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Nanoencapsulation of the flavonoid dihydromyricetin protects against the genotoxicity and cytotoxicity induced by cationic nanocapsules
Colloids and Surfaces B: Biointerfaces 173 (2019) 798–805
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Nanoencapsulation of the flavonoid dihydromyricetin protects against the genotoxicity and cytotoxicity induced by cationic nanocapsules
T
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Ana Julia F. Dalcina,b, , Bruno S. Vizzottoc, Guilherme V. Bochid, Naiara S. Guardae, Kátia Nascimentof, Michele R. Sagrillob, Rafael N. Morescoe, André P. Schuchc, Aline F. Ouriquea,b, Patrícia Gomesa,b a
Laboratory of Nanotechnology, Franciscan University, Santa Maria, Brazil Post Graduate Program in Nanosciences, Franciscan University, Santa Maria, Brazil c Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil d Department of Physiology and Pharmacology, Federal University of Santa Maria, Santa Maria, RS, Brazil e Department of Clinical and Toxicological Analyses, Federal University of Santa Maria Santa Maria, RS, Brazil f Laboratory School of Clinical Analyzes, Franciscan University, Santa Maria, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cytotoxicity Eudragit RS100® Genotoxicity In vitro toxicity Polymeric nanoparticles
We evaluated the influence of nanoencapsulation of the flavonoid Dihydromyricetin (DMY) in reducing the genotoxicity and cytotoxicity induced by cationic nanocapsules. Assays were conducted in order to evaluate the potential of protein corona formation, cytotoxicity, genotoxicity and the antioxidant capacity. Nanocapsules containing DMY (NC-DMY) and free DMY (DMY-F) did not demonstrate cytotoxicity and genotoxicity. However, Eudragit RS100® nanocapsules (NC-E) increased cytotoxicity and DNA damage formation. NC-DMY and NC-E presented high interaction with the DNA in vitro, suggesting DNA sequestration. These results indicate that nanoencapsulated DMY does not induce cytotoxicity or genotoxicity, and demonstrates high antioxidant capacity. This antioxidant capacity is probably associated with DMY, and occurs due to its ability to avoid the formation of free radicals, thus preventing the toxicity caused by the nanostructure with the cationic polymer Eudragit RS100®. Therefore, NC-DMY can be considered an important formulation with significant antioxidant potential to be exploited by nanomedicine.
1. Introduction Nanocapsules have attracted attention in the scientific community due to their bioavailability, biodegradability, photostability of drugs, modulation of the interaction with cells and tissues, possibility of reducing adverse effects of drugs, reduction of therapeutic doses, increased efficiency of encapsulation, increased solubility and stability in biological fluids, as well as for storage [1–5]. Nanocapsules are defined as vesicular structures consisting of a thin polymeric shell and usually an oily central cavity where the active substance is dissolved. They are therefore considered a reservoir system, which has a diameter typically between 200–400 nm [1,2]. The nanometric size provides a greater surface area, and this feature is
strongly correlated to their biological responses [3]. Eudragit RS100® is a positively charged polymer that is widely used to prepare nanoparticles due to its well-established mucoadhesive characteristics [6,7]. This co-polymer is comprised of ethyl acrylate, methyl methacrylate, and a methacrylic acid ester content of about 4.5–6.8% quaternary ammonium groups [8]. Ammonium groups are present in the form of salts and are responsible for the permeability of the polymer and for providing a positive surface to the polymer. This positive surface is important for the interaction with negatively charged drugs, cell surfaces, and target tissues, maximizing cellular uptake of the polymer-drug complex [6,8–10]. Although this polymer is not biodegradable, it has adequate biocompatibility for its role in increasing the interaction with the
Abbreviations: DMY, Dihydromyricetin; DMY-F, Free DMY; NC-E, Eudragit RS100® nanocapsules; NC-DMY, Nanocapsules containing DMY; FRAP, Ferric Reducing Antioxidant Power; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tretazolium bromide ⁎ Corresponding author at: Laboratory of Nanotechnology, Franciscan University, Rua dos Andradas 1614, Santa Maria, RS, 97010-032, Brazil. E-mail addresses: [email protected] (A.J.F. Dalcin), [email protected] (B.S. Vizzotto), [email protected] (G.V. Bochi), [email protected] (N.S. Guarda), [email protected] (K. Nascimento), [email protected] (M.R. Sagrillo), [email protected] (R.N. Moresco), [email protected] (A.P. Schuch), [email protected] (A.F. Ourique), [email protected] (P. Gomes). https://doi.org/10.1016/j.colsurfb.2018.10.066 Received 26 July 2018; Received in revised form 11 October 2018; Accepted 24 October 2018 Available online 27 October 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved.
