, at the same time, small enough to escape capture by phagocytes of the RES [249]. Small particles allow a rapid drug release because of their high surface area/volume ratio, while relatively larger particles encapsulate more drug and release it more slowly [250].

Surface charge of nanoparticles is defined in terms of zeta potential: it reflects the electrical potential of particles and is influenced by their composition and by the medium in which they are dispersed. Nanoparticles with a zeta potential above +30 mV are stable in suspension, as the surface charge prevents particle aggregation [251]. 4.4.2. Biodistribution

Nanomaterials cannot readily penetrate membranes and their distribution depends on a variety of anatomic features. In case of oral or cutaneous administration the epithelium is the first barrier. With parenteral administration, hydrophobic particles are rapidly coated by serum proteins, termed opson-ins, which facilitate particle uptake by the cells of RES into the liver, spleen and lymph nodes. Usually, most of the op-sonized particles are cleared from the circulation in less than 5 min, but coating nanoparticles with PEG can reduce and/or delay opsonization, thus significantly extending their half-life in the blood circulation [252].

Another barrier to tissue distribution of nanoparticles is the vascular endothelium. The latter is of the “continuous” type in most of the circulatory districts, with tight junctions between endothelial cells and basement membrane that cre-ate gaps of less than 2 nm. In this connection, the BBB is particular, as in this barrier the endothelial junctions are par-ticularly tight and effective. However, in some specialized tissues like the liver and spleen, endothelium is of the “fenes-


trated” type and allows the transit of material of up to 100 nm [253].

It is noteworthy that during vascular inflammation or damage, due to various agents or conditions, the endothelium becomes leaky and allows enhanced transit of particulate materials; moreover, in some cancers, the endothelium un-dergoes a process known as enhanced permeability and re-tention (EPR). Nanotechnology may exploit this EPR effect to improve delivery of drugs into neoplastic tissue [254].

Once they have passed the vascular endothelium barrier, nanomaterials reach the extracellular matrix, which is com-posed mainly of collagen, polysaccharides and glycopro-teins, that in principle tend to impede movement of exoge-nous molecules. However, water channels are present in this tangled matrix that may allow the transit of macromolecules. 4.4.3. Cellular Uptake

Nanocarriers are taken over by the cells through endocy-totic mechanisms. Large particles (0.25-10 µm) are taken up by phagocytosis, operated by macrophages and neutrophilis. Smaller particles are incorporated either by receptor-mediated endocytosis, namely clathrin-mediated endocytosis and potocytosis, or by pinocytosis [255].

Following their uptake, which is concentration-, time- and cell-type-dependent, nanovehicles are transported in primary endosomes and then in sorting endosomes. From the sorting endosomes, a fraction of the nanovehicle is extruded by exocytosis, while the remaining fraction is transported to secondary endosomes and fused with lysosomes. After par-tial degradation of their content by lysosomal hydrolytic en-zymes, nanocarriers eventually enter the cytosolic compart-ment. However, in the case of caveolae-mediated internaliza-

tion (potocytosis), particles are directly deposited into the cytoplasm.

Importantly, the cellular uptake of nanoparticles through endocytotic routes avoids the involvement of membrane-associated and intracellular transporters, like MRP. How-ever, the efficient internalization of these carriers greatly much depends on their interaction with endocytotic machin-ery, the cytoskeleton and various subcellular organelles [256]. 4.4.4. Intracellular Targeting

Effective intracellular drug delivery is important for therapeutic agents which have specific molecular targets inside a cell. Targets are mainly located in the lysosomes, cytosol, mitochondria or nucleus [256]. The capacity of nanovehicles to be retained within the lysosomes depends on their surface charge: nanoparticles which are negatively charged at pH 4-5 remain in the lysosomes, while those with a cationic charge escape the endosomal compartment and reach the cytoplasm [257]. Hence, by changing the surface charge of nanoparticles, it is possible to target either the lysosomes or the cytoplasm. Lysosomal targeting is ex-ploited in the therapy of lysosomal storage diseases (e.g., Tay-Sachs, Gaucher’s, Fabry’s diseases), by exogenous de-livery of enzymes or genes. A short endo-lysosomal step followed by rapid cytosolic delivery is crucial for carriers whose transported drug is susceptible to lysosomal degrada-tion.

