Flavonoid Nanoparticles: A Promising Approach for Cancer Therapy

Flavonoids, a ubiquitous group of naturally occurring polyphenolic compounds, have recently gained importance as anticancer agents. Unfortunately, due to low solubility, absorption, and rapid metabolism of dietary flavonoids, their anticancer potential is not sufficient. Nanocarriers can improve the bioavailability of flavonoids. In this review we aimed to evaluate studies on the anticancer activity of flavonoid nanoparticles. A review of English language articles published until 30 June 2020 was conducted, using PubMed (including MEDLINE), CINAHL Plus, Cochrane, and Web of Science data. Most studies determining the anticancer properties of flavonoid nanoparticles are preclinical. The potential anticancer activity focuses mainly on MCF-7 breast cancer cells, A549 lung cancer cells, HepG2 liver cancer cells, and melanoma cells. The flavonoid nanoparticles can also support the anti-tumour effect of drugs used in cancer therapy by enhancing the anti-tumour effect or reducing the systemic toxicity of drugs.


Introduction
Flavonoids are the most common and widely distributed group of plant compounds, occurring virtually in all plant parts. This group can be divided into several subfamilies such as flavones, flavanols, flavanones, flavonols, and isoflavones [1]. As a dietary component, flavonoids are thought to have beneficial effects on human health [2]. Their health-promoting properties are associated with antioxidant [3], anti-inflammatory [4], and anticancer properties [5]. Nowadays attention is drawn to the anticancer activity of flavonoids. Many studies have documented flavonoids, for example, epigallocatechin-3-gallate, quercetin, genistein, apigenin, naringenin, silibinin, and kaempferol, to be effective against various types of cancer. Unfortunately, due to low solubility [2], poor absorption [6], and rapid metabolism [7], use of flavonoids in cancer treatment is not satisfactory. In this regard, modern nanotechnology may be used. Nanocarriers can improve the bioavailability of flavonoids [1]. In vitro and in vivo studies have shown potential anticancer activity of flavonoid nanoparticles against A549 lung cancer cells, B16F10 melanoma cells, MCF-7 breast cancer cells, HepG2 liver cancer cells or CT26 colorectal cancer cells [8][9][10][11][12]. There are several different types of flavonoid nanocarriers currently used in cancer therapy. These include polymeric nanoparticles [13], nanocapsules [14], metallic nanoparticles (gold) [15] or solid lipid nanocarriers [16].
This review aims to evaluate studies on the anticancer activity of flavonoid nanoparticles. A review of English language articles published until 30 June 2020 was conducted, using PubMed (including MEDLINE), CINAHL Plus (Cumulative Index to Nursing and Allied Health Literature), Cochrane, G2/M phase and reducing the potential of the mitochondrial membrane [34]. The anticancer effect of epigallocatechin-3-gallate nanoparticles is represented in Table 1.
The most commonly used nanoparticles with quercetin are PEG, poly(lactic co-glycolic acid) nanoparticles (PLGA) and PLA. It has been reported that PEG nanoparticles prolong the circulation time of quercetin in the bloodstream and increase its solubility and stability [47]. The Tan et al. study showed that the PEG-derivatized phosphatidylethanolamine nanomicelles improved the anticancer activity of quercetin. It has been observed that these nanoparticles were more effective against A549 lung cancer cells than free quercetin [9]. Another study by Xing et al. showed that de-PEGylated nanoparticles based on triphenylphosphine-quercetin (TPP-PEG) were more effective therapeutic agents compared to pure quercetin in A459, MCF-7, and HepG2 cancer cells [36]. The anticancer activity of quercetin loaded in PEG was observed in the study of Dora et al. The results indicated that oral administration of nanosized PEG emulsion containing quercetin had cytotoxicity activity against B16F10 melanoma cells [48]. Similarly, by using quercetin loaded PEG-liposomal nanoparticles, inhibition of angiogenesis of ovarian cancer was achieved [49]. The Zhao et al. study showed that the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) (DSPE-MPEG) is a good adjuvant nanocarrier for anticancer drug delivery [50]. A few studies on the treatment of hepatocellular cancer with PLGA-loaded quercetin nanoparticles have been shown. In the Ghosh et al. study, the PLGA nanoparticles loaded in quercetin completely protected the mitochondrial membrane of the liver against cancer induced by diethylnitrosamine [42]. After oral administration, PLGA nanoparticles encapsulated with quercetin and tamoxifen (TMX) controlled tumour angiogenesis in MCF-7 breast cancer cells [10]. The Panday et al. study has proven that oral administration rutin-loaded PLGA nanoparticles of quercetin improved hepatic parameters and increased superior inflammatory markers [45]. Quercetin encapsulated in monomethoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) nanoparticles inhibited ovarian tumour growth by the mitochondrial apoptotic pathway [51,52]. Moreover, the anticancer efficacy of GeluPearl comprising of Precirol ATO 5 lipid (GPSLN) nanoparticles loaded with quercetin against B16F10 melanoma cells was proved [53]. In another study (Mandal et al.), inhibition of development of hepatocellular carcinoma by PLA nanoparticles has been shown [43]. The gold-quercetin in PLA nanoparticles inactivated the caspase/Cyto-c pathway in hepatocellular cells [44]. The da Luz et al. study has shown that poly-lactic acid nanoparticles have an anticancer effect on A549 lung cancer cells [54]. In the Li et al. study, quercetin-loaded soybean phosphatidylcholine-cholesterol (SPC-CHOL) inhibited U14 cervical cancer cells [41]. The anticancer activity of quercetin against breast cancer was observed by using PVP [38]. To improve the effect of quercetin, gold-PLA nanoparticles were also used [39,44,55]. The in vivo studies have confirmed that quercetin nanoparticles have potential in cancer treatment ( Table 2).