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and conditions were the following: Shimadzu HPLC system (Kyoto, Japan) was used and equipped with an LC-20ATpump, an SPD-M20 A photodiode array (PDA) detector, a CBM-20 A system controller, a C18 Phenomenex (4 × 3.0 mm) pre-column and RP-C18 Phenomenex column (150 mm × 4.0 mm, 5 μm particle size, 100 Å pore diameter). The mobile phase was acetonitrile–water (20:80 v/v) at pH 4.0 (adjusted with acetic acid) at an isocratic flow rate (0.6 mL min−1). Each run lasted 10.0 min at room temperature, and the retention time was 5.6 min. The injection volume was 20 μL. Detection was conducted at 290 nm. The encapsulation efficiency was determined by methodology provided by Santos et al. [19], using ultrafiltration/centrifugation devices (Amicon®10 kDa, Millipore) at 7000 × g for 10 min, followed by determination by HPLC according to the method previously described by Dalcin et al. [17].
mucosa [3,6,11] Eudragit RS100® has been widely employed in the production of oral and topical formulations, and several authors report its good biocompatibility in different applications [7,12–22]. Its use has been accepted by regulatory agencies of the United States, Brazil, Europe, and Japan for oral and topical administration [6,22,23]. Eudragit RS100® can also be used in the development of thermo-sensitive drugs [24], and represents an adequate vehicle for drug dispersion [16]. Nanoencapsulation containing Eudragit RS100® has been reported as an alternative to increase the aqueous solubility of Dihydromyricetin (DMY) [17], a flavonoid extracted mainly from stems and leaves of Ampelopsis grosedentata, a vine from southern China [25]. DMY is known for its different pharmacological properties, including: antioxidant [26,27], anti-inflammatory [25,28], antitumor [25,29], photoprotective [30], depigmenting [31], antimicrobial [17,25,32], hypoglycemic [25,33], hepatoprotective, [25,34,35], neuroprotective [36] among others [25]. Although one of the major advantages of nanomedicine is to provide drugs targeted to diseased organs, the control and prediction of nanocapsules distribution in the human body requires further investigation [37,38]. The relationship between the structure of nanoparticles and behavior in biologically complex environments, such as the human body, is far from being completely understood, making it difficult to predict its biodistribution, as well as possible undesirable cellular interactions and adverse effects [39,40].Therefore, many studies have intended to evaluate and better understand the cytotoxic and genotoxic effects of nanoparticles in in vitro models [39,40]. In this study, we investigated if the nanoencapsulation of the DMY flavonoid produced with the Eudragit RS100® polymer was able to protect against the genotoxicity and cytotoxicity induced by cationic nanocapsules. Additionally, we evaluated the effects of the nanoencapsulation on the formation of the protein corona effect and antioxidant activity of DMY flavonoid. In this study, we evaluated the influence of the nanoencapsulation of the flavonoid DMY on the cytotoxic and genotoxic effects provided by cationic nanocapsules produced with the Eudragit RS100® polymer. Additionally, we evaluated the impact of the nanoencapsulation on the formation of the protein corona effect and antioxidant activity of DMY flavonoid.
2.2. Effect of the protein corona This experimental protocol was conducted in order to predict the possibility of the formation of the protein corona around the nanoparticles when in contact with biological fluids. RPMI (Embriolife, Brazil) and DMEM (Himedia, India) culture media were supplemented with 10% fetal bovine serum and added to the formulations of NC-DMY, NC-E, and free DMY (F-DMY, the non-nanoencapsulated form) at concentrations of 1 μM and 150 μM. Subsequently, they were evaluated for the characterization of particle size, polydispersity index, zeta potential, and pH. The formulations were characterized before contact with the culture medium (after the preparation of the nanocapsule suspensions), after initial contact with the medium (without incubation), 24, and 72 h after incubation at 37C in 5% CO2. The F-DMY samples were analyzed only for pH values. 2.3. Blood collection and cell culture Peripheral blood mononuclear cells (PBMC) were used to assess the cell viability by MTT and comet assays. The peripheral blood samples were obtained of healthy volunteers in Santa Maria, RS, Brazil. The study was approved by the Institucional Ethics Committee (Number: 654264). Samples were obtained by venipuncture using heparin-like Vacutainer tubes. The separation of PBMC by density gradient was done (Histopaque -1077) by centrifugation, and 2000 cells were plated per well (in 96-well plates) for each treatment. Both MTT and comet assay were conducted after treatment with NCE, NC-DMY, and F-DHM. The concentrations used were 1 μM, 10 μM, 50 μM, 100 μM, and 150 μM, and the incubation period with cells was 24 h. In addition, control samples used for the tests consisted of a negative control (C−) only containing culture medium and PBMC, and a positive control (C+) containing culture medium, PBMC and 100 mM hydrogen peroxide (H2O2).
