Anticancer Activity of Rutin and Its Combination with Ionic Liquids on Renal Cells

The renal cell carcinoma (RCC) is the most common type of kidney cancer. Identifying novel and more effective therapies, while minimizing toxicity, continues to be fundamental in curtailing RCC. Rutin, a bioflavonoid widely found in nature, has shown promising anticancer properties, but with limited applicability due to its poor water solubility and pharmacokinetics. Thus, the potential anticancer effects of rutin toward a human renal cancer cell line (786-O), while considering its safety in Vero kidney cells, was assessed, as well as the applicability of ionic liquids (ILs) to improve drug delivery. Rutin (up to 50 µM) did not show relevant cytotoxic effects in Vero cells. However, in 786-O cells, a significant decrease in cell viability was already observed at 50 µM. Moreover, exposure to rutin caused a significant increase in the sub-G1 population of 786-O cells, reinforcing the possible anticancer activity of this biomolecule. Two choline-amino acid ILs, at non-toxic concentrations, enhanced rutin’s solubility/loading while allowing the maintenance of rutin’s anticancer effects. Globally, our findings suggest that rutin may have a beneficial impact against RCC and that its combination with ILs ensures that this poorly soluble drug is successfully incorporated into ILs–nanoparticles hybrid systems, allowing controlled drug delivery.


Introduction
Renal cell carcinoma (RCC) is the most common type of kidney malignancy in adults that is established in the renal proximal convoluted tubules [1,2]. RCC is highly vascularized and can

Chemicals
The reagents and solvents used for the synthesis of the ILs were choline hydroxide in methanol [Cho][OH]/MeOH 45% and methanol, both from Sigma-Aldrich (Saint Louis, MO, USA), also acetonitrile from VWR (Fontenay-sous-Bois, France) and the amino acids, L-phenylalanine and glycine, from PanReact AppliChem (Barcelona, Spain).
For the cytotoxicity studies, the following reagents were purchased from Sigma-Aldrich (Saint Louis, MO, USA), phosphate buffered saline (PBS; 0.01 M, pH 7.4), trypsin, penicillin-streptomycin (pen/strep) solution, thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO). Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were from Biowest (Nuaillé, France). The propidium iodide (PI) was purchased from Merck (Darmstadt, Germany). All the rutin solutions had a final concentration of 0.5% (v/v) and were prepared in DMSO, for all assays. The solutions containing ionic liquids were all prepared in sterile water.

Cell Culture
In this study, the Vero-E6 normal kidney cells (ATCC ® CRL-1586™), and the 786-O human renal cancer cells (ATCC ® CRL-1932™), were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The Vero-E6 and 786-O cells were cultured in low and high glucose DMEM medium, respectively, supplemented with 10% FBS and 1% pen/strep. Cells were maintained at 37 • C in a humidified air atmosphere containing 5% CO 2 .

MTT Assay
To evaluate the cytotoxicity of rutin and of the ILs, individually and in combination, cell viability was determined using the MTT reduction assay. Vero and 786-O cells were seeded at a density of 5 × 10 3 and 3 × 10 3 per well, respectively, in 200 µL culture medium in 96-well plates and incubated for 24 h. Cells were then incubated, either with rutin (0-250 µM), with [Cho][Phe] (0-0.5%, v/v) or with [Cho][Gly] (0-0.5%, v/v) individually, or, with rutin (0-250 µM) in combination with [Cho][Phe] (0.3%, v/v) or with [Cho][Gly] (0.2%, v/v) for 48 h. The MTT reduction assay was then carried out according to previously published procedures [24,35]. Absorbance values for untreated control cells correspond to 100% of cell viability. For this assay, two to seven independent experiments were carried out and at least four replicate cultures were used in each independent experiment.
The half-maximal inhibitory concentration (IC 50 ) was calculated using the GraphPad Prism 7 ® Statistical Software (San Diego, CA, USA).