Suitable modification of the nanovehicles surface makes possible to localize them in the mitochondria. Targeting the mitochondria appears very promising because of the central role of mitochondrial dysfunction in many human diseases. Permanently positive motifs with cationic charge allow

Table 1. In Vivo and In Vitro Studies of Flavonol Administration Using Different Nanovehicles

Flavonols Nanovehicle Experimental Model Therapeutic Application Refs.

PC- liposomes human cancer cells growth inhibition [264]

liposomes rats with permanent middle cerebral artery occlusion

treatment of ischemic brain damage [265]

PEG-liposomes oral and intranasal administration in rats anxiolitic and cognitive ef-fect [266]

unilamellar PEG-liposomes

intravenous injection in tumor-bearing mice

inhibition of solid tumor growth and angiogenesis [267]

multilamellar galactosylated liposomes intravenous injection in arsenite-or CCl4-treated rats treatment of liver fibrosis [268]

unilamellar mannosylated liposomes intravenous injection in young and aged rat model of ischemia-reperfusion injury

treatment of cerebral ische-mia-reperfusion damage [269]

PEG-polyester-PEG triblock core-shell nanoparticles cell-free in vitro system n.a. [275]

PLGA-nanocapsules oral administration in arsenite-treated rats treatment of hepatic and neuronal oxidative damage [276]

Eudragit® E-PVA-nanoparticles cell-free in vitro system antioxidant [277]

polylactide nanocapsules; multilamellar PE-liposomes; niosomes

subcutaneous injection in amistogote-infected hamsters antileishmanial agent [278]

Que

rcet

in

PEG-SLNs stomach injection or intestine perfusion in rats n.a. [272]

(n.a.: not analized).


nanocarriers to penetrate the mitochondria, exploiting their ability to accumulate lipophilic cations or their pro-tein/peptide import machinery [258].

With regard to drugs that target the nucleus of the cell, molecules smaller than 100 nm can pass through the nuclear pore by passive diffusion while larger ones are generally transported by an active signal-mediated process, known as the nuclear localization signal [259].

5. NANOVEHICLE-BASED DELIVERY OF FLAVONOIDS

Despite the growing interest in nanotechnology as a new strategy for drug delivery, with regard to its application to flavonoid supplementation few reports are yet available (Ta-bles 1-4). In particular, most of the relevant studies are in vitro studies, a small number were carried out in experimen-tal animals, while no human studies have yet been reported. Nevertheless, the available data all point to nanocarriers as very suitable tools to overcome many of the problems asso-

ciated with flavonoid pharmacology, such as their low solu-bility, short half-life, and generally poor bioavailability.

5.1. Liposomes and Flavonoids

Liposomes are currently the nanocarrier system most widely studied for use as a vehicle for flavonoids. Multi-lamellar phosphatidylcholine (PC) liposomes, with or with-out the addition of surfactant agents, have been characterized for topical and intratumor administration of catechin, EC and EGCG in nude mice [260]. The anionic species deoxycholic acid (DA) increased the encapsulation of all tested catechins, but in particular that of EGCG, while the anionic species dicetyl phosphate was effective only in the case of catechin. Intratumoral nanodelivery, in particular using surfactant-free vesicles, enhanced the accumulation of catechin and EC in neoplastic tissue, while topical application of the same for-mulations did not increase deposition of the two flavanols in the skin. EGCG performed anomalously, likely due to the higher lipophilicity of this flavonoid: its deposition in the

Table 2. In Vivo and In Vitro Studies of Flavanol Administration Using Different Nanovehicles

Flavanols Nanovehicle Experimental Model Therapeutic Application Refs.

chitosan-tripolyphosphate nanoparticles cell-free in vitro system n.a. [283]

multilamellar PC-liposomes topical and intratumor administration in mice n.a. [260]

ethanol enriched liposomes stratum corneum model; topical application in mice n.a. [262] Te

a C

atec

hins

ethanol enriched liposomes intratumor injection in mice anti-tumoral [261]

PEG-PLA-nanoparticles PC3 cells; tumor xenograft mouse model antitumoral [281]

EGC

G

PLGA-nanoparticles cyclosporine-treated rats treatment of chronic nephro-toxicity [282]

Cat

echi

n

liposomes entrapped in calcium pectinate gel beads intratumor injection in mice n.a. [263]

(n.a.: not analized). Table 3. In Vivo and In Vitro Studies of Isoflavone and Flavanone Administration Using Different Nanovehicles