2. Material and methods 2.1. Preparation and physicochemical characterization of nanoparticle suspensions Nanocapsule suspensions containing DMY (NC-DMY) were prepared by interfacial deposition of a pre-formed polymer, according to the method described previously [17]. Eudragit RS100® (Evonik, Germany) was solubilized in acetone (Synth, Brazil), DMY (Sigma-Aldrich, USA) and medium-chain triglycerides (Alpha Quimica, Brazil). Subsequently, this organic phase was added to an aqueous phase containing polysorbate 80 (Synth, Brazil). Magnetic stirring was maintained and the organic solvent was eliminated by evaporation under reduced pressure. Eudragit RS100® nanocapsule suspensions (NC-E) were similarly prepared, omitting the presence of DMY. After the preparation of the nanocapsule suspensions, the average diameter and polydispersity index (1:500 v/v in ultrapure water) were determined by dynamic light scattering (Zetasizer® Nano-ZS model ZEN 3600, Malvern Instruments, UK), and the zeta potential (500 v/v in NaCl 10 mM solution) by electrophoretic mobility (Zetasizer® Nano-ZS model ZEN 3600, Malvern Instruments, UK). In addition, the pH was measured directly from the formulations using a previously calibrated potentiometer (Digimed® DM – 20, Brazil). The DMY content was determined (n = 3) by High Conductance Liquid Chromatography (HPLC) using a method previously validated by validated by our research team25. The chromatographic instruments
2.3.1. Cell viability assay Cell viability was evaluated [44] using RPMI culture medium. In short: a solution of, the 3-(4,5-Dimethyl-2-thiazolyl)-2-5-diphenyl-2Htretazolium bromide (MTT) (Sigma-Aldrich, USA) was dissolved in phosphate buffer (pH 7.4), added to the cells, and incubated for 24 h at 37C in 5% CO2 protected from light. The analyses were conducted in triplicate in a spectrophotometer at 570 nm, and the results were expressed as percentage of viable cells in relation to control samples. Cytotoxicity was defined as [45]: Non-cytotoxic > 90% of cell viability; Slightly cytotoxic = 60–90% of cell viability; Moderately cytotoxic = 30–59% of cell viability; Severely cytotoxic ≤ 30%. 2.3.2. Comet assay The evaluation of genotoxicity was carried out by Comet assay following the protocol previously described [46]. After the incubation, on a glass slide covered previously with a 1.5% agarose layer, the PBMC 799
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were deposited and suspended in low-melting point agarose (Low Melting). The material was immersed in a lysis solution in order to remove the membranes and cytoplasms. The slides were incubated in an alkaline electrophoresis buffer and subjected to electrophoresis for 30 min at 25 V and 300 mA. In order to analyze the genetic material, neutralization, fixation, and coloring processes were carried out. Each sample was evaluated by optic microscopy, and the cells were classified according to the shape of the nuclei into four damage classes, varying from 0 (no damage) to 4 (maximum damage), including the classification of cellular apoptosis. The calculation of the damage index was conducted through the sum of the damages 1, 2, 3, 4 and apoptosis, divided by 100.
Table 1 Physicochemical characterization of nanoparticles suspensions. Parameter
NC-DMY
NC-E
Particle size (nm) Polydispersity index Zeta Potential (mV) pH Value Drug content (%) Encapsulation efficiency
146.9 ± 0.7 0.107 ± 0.01 +10.3 ± 0.7 4.0 ± 0.02 100.2 ± 0.5 80.88% ± 2.88
140.7 ± 0.4 0.097 ± 0.01 +10.2 ± 0.1 4.7 ± 0.12 – –
efficiency was about 80%, drug content was 100%, and pH was slightly acid due to the characteristics of the DMY.
2.3.3. DNA interaction The assay to evaluate DNA interaction was conducted according to the methodology previously described [47]. All samples (NC-DMY, NCE, and F-DMY) were tested at concentrations of 1 μM, 10 μM, 50 μM, 100 μM, 150 μM, 500 μM, 750 μM, and 1000 μM, and incubated overnight with 300 ng/μL plasmidial DNA pCMUT48 at room temperature, protected from light. After incubation, the samples were electrophoresed in 0.8% agarose gel in 0.5X TBE buffer. In order to determine the interaction of the formulations with the DNA and possible changes in the electrophoretic migration pattern, the samples were evaluated by densitometry analysis (Amersham Imager 600 GE®, United Kingdom).