Solubility Studies
The solubility studies were performed according to a previously published procedure [23]. Briefly, several saturated solutions of rutin were prepared in triplicate, in water and water:IL mixtures, containing 0.3% of [Cho][Phe] or 0.2% of [Cho][Gly] IL. Then, in a horizontal shaker (IKA VIBRAX VXR ® , LTF Labortechnik GmbH & Co., Bodensee, Germany), the solutions were shaken during 72 h at 25 • C. Next, all solutions were filtrated and analyzed using an UV-visible spectrophotometry Evolution ® 300 from Thermo Scientific (Hertfordshire, England) at 353 nm, the maximum absorption wavelength of rutin in water.

Flow Cytometric Analysis of DNA Cell Cycle
The cell cycle distribution of cells treated with rutin and ILs, either individually or in combination, was determined by flow cytometry. This assay was carried out according to previously published procedures [6,36]. Briefly, for cell-cycle analysis, 786-O cells were seeded at a density of approximately 5 × 10 4 per well in 6-well plates and cultured for 24 h. Afterwards, rutin (50 µM), [Gly] (0.2%, v/v) were added and cells were incubated for a further 48 h period. Cells were then harvested using EDTA (5 mM) in PBS, washed with cold PBS and fixed with 80% ethanol. Then, the cells were treated with RNase A (20 µg/mL) and stained with propidium iodide (10 µg/mL) for 20 min and analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data acquisition and analysis were executed using CellQuest software (Becton Dickinson, San Jose, CA, USA) and FlowJo (Tree Star, San Carlos, CA, USA), respectively. Three independent experiments were executed.

Production of the IL-Nanoparticles Hybrid System
The IL-nanoparticle hybrid systems were prepared using a water-in-oil-in-water (W/O/W) double emulsion method, according to previously published procedures [37]. Briefly, an aqueous solution of rutin with IL was produced using the highest concentration of drug dissolved by each IL ( This mixture was sonicated for 30 s at 70% of amplitude using a Q125 Sonicator (QSonica Sonicators, Newtown, CT, USA), obtaining the first emulsion. The latter was then poured into 25 mL of a PVA 2% (w/v) solution and sonicated at the same previous conditions, obtaining the secondary emulsion. The formulation was then placed under magnetic stirring until evaporation of the organic solvent. All formulations were prepared in triplicate.

Particle Size, Polydispersity Index and Zeta Potential Analysis
The IL-nanoparticle hybrid system was characterized in terms of its polydispersity index (PdI) and particle size by dynamic light scattering using the Delsa™ Nano C (Beckman Coulter, Inc., Brea, CA, USA) and zeta potential by the electrophoretic mobility analysis using Malvern ® NanoSizer (Worcestershire, United Kingdom). All samples were prepared in triplicate and analyzed at 23 ± 2 • C.

Association Efficiency and Loading Capacity of Rutin
For the evaluation of the association efficiency (AE) and loading capacity (LC) of rutin, all formulations were centrifuged at 16,350× g for 15 min at 4 • C and then the supernatant was collected. Through UV spectroscopy, rutin was quantified in the supernatant at 353 nm (maximum absorption wavelength in the PVA solution). The pellet resuspended in water and freeze-dried in a LABCONCO FreeZone 25 ® freeze dryer (Kansas City, MO, USA) at 400 mTorr for 24 h and −50 • C of condenser surface temperature.

In Vitro Release Study
For the release study, the nanoparticle suspension was centrifuged at 12,600× g for 20 min at 4 • C. The supernatant was removed, and the pellet was resuspended in 10.0 mL of a PBS solution. Then, the solutions were incubated at 37 • C and stirred at 100 rpm in a Heidolph ® 1000 incubator with motor Heidolph ® Unimax 1010 (Schwabach, Germany). Next, at predetermined time intervals (30 min, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h), aliquots of each sample (1 mL) were taken and replaced by the same volume of PBS. The samples were centrifuged at 12,600× g for 15 min at 25 • C and the drug present in the supernatant was quantified at 353 nm in the UV-visible Spectrophotometer Evolution ® 300 from Thermo Scientific (Hertfordshire, England).