Isoflavones and Flavanones

Nanovehicle Experimental Model Therapeutic Application Refs.

hyper-branched polyester nanoparticles cell-free in vitro system n.a. [280]

Dai

dzei

n

PEG-SLNs intravenous administration in dogs and middle cerebral artery occlusion

treatment of cardio-cerebrovascular diseases [273]

Gen

iste

in

nanoemulsion pig ear skin n.a. [288]

Nar

inge

nin

Eudragit® E-PVA-nanoparticles oral administration in CCl4-treated rats treatment of liver oxidative dam-

age [279]



skin was improved by both surfactant-free and DA-enriched liposomes compared to aqueous systems, while no signifi-cant difference emerged in its tumor accumulation with the various carriers adopted in the study [260]. The same authors reported even higher EGCG encapsulation for DA-liposomes prepared in the presence of 15% ethanol [261], as well as an enhanced catechin in vitro and in vivo skin permeation [262] and deposition in basal cell carcinomas, compared to both the free form and ethanol-free liposomes [261]. It was hy-pothesized that ethanol-enriched vesicles may penetrated the skin more easily because of the elasticity conferred to them by insertion of alcohol into the PC membranes [262].

Cholesterol also modulated liposome properties, as shown for catechin, whose encapsulation and release to the skin was decreased in particles containing the sterol, as a consequence of their more rigid structure [262]. In addition, liposomal systems appeared to strengthen the EGCG anti-proliferative activity against tumoral cells, probably by pro-tecting the flavanol from oxidative degradation [261].

Calcium pectinate gel (CPG) beads, entrapping catechin-loaded liposomes, with or without hydroxypropylmethylcel-lulose (HPMC) as coating agent, were considered for cate-chin supplementation in simulated intestinal and gastric flu-ids. The entrapment efficiency (EE) for catechin, i.e. the percent amount of entrapped drug compared to that initially administered, was higher for CPG-liposomes in the presence of HPMC, that likely bound free catechin and prevented its leakage during bead preparation. Compared to CPG beads alone, CPG-liposomes delayed catechin release into the acid gastric fluid, and even more efficiently delayed its release into the neutral intestinal fluid, while HPMC coating was effective in regulating catechin release only in the intestine, undergoing rapid degradation in an acidic environment like that present in the stomach [263].

PC liposomes have been used to deliver quercetin and ru-tin to human cancer cells in vitro [264]; liposomal quercetin has been shown to reduce ischemic brain damage in rats, presumably by restoring the brain GSH pool [265]. A pegy-lated liposome system has also successfully been employed, in male Wistar rats, for intranasal administration of quer-cetin. This flavonol, a planar molecule, can easily intercalate into the liposomal phospholipids and is more easily trans-ported by this semilipophilic vehicle through the olfactory epithelium up to the cerebrospinal fluid and eventually to the brain, with an efficient amplification of its therapeutic effects [266].

The chemiotherapeutic properties of quercetin are em-powered if it is injected intravenously in tumor-bearing mice after being incorporated in unilamellar PEG-coated liposomes, as has been confirmed in vivo by the inhibition of tumor growth and angiogenesis, and in vitro by the induction of apoptosis. In particular, quercetin encapsulation in the non-aqueous core of liposomes appeared, compared to free quercetin, to significantly prolong the permanence of this flavonoid in the blood circulation, and to facilitate its spe-cific accumulation in tumor tissues [267]. A further optimi-zation in quercetin supplementation, notably for its delivery to hepatic tissue, was achieved using multilamellar galacto-sylated liposomes, that can specifically interact with the ga-lactosyl receptors present on the hepatocyte surface [268]. Indeed, arsenite-induced liver fibrosis in rats was efficiently prevented by the injection of a galactosylated liposomal for-mulation of flavonoids, that likely exerted cellular and mem-brane protection against oxidative attack and avoided arsenic deposition in the mitochondria. On the contrary, free quer-cetin was unable to counteract hepatocellular damage [268]. Small unilamellar mannosylated liposomes were also suc-cessfully employed for site-specific quercetin delivery to the brain, where mannosyl receptors are localized. Again, whereas free and liposomal quercetin did not protect aged or young rat brain from ischemia-reperfusion related oxidative injury, treatment with the flavonoid encapsulated in manno-sylated liposomes, a lipophilic vehicle that in ischemic con-dition may diffuse into the hyperpermeable BBB, fully pre-vented lipid peroxidation in neuronal cells and inhibited edema formation [269].