3.2. Protein corona effect The results of the protein corona effect are presented in Fig. 1. In the initial contact of the nanocapsules with both media, there was a significant increase of the diameter for NC-DMY in the lowest concentration (1 μM). However, this increase in diameter was not found in the highest concentration evaluated (150 μM). For NC-E, a significant increase was only found in the RPMI culture medium, and in the lowest concentration (1 μM) after initial contact, as well as after 24 h of incubation with the medium. Further incubation times did not provide significant increase in particle size. The stability of the nanocapsules was also evaluated by monitoring the polydispersity changes of the formulations evaluated in the culture medium (Fig. 1B). After contact with the culture medium, the results demonstrated a significant increase in all incubation times both for RPMI and DMEM medium for all samples at the lowest concentration of 1 μM, for both NC-DMY and NC-E. However, no significant differences were demonstrated for the highest concentration of 150 μM. Fig. 1C shows that after contact with the culture medium, all samples presented a change in charge from the positive zeta potential into negative, regardless of time and culture medium evaluated. The initial pH values for the NC-DMY, NC-E, and F-DMY formulations were 4.03 ± 0.02, 4.70 ± 0.02, and 4.45 ± 0.02, respectively (data not shown). The culture media have pHs of 7.42 ± 0.02, and 7.82 ± 0.02 for RPMI and DMEM, respectively. After contact with the media it was possible to verify an increase in the pH values for all samples, independent of the medium and incubation time. The pH values increased and were similar to the pH values of the individualized medium.
2.4. Determination of the antioxidant capacity For the determination of the antioxidant capacity of the formulations, the Ferric Reducing Antioxidant Power (FRAP) assay was used according to the methodology previously described [49], adapted to the automated system BS 380 (Mindray®, China). In short, NC-DMY, NC-E, and F-DMY were evaluated at different concentrations (1, 10, 50, 100 and 150 μM), diluted in distilled water. The FRAP reagent was prepared from the combination of acetate buffer with 2,4,6-Tris(2-Pyridyl)-striazine (TPTZ) (Sigma-Aldrich, USA) solution and an aqueous solution of ferric chloride (Sigma-Aldrich, USA). At acid pH, the Fe3+ complex is reduced to Fe2+ and forms an intense blue solution. The FRAP solution was used as the reference reagent, and the absorbance was measured at 605 nm. For the calibration curve, ferrous sulphate was used as standard at concentrations ranging from 0 to 1000 μmol/L. Distilled water was used as the negative control. The assay was conducted in triplicate (n = 3), and the results were expressed in μmol/L. 2.5. Statistical analysis
3.3. Cell viability assay The results of the characterization were expressed as a mean ± standard deviation. For the analysis of the protein corona effect, evaluation of cytotoxicity, genotoxicity, and determination of antioxidant capacity, data were expressed as mean ± standard deviation and analyzed by One-Way ANOVA, followed by the Dunett´s test. Values with p ≤ 0.001 were considered statistically significant. For determination of the antioxidant capacity, the t-test was conducted to compare NC-DMY and F-DMY. Values with p ≤ 0.05 were considered statistically significant. All graphs and statistical analyzes were done on GraphPad Prism® software version 4.0 (GraphPad, USA). 3. Results
The MTT cell viability assay demonstrated that the different concentrations of NC-DMY did not show significant difference as compared to the negative control (Fig. 2). This indicates that the nanocapsules containing the DMY drug were not cytotoxic. NC-E showed a significant decrease in viability for the highest concentrations of 100 and 150 μM. NC-E at the concentration of 100 μM demonstrated mild cytotoxicity (62%), and at the concentration of 150 μM (59%), moderate cytotoxicity. F-DMY showed no reduction in cell viability at the concentrations evaluated; however, there was increased cell viability at the highest concentrations (50, 100 and 150 μM).