Statistical Analyses
Differences in mean values of the results were evaluated with one-way analysis of variance (ANOVA) and then followed by Tukey's multiple comparison test, after assessing normality. The analyses were performed with SPSS statistical package (version 25, SPSS Inc. Chicago, IL) and GraphPad Prism 7 ® from GraphPad Software (San Diego, CA, USA).

Effect of Rutin on the Viability of Renal Cells
In the present study, the MTT assay was used to evaluate the impact of rutin (0-250 µM; 48 h) treatment on the cell viability of two renal cell lines, the Vero normal kidney cells, and the 786-O human renal cancer cells.
The results with Vero cells only showed a significant decrease in cell viability at the two highest studied concentrations of rutin (100 and 250 µM), with the respective viabilities being 65.6% and 52.1% ( Figure 1A).
On the other hand, for the 786-O cancer cells, a concentration-dependent decrease of cell viability at much lower concentrations than in Vero cells was observed, with an IC 50 value of 45.2 µM ( Figure 1B). The results showed that rutin induced a remarkable decrease in cell viability, compared with non-treated control cells at concentrations of 50, 100, and 250 µM, reaching 56.1%, 32.4%, and 25.3%, respectively. 52.1% ( Figure 1A).
On the other hand, for the 786-O cancer cells, a concentration-dependent decrease of cell viability at much lower concentrations than in Vero cells was observed, with an IC50 value of 45.2 μM ( Figure  1B). The results showed that rutin induced a remarkable decrease in cell viability, compared with non-treated control cells at concentrations of 50, 100, and 250 μM, reaching 56.1%, 32.4%, and 25.3%, respectively.

Effect of the Two Choline-amino Acid ILs on the Viability of Renal Cells
Since ILs may be used as functional excipients to allow the incorporation of rutin in delivery systems, it was also relevant to understand their influence on cell viability. Hence, the effect of the two prepared choline-amino acid ILs (0%-0.5% (   It was also important not only to evaluate the effect of rutin and ILs individually but also of the combined treatment of rutin (0-250 μM) with each IL (0.3% of [Cho][Phe] or 0.2% [Cho][Gly] (v/v)), using the same experimental conditions. In this case, the results showed that in general, the presence of each IL did not lead to significant differences in the cell viability when compared with the viability of rutin-treated cells in both cell lines ( Figure 3). Subsequently, considering all the results up to this point, for the following studies, the concentration of 50 μM for rutin and the concentrations of 0

Cell Cycle Distribution of 786-O Cells Treated with Rutin Individually and in Combination with ILs
The influence of the individual treatment with rutin or ILs and the combination of rutin with ILs in the human renal cancer cell cycle progression was studied by assessing the cellular DNA content.
The ILs (0.3% and 0.2%, respectively), no relevant and statistical differences were observed when compared to rutin-exposed cells (Figure 4).

Cell Cycle Distribution of 786-O Cells Treated with Rutin Individually and in Combination with ILs
The influence of the individual treatment with rutin or ILs and the combination of rutin with ILs in the human renal cancer cell cycle progression was studied by assessing the cellular DNA content.

Solubility of Rutin in the Presence of Choline-amino Acid ILs
The impact of the two choline-amino acid ILs on the solubility of rutin was investigated through solubility studies in aqueous solutions in the presence of 0

Solubility of Rutin in the Presence of Choline-amino Acid ILs
The impact of the two choline-amino acid ILs on the solubility of rutin was investigated through solubility studies in aqueous solutions in the presence of 0

Particle Size, Polydispersity Index and Zeta Potential Analysis
Regarding the physicochemical proprieties, the IL-nanoparticle hybrid systems showed a diameter ranging between 250-300 nm, with a PdI around 0.2 and a zeta potential of about −35 mV ( Figure 5). No relevant differences were found on these parameters between loaded and unloaded delivery systems. Also, the results obtained for the formulations with the two different ILs were very similar.

