**7. Perspectives in the formulation of flavonoids metal complexes**

In order to enhance the water solubility and to control the release of flavonoids metal complexes, many efforts have been focused on the preparation of cyclodextrin complexes, or of novel micro‐ and nano‐carriers, such as liposomes and organic compounds‐inorganic particles hybrid materials (HMs). In the following subchapter, the most important systems developed for the inclusion of flavonoids metal complexes with antioxidant or antitumor activity will be reviewed, as well as the systems that include flavonoids with antitumor activity based on the complexation process between flavonoid and metal ions.

#### **7.1. Liposomal systems**

Flavonoids' ability to penetrate into the hydrophobic regions of lipid bilayers in biological membranes is a key factor to prevent peroxidation of unsaturated double bonds. In this regard, the lipophilicity of flavonoids is essential for an adequate penetration. According to Tweedy's chelation theory, the polarity of the metal ion is reduced, as a consequence of chelation, mainly because of the partial sharing of its positive charge with the ligand's donor groups and possible electron delocalization over the entire ring. Consequently, the lipophilic character of the chelates increases, favoring their permeation through the lipid layers of biological membranes [112]. For two complexes of quercetin and taxifolin with Fe(II), it was assumed that the oxygen charges are generally decreases, while the main negative charge is localized on the iron atom. Thus, after chelating the metal ion, the polarity of flavonoid molecules generally decreased, while the iron atom becomes the most polar and hence hydrophilic part of the molecule. Overall, the lipophilicity of the complexes is considerably larger than that of the corresponding free flavonoids [113, 114].

On the other side, the hydrophobicity of flavonoids chelates dramatically reduces the water solubility, which restricts their medical applications. Integration of these complexes into liposomes may increase their bioavailability and improve the therapeutic effect.

The interaction study of quercetin‐iron complexes with dimyristoylphosphatidylcholine (DMPC) or palmitoyl‐oleoyl phosphatidylethanolamine (POPE) multilamellar liposomes revealed that, during preparation, quercetin should be added first to the suspension of liposomes [115]. It was presumed that quercetin can increase the permeability of lipid bilayers to iron cations, showing ionophore activity toward iron cations.

#### **7.2. Cyclodextrins**

The inclusion into cyclodextrins is a convenient alternative to solve the problems related to the low solubility of hydrophobic drugs, such as flavonoid complexes. Inclusion of Cu(II) and Cr(III) complexes of flavonoids morin, quercetin, and 6‐hydroxyflavone into β‐cyclodextrin led to an enhancement of aqueous solubility [116]. The anticancer activity of metal‐flavonoid complexes was evaluated in terms of dsDNA binding in the environment of beta‐cyclodextrin, and it was revealed that DNA could bind Cu‐flavonoid‐βCD through intercalation and Cr‐flavonoid‐βCD via an electrostatic‐binding mode.

#### **7.3. Organic/inorganic hybrid nanosystems**

Undisputedly, there is a consistent amount of experimental evidence regarding the interaction of flavonoids and their metal complexes with nucleic acids. However, some details with respect to the binding sites in the DNA structure need further investigations. There appears that the complexes possess higher affinity toward GC‐rich sequences in DNA [111], but this

In order to enhance the water solubility and to control the release of flavonoids metal complexes, many efforts have been focused on the preparation of cyclodextrin complexes, or of novel micro‐ and nano‐carriers, such as liposomes and organic compounds‐inorganic particles hybrid materials (HMs). In the following subchapter, the most important systems developed for the inclusion of flavonoids metal complexes with antioxidant or antitumor activity will be reviewed, as well as the systems that include flavonoids with antitumor activity based

Flavonoids' ability to penetrate into the hydrophobic regions of lipid bilayers in biological membranes is a key factor to prevent peroxidation of unsaturated double bonds. In this regard, the lipophilicity of flavonoids is essential for an adequate penetration. According to Tweedy's chelation theory, the polarity of the metal ion is reduced, as a consequence of chelation, mainly because of the partial sharing of its positive charge with the ligand's donor groups and possible electron delocalization over the entire ring. Consequently, the lipophilic character of the chelates increases, favoring their permeation through the lipid layers of biological membranes [112]. For two complexes of quercetin and taxifolin with Fe(II), it was assumed that the oxygen charges are generally decreases, while the main negative charge is localized on the iron atom. Thus, after chelating the metal ion, the polarity of flavonoid molecules generally decreased, while the iron atom becomes the most polar and hence hydrophilic part of the molecule. Overall, the lipophilicity of the complexes is considerably larger than that of the corresponding free flavonoids [113, 114].

On the other side, the hydrophobicity of flavonoids chelates dramatically reduces the water solubility, which restricts their medical applications. Integration of these complexes into lipo-

The interaction study of quercetin‐iron complexes with dimyristoylphosphatidylcholine (DMPC) or palmitoyl‐oleoyl phosphatidylethanolamine (POPE) multilamellar liposomes revealed that, during preparation, quercetin should be added first to the suspension of liposomes [115]. It was presumed that quercetin can increase the permeability of lipid bilayers to

The inclusion into cyclodextrins is a convenient alternative to solve the problems related to the low solubility of hydrophobic drugs, such as flavonoid complexes. Inclusion of Cu(II) and

somes may increase their bioavailability and improve the therapeutic effect.

iron cations, showing ionophore activity toward iron cations.