3.1. Physicochemical characterization of nanocapsule suspensions
3.4. Comet assay
The results of the physicochemical characterization of NC-DMY and NC-E are described in Table 1. The mean particle diameters were around 140 nm for both formulations. The samples presented low polydispersity index, which demonstrates good homogeneity of the systems, as well as positive zeta potential around + 10 mV due to the presence of the Eudragit RS 100® cationic polymer. The encapsulation
The levels of DNA damage induced by the formulations are depicted in Fig. 3. NC-DMY and F-DMY did not show significant difference as compared to the negative control. This indicates that there was no damage to the cellular DNA and, according to this test, that the formulations of NC-DMY and F-DMY were not genotoxic. For the NC-E, concentrations of 50, 100 and 150 μM caused 800
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Fig. 1. Results of protein corona effect for the formulations NC-DMY and NC-E characterized in particle size (A), polydispersity index (B), zeta potential (C), before and after contact with RPMI and DMEM culture medium in the different incubation periods. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by variance One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
Surprisingly, the results found for NC-DMY and NC-E indicate a high interaction with DNA in concentrations higher than 10 μM (Fig. 4B and C), demonstrating the occurrence of DNA sequestration by these formulations at the concentrations of 50, 100, 150, 500, 750, and 1000 μM (DNA retained on the upper part of the gel).
significant DNA damage to the cells in a concentration-dependent manner. This result is consonant to the MTT viability test, where for the same concentrations there was a decrease in cell viability, indicating a possible toxic effect of the NC-E in the highest concentrations.
3.4.1. Interaction with DNA We evaluated 8 concentrations, 3 of them higher than those evaluated in previous tests, in order to verify its interaction with DNA. The analysis of a plasmid DNA sample interaction with NC-DMY, NC-E and F-DMY can be seen in Fig. 4, and these results indicate that F-DMY does not interact with DNA.
3.5. Determination of the antioxidant capacity The antioxidant capacity was evaluated for all formulations at the same concentrations used for the assessment of cytotoxicity and genotoxicity (Fig. 5). 801
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Fig. 2. Cell viability assay by MTT with 24 h incubation. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
(4 ± 2C) [17]. Our group also verified the efficacy of the anti-biofilm effect of dihydromyricetin-loaded nanocapsules on urinary catheter infected by Pseudomonas aeruginosa. NC-DMY was more effective when compared with free DMY, demonstrating that NC-DMY can potentially be used as an innovative approach to urinary catheter biofilm treatment or prevention. [17] However, few in vitro and in vivo studies are devoted to the toxicity of nanoparticulate systems, in particular to the nanocapsules produced with the Eudragit RS100® polymer. In addition, most of these studies used only one assay (usually cell viability by MTT) to assess the toxicity of nanoparticles. Regarding the results of the protein corona assay, the initial increase in diameter can be explained by the fact that the analysis was conducted after the nanocapsules were in immediate contact with the medium, what may not have granted sufficient time for the nanoparticles to stabilize in the solutions containing the culture medium. The significant increase in particle diameter after the initial period was verified only at the 24-hour incubation for NC-E at the concentration of 1 μM for the RPMI medium. The other concentrations of NC-DMY or NC-E did not demonstrate increase in the mean diameter of the particles, regardless of the incubation time and culture medium evaluated. Similar results were found [49] who evaluated the diameter of white nanocapsules produced with Eudragit RL100® and PMMA (poly-methylmethacrylate), and verified that there was no increase in particle diameter after contact with the RPMI medium. The increased polydispersity probably occurred due to the heterogeneity of the media, and to the fact that the graphical profile of the size distribution of the culture media alone shows more than one peak,
As expected, the F-DMY formulations demonstrated high dose-dependent antioxidant capacity, and these results corroborate previous studies demonstrating the antioxidant activity of F-DMY [31,34,65–68]. NC-E did not demonstrate significant antioxidant capacity when compared to the drug in the free or nanostructured form. The values found ranged from 13 to 390 μM/L FRAP, demonstrating that the constituents of the formulation and the structure of the nanocapsule do not have antioxidant potential. As verified for F-DMY, NC-DMY demonstrated an important antioxidant capacity and in a dose-dependent manner, reaching values higher than 5000 FRAP μmol/L. Results from the FRAP assay demonstrated that DMY inserted into polymeric nanocapsules maintained its antioxidant capacity when compared to F-DMY. 4. Discussion The present study reported at first time that the nanoencapsulation of DMY was able to protect the cytotoxicity and genotoxicity induced by cationic nanocapsules produced with the cationic polymer Eudragit RS100® in PBMCs. The results of the physicochemical characterization of the nanocapsule suspensions corroborate with results previously found by us [17], as well as by other authors who used the same nanostructure with other drugs [6,7,18–20,50–52]. Studies of the stability of NC-DMY and NC-E for 90 days under different storage conditions (refrigeration, room temperature and climatic chamber) were previously conducted and demonstrated that the best condition for suspension storage without losing physical and chemical characteristics was under refrigeration
Fig. 3. Comet assay with 24 h of incubation. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗p < 0.05, ∗∗p < 0.01, and ∗∗∗ p < 0.001. 802
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Fig. 4. Results of interaction with DNA in electrophoresis in agarose gel 0.8% for F-DHM (A), NC-DMY (B) and NC-E (C).