Genistein
Genistein is an isoflavonoid found in a number of plants including soybeans, fava beans, and lupins [56]. Some studies have shown beneficial effects of genistein nanoparticles against several cancer lines. Genistein has poor water solubility, rapid metabolism, and low oral bioavailability, which limit the clinical application of this flavonoid [57]. Genistein loaded TPGS-b-PCL (d-α-tocopheryl polyethylene glycol 1000 succinate-poly(ε-caprolactone)) inhibited HeLa cervical tumour cells growth and had a higher level of cytotoxicity compared to genistein-loaded PCL nanoparticles [58], whereas genistein-loaded M-PLGA-TPGS (poly(d,l-lactide-co-glycolide)-d-α-tocopheryl polyethylene glycol 1000 succinate) had a linear apoptotic effect against HepG2 liver cancer cells [12]. Unfortunately, the improvement in the bioavailability of genistein resulting from the use of nanoparticles is associated with high cytotoxicity in normal cells. Further in vitro studies focused on anticancer activity (apoptosis and autophagy of cancer cells) in colon cancer HT29 cells with genistein-loaded PEGylated silica hybrid nanomaterials and lung cancer A549 cells with genistein-miRNA-29b-loaded hybrid nanoparticles-GMLHN, as well as hematopoietic cancer cells with genistein-carboxymethylated chitosan nanoparticles-Fe3O4-CMC [59][60][61]. Despite low water solubility, low bioavailability, and instability of pure genistein, the genistein-loaded nanoparticles described in the presented studies have made it possible to use them in anticancer treatment [62]. Studies on the anticancer activity of genistein nanoparticles are presented in Table 3.

Silibinin
Silibinin is found in the seeds of milk thistle [63]. Studies show that silibinin has an antineoplastic potential against many cancers by promoting the cell-cycle and inhibiting proliferation [64]. However, due to its hydrophobic structure, it has poor water solubility and permeability across intestinal epithelial cells. To improve the effect of silibinin, nanoparticles, i.e., PEG, polyvinyl alcohol (PVA), and poly-N-(2-hydroxypropyl) methacrylamide (pHPMA)-coated wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles have been used [65]. The study of Xu et al. on silibinin nanoparticles described their effect on cancer cells and the blocking of metastasis of breast cancer. These silibinin-loaded lipid nanoparticles (SLNs) containing TPGS and phosphatidylcholine were designed and prepared by a thin-film hydration method [66]. In another study (Gohulkumar et al.), silibinin encapsulated in PVA-Eudragit nanoparticles showed anticancer efficacy in oral carcinoma cells [67]. Another study showed that silibinin encapsulated in PEG nanoparticles had a cytotoxic effect on breast cancer (MCF 10A) in vitro [68]. Free silibinin has low solubility and inadequate dissolution, which cause low oral bioavailability. The Sahibzada et al. study shows two methods for manufacturing nanoparticles of silibinin (APSP-anti-solvent precipitation with a syringe pump and EPN-evaporative precipitation of nanosuspension), which increase its solubility, making this flavonoid a potential oral drug in cancer therapy [69]. Huo et al. showed that the combination therapy of silibinin and paclitaxel (PTX) loaded in dextran-deoxycholic acid (Dex-DOCA) nanoparticles effectively accumulate in tumour sites by passive targeting and inhibit tumour growth through an enhanced intratumoural penetration in mice [70]. Changes in the tumour microenvironment were observed in another study where silibinin and IPI-549 nanoparticles (AEAA-PEG-PCL-aminoethyl anisamide-polyethylene glycol-polycaprolactone) inhibited 4T1 breast cancer cells [71]. It is also worth noting that silibinin demonstrates an anti-metastasis effect. Research on the poly-N-(2-hydroxypropyl) methacrylamide (pHPMA)-coated wheat germ agglutinin-modified lipid-polymer hybrid nanoparticles, co-loaded with silibinin and cryptotanshinone (S/C-pW), showed inhibition of tumour growth in 4T1 tumour-bearing mice and presented anti-metastasis activity in the lung [72]. In vitro and in vivo studies concerning the potential anticancer activity of silibinin nanoparticles are presented in Table 4.