Silymarin bioavailability can be significantly increased by oral spray administration of a suspension of stable hybrid liposomes (composed of soybean lecithin, cholesterol, sur-factant agents and charge inducers) incorporating the drug molecule [270, 271]. The main advantages of this delivery system, that showed good hepatoprotective activity against CCl4-induced oxidative damage in rat liver, are the ease of its utilization and the prevention of drug degradation in the gastrointestinal tracts. Even more importantly, because of their mucoadhesive properties that favor silymarin’s permea-tion through the buccal mucosa, liposome vesicles might enhance the drug’s body absorption. This permeation was particularly evident for positively charged liposomes, namely those synthesized with the addition of the cationic lipid stea-rylamine, that not only showed higher silymarin EE, but also interacted with the negative charges of the mucus layer [270].

Table 4. In Vivo and In Vitro Studies of Flavonolignan Administration Using Different Nanovehicles

Flavonolignans Nanovehicle Experimental Model Therapeutic Applica-tion Refs.

hybrid liposomes oral spray administration in CCl4-treated rats hepatoprotective activity [270, 271] Si

lym

arin

SEDDS/SMEDDS gavage administration in rabbits n.a. [291]

Sily

bin

stealth SLNs cell-free in vitro system n.a. [274]



With regard to the effects induced by the presence in liposomal carrier of surfactant agents, such as Tween 20, the entrapment efficiency of flavonoids appeared to be some-what reduced, but on the other hand their permeation through the mucosa was significantly improved, likely because of a higher deformability conferred to the liposomes by the sur-factant [270, 271].

5.2. Solid Lipid Nanoparticles and Flavonoids

Few reports have addressed the application of SLN to flavonoid supplementation. SLN coated with PEG were pro-duced for quercetin encapsulation and their gastrointestinal adsorption was analyzed after oral administration in rats. These SLN in in vitro tests produced a good EE for quercetin and a sustained release profile. The in vivo absorption of SLN was good at the intestinal level and especially favored by the presence of surfactants like Tween 80 or lecithin. Moreover, quercetin encapsulation in the lipid matrix of SLN reduced its exposure to intestinal bacteria and enzymes and gave the drug good adhesion to mucosal surface, further im-proving its bioavailability [272].

Daidzein-loaded SLN bearing pegylated phospholipids as stabilizer have been tested in animal models as an injectable delivery tool for treatment of cardio- and cerebro-vascular diseases. This kind of drug delivery exhibited high efficacy in organ targeting, while oral or intravenous administration of daidzein in dimethyl sulfoxide solution mainly concen-trated in the kidney, and from the kidney was promptly eliminated [273].

SLN composed of stearic acid and surfactant polyoxyethilene 20 stearyl ether (Brij 78) have recently been considered for parenteral administration of silibinin, one of the molecules present in the active silymarin extract, because of the slow flavonoid release from SLN and their stability in storage [274].

5.3. Polymeric Nanoparticles and Flavonoids

Polymeric nanoparticles have very recently been evalu-ated as potential vectors for flavonoid delivery, since their colloidal nature may help in overcoming diverse barriers in the body, including the gastrointestinal mucosa and the BBB. Sustained in vitro release of quercetin was achieved by its entrapment in triblock core-shell nanoparticles consisting of copolymers of hydrophilic PEG intercalating different hy-drophobic polyesters [275]. Either PLGA-nanocapsules [276] or Eudragit® E (a cationic aminoalkyl methacrylate copolymer) plus PVA-nanoparticles [277] have been de-signed for quercetin inclusion and have demonstrated very good drug EE. In addition, both systems strengthened the antioxidant activity of the flavonoid with respect to the free forms, an effect that was quite closely correlated either to the marked increase in drug release or to the target tissue accu-mulation observed for Eudragit® E-particles and PLGA-capsules, respectively.