**7. Perspectives in the formulation of flavonoids metal complexes**

on the complexation process between flavonoid and metal ions.

assumption needs to be backed up by more data.

320 Flavonoids - From Biosynthesis to Human Health

**7.1. Liposomal systems**

**7.2. Cyclodextrins**

Based on the property of flavonoids to reduce metal ions such as Ag(I) and Au(III), some hybrid systems of *flavone/metal nanoparticles* have been developed. It was suggested that flavanones can be adsorbed on the surface of metallic nanoparticles through the interaction of the metals with the carbonyl groups or π electrons in the flavonoid structures [117].

The effect of 3‐hydroxyflavone (3‐HF) in a silver nanoparticles (SNPs) complex on the cell viability and on the cell morphology of L929 mouse fibroblast cells was studied *in vitro*. The contribution of the carrier protein BSA to 3‐HF properties has also been investigated. Determination of the cell viability using MTT assay revealed that 3‐HF in BSA/SNPs systems presented no cytotoxic effect in L929 mouse fibroblast cells at any of the tested concentrations [118].

In order to enhance the interaction efficacy with biomacromolecules and therefore increase its therapeutic potential, morin was conjugated with citrate‐coated Au nanoparticles (M‐C‐ AuNPs). Interactions of M‐C‐AuNPs and C‐AuNPs with BSA were studied in order to compare the efficiency of M‐C‐AuNPs and C‐AuNPs in biological systems. It was found that the binding affinity toward BSA of M‐C‐AuNPs is significantly higher than that of C‐AuNPs', indicating that M‐C‐AuNPs might show better BSA interaction efficiency, better biocompatibility, and chemical stability than C‐AuNPs [119].

Taking into account the potential biomedical applications, for example, targeted drug delivery, several flavonoids with antioxidant and antitumor activities have been conjugated with *magnetic nanoparticles*, mainly Fe3 O4 . Flavonoid molecules can bind to Fe<sup>3</sup> O4 via the hydroxyl substituents in their deprotonated form. Most of the biomedical applications of magnetic nanoparticles require surface modification for drug loading or anchoring linkers in support of sustained drug release. Surface functionalization with PEG (polyethylene glycol), PVP (polyvinylpyrrolidone), PVA (polyvinyl acetate), peptides, carbohydrates, proteins, and so on facilitates the drug loading and controlled release, and also controls the stability of the system [120].

Quercetin‐conjugated superparamagnetic Fe3 O4 nanoparticles were investigated for the *in vitro* cytotoxic activity on breast cancer cell lines. The MTT assay revealed that the dextran‐ coated Fe<sup>3</sup> O4 nanoparticles did not exhibit notable toxicity against MCF7 cells, whereas the cytotoxicity of quercetin‐conjugated Fe3 O4 nanoparticles increased significantly in comparison with the free quercetin [120]. The results sustain that the quercetin‐conjugated Fe3 O4 nanoparticles are promising anticancer agents for targeted drug delivery.

A drug delivery methodology was proposed to study a new quercetin release system in the form of magnetite‐quercetin‐copolymer (MQC), as perspective of targeting specific organs within the body. The quercetin‐magnetite nanoparticles (Fe3 O4 ) system was incorporated into a triblock copolymer of ethylene oxide and oxyphenylethylene, as a model of drug carrier system for anticancer agents [121].

Furthermore, quercetin loading on *mesoporous carriers* was performed in order to enable the sustained delivery of the bioactive compound. Mesoporous nanosized silicas are widely used as carriers for drug delivery. However, the appropriate chemical surface modification of the mesoporous matrix is essential, taking into account that the silanol groups of the silica surface do not possess sufficient selectivity to adsorb drug molecules with different functionalities [122]. Quercetin was loaded on the pure silica and Zn‐modified mesoporous MCM‐41 and SBA‐16 supports, and the formation of complexes between quercetin and pure siliceous or Zn‐modified MCM‐41 and SBA‐16 mesoporous silica was determined. Quercetin has a higher binding affinity for the Zn2+ cation than to the silanol groups. Therefore, the release of quercetin is easier from the silicate samples containing only superficial silanol groups than from the Zn‐modified samples. The obtained mesoporous delivery systems with Zn‐quercetin complex showed promising results for further use in dermal formulations [123].

Natural zeolite of the clinoptilolite type (CT, particle size of up to 200 µm) and its modified forms with different concentrations (0.06–5%) of the pharmaceutically active compounds quercetin and quercetin dihydrate (QD) have been investigated for their anticancer activity. Carcinoma cell lines Jurkat, CEM, HeLa, MCF7, A549, and MDA were treated with various amounts of natural clinoptilolite and their modified forms CTQ and CTQD. Incorporation of the flavonoids quercetin and quercetin dihydrate with antiproliferative activity had no synergic effect on the zeolite cytotoxicity, but the protective effect of cancer cells. The tumor cell lines studied after the application of modified zeolite CTQ or CTQD had lower antiproliferative activity in comparison with the natural zeolite of the clinoptilolite‐type CT. The modified zeolite CTQD had greater antiproliferative effects than modified zeolite CTQ [124].