Apigenin
Apigenin is found in several types of vegetables and fruits, especially berries. This flavonoid is involved in regulating signalling pathways in hepatocellular carcinoma and skin cancer. The beneficial effect of free apigenin in cancer treatment is relatively low because of its low lipid and water solubility [73]. Nowadays, to improve the bioavailability of flavonoids, PLGA nanoparticles are mainly used. A study by Das et al. showed an anti-proliferative effect of apigenin loaded in PLGA nanoparticles on A475 skin cancer cells. It is worth emphasizing that these nanoparticles were effective in maintaining photodegradation through ultraviolet light [74]. Other study on apigenin encapsulated in PLGA nanoparticles showed that intravenous administration of this molecules successfully reached HepG2 and Huh-7 cells in vitro as well as the liver of carcinogenic animals and delayed development of hepatocellular carcinoma in rats [75]. Novel nanomaterials have recently been investigated, which contribute to enhanced solubility and bioavailability of apigenin via preparation of solid dispersions of mesoporous silica nanoparticles [76]. The anticancer effects of apigenin nanoparticles are reported in Table 5.

Naringenin
Naringenin is widely distributed in fruits, especially citrus fruits, bergamot, and tomatoes [77]. Its anticancer properties are associated with anti-inflammatory and antioxidant activities. The clinical effect of cancer treatment with naringenin is limited by its low solubility and minimal bioavailability, related to its hydrophobic ring structure [78].
Current research has shown that naringenin nanoparticles can inhibit carcinogenesis in oral squamous cell carcinoma [79], lung cancer (A549) [80], and colorectal cancer (colon-26) [81]. Due to their anti-proliferative and antioxidant potential, nanoparticles can be potentially useful in oral cancer chemoprevention [79,82]. In the Sulfikkarali et al. study, PVA-EE (polyvinyl alcohol and Eudragit 500) loaded with naringenin had a positive effect on DMBA-induced oral squamous cell carcinoma in hamsters. The oral administration of PVA-EE-naringenin completely prevented the tumour formation as compared to the free naringenin [79]. Chitosan nanoparticles encapsulating naringenin were used in an in vitro lung cancer model (A549), reporting cytotoxic effects on the cancer cells while having a nontoxic effect on normal 3T3 fibroblast cells [80]. The Chaurasia et al. study has described tumour suppression in BALB/c mice bearing colon-26 cells. Both naringenin-encapsulated soluthin-maltodextrin nanoparticles and EE-naringenin improve bioavailability and have a cytotoxic effect against colorectal cancer cells [32,33]. The Fuster et al. study reported that naringenin loaded in silk fibroin nanoparticles had anticancer potential for treatment of cervical cancer HeLa cells [78] (Table 6).

Luteolin
Luteolin is found in various types of plants such as fruits, vegetables, and medicinal herbs [84]. It induces apoptosis and inhibits cancer cell migration, invasion, and angiogenesis [85]. Due to its hydrophobic structure, it has poor water solubility, poor systemic delivery, and low efficacy. To improve the effect of luteolin, nanoparticles, i.e., PLA-PEG and folic acid-PEG-PCL have been used [86].
One in vitro study concluded that water-soluble polymer-encapsulated nano-luteolin from hydrophobic luteolin (PLA-PEG) inhibited the growth of lung cancer cells (H292 cell line) and squamous cell carcinoma of head and neck (SCCHN) cells (Tu212 cell line) [86], which are among the most frequent cancers worldwide [87][88][89]. Moreover, in vivo study using a tumour xenograft mouse model demonstrated that nano-luteolin has a significant inhibitory effect on the tumour growth of SCCHN in comparison to free luteolin [86]. The study by Wu et al. study showed that luteolin encapsulated in folic acid modifiedpoly(ethylene glycol)-poly(ecaprolactone) (Fa-PEG-PCL) nano-micelles induced glioblastoma multiforme growth of GL261 cells [90]. Additionally, in the safety assessment of nanoparticles used in the tested mice, there were no obvious side effects [90]. Due to the fact that luteolin is hydrophobic [91] and has low biocompatibility [90], it still requires further studies to improve its bioavailability (Table 7).