Different vesicular delivery models have been investi-gated for quercetin supplementation as an anti-leishmanial agent in hamsters. The species included in the study were: polylactide polymer-made nanocapsules; multilamellar phosphatidyl ethanolamine-liposomes; niosomes, i.e. non-

ionic surfactant based liposomes, in this case composed of sorbitan monopalmitate; microspheres prepared from poly-lactic coglycolic acid polymers. At equivalent quercetin doses, nanocapsules exhibited the highest EE and the highest potency in counteracting the parasite burden in the spleen, without exerting liver nor kidney toxicity. The efficacy ap-peared to be inversely correlated to the vesicle size, which can be partly explained by the fact that the bigger the parti-cles, the easier is their uptake by reticuloendothelial cells. Another advantage of nanocapsules over other carriers is the absence of lipids in their structure, avoiding attack by circu-lating lipases and thus ensuring greater stability of the encap-sulated drug and longer permanence in the blood circulation [278].

Eudragit® E-nanoparticles might make oral administra-tion of the hepatoprotective agent naringenin successful: the presence of basic dimethylamino groups on the polymer fa-vors the particle dissolution in the acid gastric environment, thus also affording the solubilization of naringenin, which otherwise is extremely insoluble in aqueous systems. The strong hydrophobic affinity between naringenin and Eu-dragit® E, and the intercalating property of PVA, added as an emulsion stabilizer, led to the formation of a very stable par-ticle characterized by a high drug EE and efficient drug re-lease. All these features would facilitate naringenin’s uptake and transport into the hepatic tissue, enhancing its therapeu-tic properties as antioxidant, anti-apoptotic and hepatoprotec-tive agent, as has been confirmed by experimental data ob-tained with CCl4-intoxicated rats [279].

A novel hyper-branched polyester, i.e. the aliphatic poly-ester Boltorn® H120 modified by reaction with succinic an-hydride and glycidyl methacrilate, has been characterized for the oral administration of daidzein encapsulated in nanopar-ticles. Not only does this system enhance the hydrosolubility of daidzein, but it also appears to act as a controlled drug release carrier, the release rate of daidzein from this complex being relatively slow over a few days [280].

A marked increase of EGCG’s anti-proliferative, anti-angiogenic and pro-apototic activities was observed in PC3 cells treated with the drug encapsulated in pegylated PLA-nanoparticles. The same preparation also prolonged drug permanence in the circulation of a xenografted mouse model and significantly reduced tumor growth [281]. The therapeu-tic efficacy of EGCG in the oral treatment of nephrotoxic rats was also strongly enhanced by its encapsulation in PLGA-nanoparticles [282].

Alternative delivery of tea catechins was achieved with chitosan-tripolyphosphate nanoparticles. In this case, drug release appeared to be favored by the presence of relatively low chitosan concentrations [283]. A nanoparticle formula-tion was also evaluated for oral delivery of an ethanol extract of Cuscuta Chinensis, a traditional Chinese herbal medicine commonly used as a tonic for liver and kidney, in which more than 20 different flavonoids are present including quer-cetin, kaempferol and their corresponding glycosides. The nanoparticles were prepared with PF68, a water-soluble non-ionic surface-active copolymer that penetrates the herbal alcoholic extract during the nanonization process, forming very stable and small nanospheres that improved the drug’s bioavailability. Consequently, the therapeutic dose of the


medicine could be reduced, thanks to the stronger antioxi-dant and hepatoprotective effects obtained with the vehicu-lated extract compared to the free extract [284].

5.4. Other Delivery Systems

Absorption of orally-administered rutin, the glycoside of quercetin, might be improved by using a nanocrystal formu-lation that can be incorporated into solid dosage forms such as tablets or capsules [285, 286]. The nanocrystal approach is based on the principle that in vivo kinetic solubility of a given substance is facilitated by diminishing its size, which increases saturation solubility, and provides a larger surface area and a faster dissolution velocity [287]. Rutin nanocrys-tals have been confirmed to have a low but long-lasting in-crease in kinetic solubility and a more marked enhancement in dissolution velocity, properties that make their employ-ment much more effective than that of the raw drug in the case of oral administration. Rutin nanocrystals can be ob-tained by lyophilization and spray drying of aqueous rutin nanosuspensions in the presence of different stabilizers. The most efficient stabilizer appears to be sodium dodecylsulfate [285, 286].

Nanoemulsion formulations composed of lecithin, water and medium-chain triglycerides or octyldodecanol can be obtained by spontaneous emulsification, yielding nanosized droplets that have successfully been employed for the topical administration of genistein [288].