dissociation, as well as changes in the environment, and may subsequently change their behavior. [54] The pH change of the formulations upon contact with the medium allows us to ensure that even being acidic, when in contact with the biological medium they did not cause imbalance in pH values. This can be explained by the characteristic of the constituents of the culture media, which have buffering systems in their composition in order to maintain pH regulation and to establish adequate culture conditions. [55] The results of the corona protein effect show that the nanocapsules were stable when in contact with the culture media. In addition, it was possible to evaluate that there was no particle agglomeration by the average particle diameter measurement when in contact with culture media; or if the formation of the protein corona occurred, this was not sufficient to provide an increase in particle size. This indicates the absence of protein corona formation for the nanoparticles evaluated in the RPMI and DMEM culture media. The results of the cell viability assay by MTT for F-DMY demonstrated no reduction in viability and corroborate with the findings [56,57] who evaluated it in other cell lines, such as human liver cancer cells (HepG2), and human umbilical vein endothelial cells (HUVECs), respectively. However, our results demonstrated increased F-DMY viability (above the concentration of 10μM), suggesting a proliferative effect. Ferreira et al. [58], attributed this effect to the stimulation of the immune system by the flavonoid, which has antioxidant activity and can provide immunostimulatory effects modulating gene expression, which may be related with increased cell metabolism.59 It is important to mention that NC-DMY showed no changes in cell viability. Therefore, the results of the present study demonstrated that NC-DMY and F-DMY did not induce cytotoxicity. However, for NC-E, a decrease in cell viability was noted in a dose-dependent manner with moderate
consequent to the various nutrients contained in the culture media. In addition, this changed the profiles of particle size distribution of the formulations from unimodal to bimodal or multimodal (supplementary information - S1) when inserted into the culture media. Increases in the polydispersity index values were also found [7] when evaluating nanocapsules containing carvedilol in the porcine sublingual mucosal medium. This increase in polydispersity values indicated an increase in heterogeneity of the size distribution [7]. It is important to consider that the culture media consist of various nutrients which provide diversity in sample composition and results in increased heterogeneity and consequently, increase in the polydispersity index. The results found suggested that the most affected physicochemical parameter by the interaction of nanocapsules with the components of the cellular medium was the charge. As of today, there are no reports in the literature evaluating the stability of nanocapsules containing Eudragit RS 100® in contact with culture media RPMI and DMEM. Nevertheless, researchers [7] noted a similar behavior in porcine sublingual mucosal medium, as well as verified this charge changes for their formulations. The results were attributed to the high capacity of the nanoencapsulated drug to interact with mucin molecules when compared to the drug in the free form, suggesting that the Eudragit RS100® polymer can interact with negative mucin molecules by electrostatic attraction. We believe that the same effect found can be justified in our experiment. Graça et al., [53] also noted changes in positive to negative charge when evaluating nanocapsules produced with Eudragit RS100® and poly methyl-methacrylate (PMMA) on immediate dilution in the RPMI medium. This was justified as a probable consequence of the dynamic formation of a protein corona, which may be coated by biomolecules present in the biological environment that bind to the surface of nanocapsules. In contrast, the protein corona composition varies over time due to continuous protein association and
Fig. 5. Result of antioxidant capacity by FRAP. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. 803
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[25–27], and the reason for this antioxidant activity is the chemical structure of flavonoid compounds, which can scavenge free radicals, chelate metals, and donate electrons and hydrogen atoms. Its chemical structure consists of 15 carbons arranged in a configuration of 6C-3C6C, having 6 hydroxyl groups, and the ortho-trihydroxy group (ring B) in DMY mainly conducts antioxidant activity [64]. NC-DMY maintained its antioxidant capacity when compared to FDMY. Therefore, we believe that the incorporation of DMY into a nanostructured carrier did not interfere in its antioxidant capacity. In addition, it can be noticed that NC-DMY suggest potentiation in the antioxidant effect when compared to F-DMY (p = 0.0158 for the concentration of 150 μM, and p = 0.0052 for the concentration of 100 μM). It is important to mention that the encapsulation efficiency values for NC-DMY were 80%. That is, 80% of the DMY is inside the nanocapsule, what suggests that the remaining 20% are adhered to the polymeric membrane. We may therefore imply that nanoencapsulated DMY in addition to providing the effect of the drug sustained release [17], may provide the effect of protection against the toxicity generated by NC-E through an antioxidant effect generated by DMY adhered to the surface of the nanostructure that acts against the generation of free radicals. We suggest that DMY also can be added to nanocapsules of Eudragit RS100® as an ingredient of the nanocapsule to prevent the cytotoxicity induced by Eudragit in order to prepare other drug formulations and also execute its antioxidant effect.