Kaempferol
Kaempferol is widely distributed in vegetables (broccoli, spinach), fruits (strawberries, apples), and herbal medicines [92]. The limitation of free kaempferol is inefficient systemic delivery and limited bioavailability [93]. Promising nanoparticles to improve the anticancer efficacy of this flavonoid are chitosan, gold, and PLGA. The potential anticancer effect of this flavonoid is associated with inhibition of phosphatidylinositol-3-kinase (Pl-3) and ribosomal s6 kinase (rsk) and cell cycle arrest in various cancer types [94]. In vitro studies have demonstrated that kaempferol nanoparticles can inhibit carcinogenesis in ovarian cancer cells (A2780/CP70 and OVCAR-3) [93], rat glioma cells (C6) [95] and lung cancer cells (A549) [96]. In the study by Luo et al. both PEO-PPO-PEO as well as PLGA nanoparticles formulations incorporating kaempferol significantly reduced viability of ovarian cancer cells (A2780/CP70 and OVCAR-3), compared with kaempferol alone [93]. The PEO-PPO-PEO nanoparticles were more effective than PLGA nanoparticles, however, PEO-PPO-PEO nanoparticles reduced the viability of ovarian cancer cells (OVCAR-3) and normal ovarian cells (IOSE397), while PLGA nanoparticles had selective toxicity and reduced only the viability of ovarian cancer cells (OVCAR-3) [93]. In another study, kaempferol-loaded mucoadhesive chitosan nanoemulsion (MNE) was used to induce glioma cell in rats [95]. The kaempferol-loaded MNE reduced C6 glioma cell viability a greater degree than free kaempferol. Therefore, kaempferol-loaded mucoadhesive chitosan nanoemulsion could be a promising alternative for brain cancer treatment [95]. The Govindaraju et al. study reported that kaempferol with gold nanoparticles had higher toxicity to A549 lung cancer cells than to normal human cells [96] ( Table 8).

Other Flavonoids (Fisetin and Myricetin)
Fisetin is found in various fruits and vegetables (e.g., apple, strawberry, grape, persimmon, onion) [97]. In the Ghosh et al. study, fisetin-loaded human serum albumin nanoparticles revealed anticancer activity against MCF-7 breast cancer cells in vitro [98]. The Feng et al. study confirmed the effect of fistein nanoparticles on 4T1 breast cancer cells in vivo. Additionally, in vitro fistein-loaded PLA nanoparticles had an anti-tumour effect against colon cancer HCT116 cells [99].
Myricetin is very common in various plants, such as vegetables, fruits, and in teas and wines [100]. In vitro myricetin encapsulated in solid Gelucire-based lipid nanoparticles in the presence of phosphate buffer provided sustained release with no signs of degradation [101]. In the Khorsandi et al. study, solid lipid nanoparticles of myricetin induced growth of lung cancer A549 cells and increased necrosis with no influence on proliferation and apoptosis [102].
The anticancer effects of fisetin and myricetin nanoparticles are presented in Table 9. Flavonoids, a ubiquitous group of naturally occurring polyphenolic compounds, have recently gained importance as anticancer agents. Unfortunately, due to low solubility, absorption, and rapid metabolism of dietary flavonoids, their anticancer potential is not sufficient. The use of nanotechnology has improved the bioavailability of flavonoids and has increased their anti-tumour activity.
Both in vitro and in vivo studies have shown that flavonoid nanoparticles are promising in cancer treatment in the near future. Most of the studies determining the anticancer properties of flavonoid nanoparticles are preclinical. The potential anticancer activity focuses mainly on MCF-7 breast cancer cells, A549 lung cancer cells, HepG2 liver cancer cells, and melanoma cells.
The anticancer activity of flavonoid nanoparticles is associated with apoptosis and antiproliferation, inhibition of the cell cycle of cancer cells, regulation of the host's immune system or an anti-inflammatory effect. A particular issue worth investigation is the influence of nanoparticles on the tumour microenvironment, which may be quite important in metastasis.
It is worth noting that flavonoid nanoparticles can also support the anti-tumour effect of drugs used in cancer therapy by enhancing the anti-tumour effect or by reducing the systemic toxicity of drugs. Funding: This review was written with no external funding.

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