Finally, two unconventional but very promising types of nanodelivery are worthy of mention, namely self-emul-sifying and self-microemulsifying drug delivery systems (SEDDS and SMEDDS). These systems consist of isotropic mixtures of drugs, lipids, surfactants and co-surfactants that, due to the motility of the gastrointestinal tract, form microe-mulsions with a particle size even smaller than 100 nm. These small emulsion droplets apparently provide powerful drug release and rapid absorption. Moreover, any excipients that may be added to SEDDS/SMEDDS can prevent both presystemic drug metabolism and intestinal efflux, and pro-mote the intestinal lymphatic transport of the drug, with a net gain in its absolute bioavailability [289]. SEDDS/SMEDDS formulations have so far been developed for oral administra-tion of the isoflavone puerarin [290] and of silymarin [291] and tested in animal models with an amelioration of flavon-oid bioavailability, even if further optimizations of the sys-tem is needed.

CONCLUSIONS

Despite the well recognized antioxidant, anti-inflam-matory and anti-proliferative properties of flavonoids, their utilization in the prevention and treatment of major human diseases has been so far quite limited, excluding oncology. On the contrary, the same pharmacological potential has been “adopted” to give a great impulse, especially in West-ern countries, to the commercial diffusion of plant extracts to cure for example menopausal side effects, chronic venous insufficiency, but also those that are called more vaguely “age-related changes”. Alongside the difficulty in defining and monitoring the exact amount of an active flavonoid ad-ministered within a plant or fruit extract, the great pharma-

cological potential of the whole class of these compounds risks not to adequately be used.

Only a net improvement in bioavailability and target’s selectivity will definitely promote a sustainable and reliable use of flavonoids in preventive medicine and therapy. Ad-ministration of flavonoids by nanovehicles might likely con-tribute to promoting their appropriate pharmacological adop-tion and use.

ACKNOLEDGEMENTS

The authors wish to thank the European Science Fonda-tion COST B35 Action, the Italian Ministry of University, Prin 2007, the Region of Piedmont (Ricerca Sanitaria Final-izzata 2007, 2008) and the University of Turin, Italy for sup-porting this work.

ABBREVIATIONS

Aβ = β-amyloid AD = Alzheimer’s disease AP-1 = activating protein-1 APP = amyloid precursor protein BBB = blood-brain barrier COX = cyclooxygenase CPG = calcium pectinate gel CYP = cytochrome P450 DA = deoxycholic acid EC = epicatechin EE = entrapment efficiency EGC = epigallocatechin EGCG = epigallocatechin 3-gallate EGFR = epidermal growth factor receptor EPR = enhanced permeability and retention ERK1/2 = extracellular signal regulated protein kinase

1/2 GSH = glutathione HPMC = hydroxypropylmethylcellulose ICAM = intercellular adhesion molecule IĸB = inhibitor ĸB IL = interleukin iNOS = inducible nitric oxide synthase JNK = c-Jun-N-terminal kinase LDL = low density lipoprotein LOX = lipoxygenase MAPK = mitogen-activated protein kinase MCP-1 = monocyte chemotactic protein-1 MIP-2 = macrophage inflammatory protein-2


MMP = matrix metalloproteases MRP = multidrug resistance-associated proteins NADPH = reduced nicotinamide adenine dinucleotide

phosphate NF-ĸB = nuclear factor ĸB NO = nitric oxide ONOO- = peroxynitrite PACA = poly(alkyl-cyanoacrylate) PBCA = poly(butyl-cyanoacrylate) PC = phosphatidylcholine PDGF = platelet-derived growth factor PDGFR = platelet-derived growth factor receptor PEG = polyethylene glycol PGA = poly(glycolic acid) PI3K = phosphatidylinositol 3-kinase PKC = protein kinase C PLA = poly(lactic acid) PLGA = poly(lactide-co-glycolide) PMMA = poly(methyl methacrylate) PVA = poly(vinyl alcohol) RES = reticuloendothelial system RNS = reactive nitrogen species ROS = reactive oxygen species SEDDS = self-emulsifying drug delivery systems SLN = solid lipid nanoparticle SMC = smooth muscle cells SMEDDS = self-microemulsifying drug delivery systems TNFα = tumor necrosis factor-α uPA = urokinase-type plasminogen activator VCAM = vascular cell adhesion molecule VEGF = vascular endothelial growth factor VEGFR = vascular endothelial growth factor receptor

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Received: September 02, 2009 Revised: December 10, 2009 Accepted: December 12, 2009

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