cytotoxicity at the highest concentration (59%), demonstrating the cytotoxic potential of the cationic nanocapsules produced with the polymer Eudragit RS100® when evaluated in the absence of the drug. This same effect was also found by Eidi et al., [10] where nanocapsules produced with Eudragit RS100® and without the drug showed concentration-dependent cytotoxicity and increased toxicity when compared to nanocapsules containing the drug in macrophage cells (NR8383). Gargouri et al., [47] also reported that nanocapsules without the DNA were more cytotoxic than nanocapsules containing DNA in pharyngeal cancer cells (FaDu) and in human breast adenocarcinoma cells (MDA-MB 231 and MCF-7). Contri et al. [52], evaluated the viability of human fibroblasts and keratinocytes against white nanocapsules produced with Eudragit RS100®, and reported a decrease in cell viability in a concentration-dependent manner of treatment, although this cytotoxicity was not significant. In contrast, the mild toxicity noted supports the hypothesis of a nonspecific cell response to stress associated to the exposure to nanocapsules at very high concentrations [60]. The results of the comet assay for NC-E were different from those found in the literature [10,52,53]. However, these authors used other cell lines, other concentrations, and other methodologies for the assessment of genotoxicity. We chose the PBMC cell line because it is the most sensitive human cell line to evaluate the toxicity of various chemical compounds, and has been applied for decades as cytotoxic and genotoxic biomarkers because it is abundant in the blood circulation. In addition, when exposed to any mutagenic agent they are able to reflect recent damage [57]. However, in assay conducted by Eidi et al. [10], on cellular apoptosis, the conclusion was that Eudragit RS100® nanocapsules can induce cell death, although the type of cell death (necrosis or apoptosis) involved should be better investigated. It should also be investigated whether cell death induced by nanocapsules is consequent to their internalization by cells or their interaction with cell membranes. Nevertheless, it is important to mention that Louro et al. [54] report that such primary lesions usually arise soon after exposure to genotoxic agents and may be easily repaired by the DNA repair machinery. As for the DNA interaction assays, we verified the disappearance of the bands for NC-E and NC-DMY in the highest concentrations. This same behavior was observed by Du et al. [62] where bovine serum albumin was synthesized with the cationic polymers tetraethylenepentamine (TEPA) and polyethyleneimine (PEI) and used as a potential gene transporter. The results demonstrated that the DNA bands did not appear on the gel in the highest concentrations tested, which meant that the formulation evaluated had the capacity to sequestrate DNA due to the agitation effect of the α-helical structure and to the positive potential of the complexes. Furthermore, the complexes demonstrated high transfection efficiency of DNA, proving to be a potential gene transporter. Lu and Liu [63] also verified this gel retargeting effect by cationic nanocapsules produced with PEG-PCL-PEI. The authors justify that this binding affinity of copolymers with the DNA occurs consequent to the electrostatic interaction between negatively charged phosphates along nucleic acid and cationic copolymers segments. Therefore, we believe that this same behavior described [64] occurred in our experiment. The disappearance of the DNA bands may indicate the use of NC-DMY and NC-E formulations as potential gene carriers. We strongly suggest, therefore, that this same effect of sequestration/retardation for NC-DMY and NC-E can be justified by the electrostatic interaction of the DNA with the quaternary ammonium groups present in the Eudragit RS100® polymer. It is important to point out that the highest concentrations of NC-E induced a decrease in cell viability and increased DNA damage. Furthermore, no cytogenotoxicity was demonstrated for the NC-DMY, since it did not decrease cell viability, as well as did not induce DNA damage. Therefore, our results demonstrate the preliminary safety of these nanoparticles. DMY, as well as most flavonoids, possess antioxidant properties