Particle Size, Polydispersity Index and Zeta Potential Analysis
Regarding the physicochemical proprieties, the IL-nanoparticle hybrid systems showed a diameter ranging between 250-300 nm, with a PdI around 0.2 and a zeta potential of about −35 mV ( Figure 5). No relevant differences were found on these parameters between loaded and unloaded delivery systems. Also, the results obtained for the formulations with the two different ILs were very similar.

Association Efficiency (AE) and Loading Capacity (LC) of Rutin
The AE and the LC of the rutin loaded into the nanoparticles were also evaluated. The AE of rutin in the delivery systems were 84.5% ± 0.3%, in the presence of [Cho][Phe], and 84.7% ± 0.3%, with [Cho][Gly] ( Table 2). The LC values were also similar for both formulations ( Table 2). All results showed that there are no significant differences in the AE and LC between both ILs.

Association Efficiency (AE) and Loading Capacity (LC) of Rutin
The AE and the LC of the rutin loaded into the nanoparticles were also evaluated. The AE of rutin in the delivery systems were 84.5% ± 0.3%, in the presence of [Cho][Phe], and 84.7% ± 0.3%, with [Cho][Gly] ( Table 2). The LC values were also similar for both formulations ( Table 2). All results showed that there are no significant differences in the AE and LC between both ILs.

In Vitro Release Study
Concerning the in vitro release study, the release profile shows an initial burst in the first 5 h of the study (Figure 6). After that, the IL-nanoparticles hybrid systems showed a sustained drug release over time (Figure 6

Discussion
The ccRCC represents the most common subtype of RCC, comprising about 75% of all RCC tumors [6]. The mechanism that regulates RCC growth remains unclear. Moreover, the low sensitivity to conventional therapies and the low response rate to targeted therapies limits the treatment of this type of renal cancer [1,4,5,38]. Thus, the discovery of new and/or more effective and safe therapies becomes essential. In previous studies, rutin has shown biological activity toward different cancer cell lines, indicating that this natural compound could be a promising anticancer agent [8,16,[18][19][20]39]. However, despite its potential anticancer activity, its low solubility represents a challenge and limits the applicability of rutin. Bearing this in mind, the present study aimed to evaluated the impact of rutin on 786-O cells, a well-established and characterized human renal cancer cell line [6] and the applicability of ILs to improve the solubility and delivery of this biomolecule, while considering the safety and applicability of this natural compound alone and when combined with the ILs.
Herein, the impact of rutin (0-250 μM; 48 h) treatment on the cell viability of two renal cell lines, the Vero normal cells and the 786-O cancer cells, was obtained using the MTT assay. It was important to evaluate the effect of rutin on the viability of a representative kidney cell model, the Vero cells [40], to understand if this compound can be used safely. Our results showed that rutin did not induce relevant cytotoxicity up to 50 μM, although at concentrations higher than 50 μM the cell viability of Vero cells significantly decreased.

Discussion
The ccRCC represents the most common subtype of RCC, comprising about 75% of all RCC tumors [6]. The mechanism that regulates RCC growth remains unclear. Moreover, the low sensitivity to conventional therapies and the low response rate to targeted therapies limits the treatment of this type of renal cancer [1,4,5,38]. Thus, the discovery of new and/or more effective and safe therapies becomes essential. In previous studies, rutin has shown biological activity toward different cancer cell lines, indicating that this natural compound could be a promising anticancer agent [8,16,[18][19][20]39]. However, despite its potential anticancer activity, its low solubility represents a challenge and limits the applicability of rutin. Bearing this in mind, the present study aimed to evaluated the impact of rutin on 786-O cells, a well-established and characterized human renal cancer cell line [6] and the applicability of ILs to improve the solubility and delivery of this biomolecule, while considering the safety and applicability of this natural compound alone and when combined with the ILs.
Herein, the impact of rutin (0-250 µM; 48 h) treatment on the cell viability of two renal cell lines, the Vero normal cells and the 786-O cancer cells, was obtained using the MTT assay. It was important to evaluate the effect of rutin on the viability of a representative kidney cell model, the Vero cells [40], to understand if this compound can be used safely. Our results showed that rutin did not induce relevant cytotoxicity up to 50 µM, although at concentrations higher than 50 µM the cell viability of Vero cells significantly decreased.
Furthermore, to understand the anticancer potential of this natural compound, a concentration-response curve of rutin in 786-O human renal cancer cells was established. It was observed a clear concentration-dependent cytotoxicity effect of rutin, with a remarkable decrease at 50-µM concentration. These results are in agreement with previously published data performed in the same or different experimental conditions but using different cancer cell lines [18,39,41]. Moreover, with this study, it was possible to demonstrate that in the presence of rutin, 786-O cells are more sensitive to its cytotoxic effects than other previously studied cancer cell lines, namely, hepatoma cells of Rattus novergicus (HTC), human breast cancer cells (MDA-MB-231 and MCF-7), and colon cancer cells (HT29 and Caco-2) [7,9,23,39].
Hence, it is relevant to point out that these results reveal that rutin exhibits a higher cytotoxic effect against the 786-O cancer cells than against the non-cancer Vero cells, showing the potential anticancer activity of rutin against RCC. In addition, the results show that in further studies, 50 µM should be the upper concentration of rutin used to ensure both efficacy and safety.
Despite these promising results, rutin has very low solubility in water, 0.2 mg/mL [23], which limits its incorporation in delivery systems and, consequently, its applicability. Recently, a previous study from our group showed that the presence of ionic liquids, at non-toxic concentrations, enhanced the solubility of rutin and its loading into O/W emulsions, thus indicating that these salts may be used as green functional excipients [23]. However, since in this previous study the experimental conditions, the cell model and one of the ILs used were different ([Cho][Gly]), it becomes relevant for the present study to assess, for the first time, the influence of [Cho][Phe] and [Cho][Gly] ILs on the cell viability of the renal cell lines under study. Our results showed that both ILs induced a concentration-dependent decrease in cell viability in both cell lines. This may be due to the fact that the choline-based cation is a quaternary ammonium compound and these compounds are known cationic surfactants [42], which may justify the decrease in cell viability. Nonetheless, this cytotoxicity is not as pronounced as for ILs containing alkyl side chains, such as imidazole-based ionic liquids [24]. Furthermore, and even though, the toxicity of ILs has been mostly attributed to the cation, particularly the type of cationic head group and the length of the alkyl side chain, it is also known that the type of anion may have some impact on this toxicity [43], which justifies the observed differences between the two studied ILs. In fact, it has been shown that different amino acids lead to different cytotoxicities, with the elongation of the amino acid side chain being one of the structural contributions to enhance their cytotoxicity, although the toxicity sequence may differ depending on the model system used [43]. Moreover, our results also revealed that in further studies, to ensure safety, the highest concentration of ILs used should be 0.3% (v/v) for [Cho][Phe] and 0.2% (v/v) for [Cho][Gly] since up to these concentrations no significant cytotoxic effects were observed in Vero cells. Then, it was crucial to assess the solubility of rutin in the presence and absence of the studied ILs. Previous reports have shown that choline-amino acid ILs are able to enhance drug solubility [23,24,27]. Hereafter, to further explore the potential applicability of rutin combined with ILs, it is crucial to assess not only the individual effects of rutin, and each IL but also to evaluate the impact of the combined treatment with rutin and each IL on the cytotoxicity. The obtained results were very promising since, at the chosen concentrations of rutin and each IL, no significant differences were observed between the rutin-exposed cells and the cells exposed to the combination of rutin with each IL. This shows the functionality of the ILs since at biocompatible concentrations, their presence allows the maintenance of the rutin's activity while allowing a higher drug solubility and delivery.
Drug-induced cytotoxicity is frequently accompanied by cell cycle modification. Thus, after the selected concentrations from the cell viability studies, the impact of rutin on the human renal cancer cell cycle was also assessed to continue to evaluate the anticancer effects of this natural product.
In this study, rutin showed an impact on the cell progression of 786-O cells, leading to a significant increase in the sub-G1 population, with a consequent decrease in the G0/G1 population. This is in accordance with the previous results obtained for the cell viability, which reinforces the possible anticancer activity of this biomolecule.
The rutin's anticancer mechanisms of action are not yet fully understand. However, besides the impact of rutin on cell viability and cell cycle, shown herein, there are also some studies in other cancer cell models showing that this compound interferes with some carcinogenesis mechanisms, such as chemotactic ability [18] and cell adhesion and migration [39]. Additionally, rutin could also reverse multidrug resistance by the inhibition of P-glycoprotein [9].
Despite the fact that rutin's mode of action is not totally comprehended, this study clearly suggests the possible applicability of rutin for renal cancer treatment. Furthermore, considering this applicability, the encapsulation in nanoparticles could be a relevant strategy to enhance therapeutic efficiency [44]. Nonetheless, the low aqueous solubility of rutin represents an impairment for this incorporation in drug delivery systems. In fact, in a previous study, we have shown that nanoparticles combined with 0.2% of [Cho][Phe] allowed us to incorporate rutin with an AE of 75.6%, which in the absence of the IL was not possible [35,37]. Hence, in this study, we prepared IL-nanoparticles hybrid systems, loaded and unloaded with rutin, using the concentrations of [ [Phe]-nanoparticle hybrid system demonstrated that an enhancement of 0.1% of the incorporated IL did not interfere with the hybrid nanosystems stability, but allowed a higher incorporation of the drug and a 10% increase in the AE when compared with the previous prepared hybrid IL-nanocarrier [35]. This shows that the amount of IL impacts drug loading and AE. Also, it should be noted that when using the [Cho][Gly], the newly developed hybrid IL-nanosystem is also stable and that even though this IL is present at a lower percentage (0.2%) compared with [Cho][Phe] (0.3%), the obtained AE is quite similar. The LC values are low for both ILs and similar to those already described in the literature for hybrid IL-nanosystems [37].
Finally, it was also assessed the release of the rutin from the freshly prepared IL-nanoparticle hybrid systems. An initial burst was observed, as expected, probably due to the rutin adsorbed in the surface of the nanoparticles [35,45]. Nonetheless, this burst release of about 45% happened only after 5 h, probably due to a higher affinity of rutin to the IL-nanoparticles hybrid system, followed by sustained release up to 72 h. This release profile is important to enhance the therapeutic effect and reduce the number of administrations required.
Hence, when considering the low solubility and bioavailability of rutin, and the possible anticancer applicability of this compound, the prepared hybrid IL-nanocarriers may represent a promising strategy to overcome these challenges, presenting a suitable structure and performance and allowing a higher drug loading and controlled drug delivery.
Globally, these results are quite promising and show it would be valuable to continue to study the rutin's mechanisms of action, to explore its role in cancer progression, and also to assess the efficacy and safety of the rutin loaded IL-nanosystem hybrid systems.

Conclusions
In summary, this study aimed to investigate the effect, safety, and applicability of rutin as a potential therapeutic agent, in the treatment of human renal cancer and to assess the functionality of ILs to allow the delivery of this poorly soluble drug in a biocompatible manner.
Our results suggest that rutin displays cytotoxic effects on 786-O human cancer cells and that at 50 µM, it may be safely used against ccRCC since, at this concentration, no significant effect was observed in normal renal cells. Moreover, we also showed that the two choline-amino acid ILs under study can act as functional excipients since their presence at non-toxic concentrations in Vero cells enhanced the drug solubility while allowing the maintenance of the rutin's activity. Furthermore, ionic liquids-nanoparticle hybrid systems containing rutin were also prepared and our results showed that the ionic liquids were crucial to increasing drug loading and that these IL-nanoparticle hybrid systems may be used as a strategy for rutin delivery, contributing to its applicability as a potential therapeutic agent. These results are promising, showing the potential use of ILs to improve drug delivery and opening a new avenue for further studies to understand the underlying mechanisms of rutin cytotoxicity in order to develop effective and safe hybrid nanocarriers capable of being targeted to the ccRCC.