5. Conclusions For the first time, different in vitro biological tests were conducted in order to evaluate the cytotoxicity and genotoxicity of cationic nanocapsules produced with Eudragit RS100® and containing DMY and NC-E in peripheral blood mononuclear cells, as well as their interaction with the DNA molecule. Our work is the first to show that the nanoencapsulation of the flavonoid Dihydromyricetin (DMY) may decrease the cytotoxicity provided by the cationic polymer Eudragit RS100® due to its potential antioxidant effect. The results of the protein corona demonstrated that there was no increase in particle diameter, suggesting absence of protein corona, or if it occurred, no interference in the mean particle diameter. NC-E at the highest concentrations demonstrated moderate cytotoxicity and increased DNA damage as compared to control, suggesting toxic potential. The results demonstrate that DMY, when nanoencapsulated, maintained its antioxidant capacity and prevented the cytogenotoxic effect of the cationic polymer Eudragit RS100®, reinforcing that its use can be better exploited by nanomedicine for different biological applications. Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. The authors also would like to thank Evonik for supplied Eudragit RS100®. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.10.066. References [1] A.D. Mali, R.S. Bathe, Updated review on nanoparticles as drug delivery systems, Int. J. Anal. Pharm. Biomed. Sci. 8 (9) (2015) 18–34. [2] C.E. Mora-huertas, H. Fessi, A. Elaissari, Polymer-based nanocapsules for drug delivery, Int. J. Pharm. 385 (2010) 113–142. [3] L.A. Frank, R.V. Contri, R.C.R. Beck, A.R. Pohlmann, S.S. Guterres, Improving drug biological effects by encapsulation into polymeric nanocapsules, WIREs Nanomed. Nanobiotechnol. 7 (2015) 623–639. [4] L. Bregoli, D. Movia, J.D. Gavigan-imedio, J. Lysaght, J. Reynolds, A. Prina-mello, Nanomedicine applied to translational oncology: a future perspective on cancer
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Nanoencapsulation of the flavonoid dihydromyricetin protects against the genotoxicity and cytotoxicity induced by cationic nanocapsules
T
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Ana Julia F. Dalcina,b, , Bruno S. Vizzottoc, Guilherme V. Bochid, Naiara S. Guardae, Kátia Nascimentof, Michele R. Sagrillob, Rafael N. Morescoe, André P. Schuchc, Aline F. Ouriquea,b, Patrícia Gomesa,b a
Laboratory of Nanotechnology, Franciscan University, Santa Maria, Brazil Post Graduate Program in Nanosciences, Franciscan University, Santa Maria, Brazil c Department of Biochemistry and Molecular Biology, Federal University of Santa Maria, Santa Maria, RS, Brazil d Department of Physiology and Pharmacology, Federal University of Santa Maria, Santa Maria, RS, Brazil e Department of Clinical and Toxicological Analyses, Federal University of Santa Maria Santa Maria, RS, Brazil f Laboratory School of Clinical Analyzes, Franciscan University, Santa Maria, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cytotoxicity Eudragit RS100® Genotoxicity In vitro toxicity Polymeric nanoparticles
We evaluated the influence of nanoencapsulation of the flavonoid Dihydromyricetin (DMY) in reducing the genotoxicity and cytotoxicity induced by cationic nanocapsules. Assays were conducted in order to evaluate the potential of protein corona formation, cytotoxicity, genotoxicity and the antioxidant capacity. Nanocapsules containing DMY (NC-DMY) and free DMY (DMY-F) did not demonstrate cytotoxicity and genotoxicity. However, Eudragit RS100® nanocapsules (NC-E) increased cytotoxicity and DNA damage formation. NC-DMY and NC-E presented high interaction with the DNA in vitro, suggesting DNA sequestration. These results indicate that nanoencapsulated DMY does not induce cytotoxicity or genotoxicity, and demonstrates high antioxidant capacity. This antioxidant capacity is probably associated with DMY, and occurs due to its ability to avoid the formation of free radicals, thus preventing the toxicity caused by the nanostructure with the cationic polymer Eudragit RS100®. Therefore, NC-DMY can be considered an important formulation with significant antioxidant potential to be exploited by nanomedicine.
1. Introduction Nanocapsules have attracted attention in the scientific community due to their bioavailability, biodegradability, photostability of drugs, modulation of the interaction with cells and tissues, possibility of reducing adverse effects of drugs, reduction of therapeutic doses, increased efficiency of encapsulation, increased solubility and stability in biological fluids, as well as for storage [1–5]. Nanocapsules are defined as vesicular structures consisting of a thin polymeric shell and usually an oily central cavity where the active substance is dissolved. They are therefore considered a reservoir system, which has a diameter typically between 200–400 nm [1,2]. The nanometric size provides a greater surface area, and this feature is
strongly correlated to their biological responses [3]. Eudragit RS100® is a positively charged polymer that is widely used to prepare nanoparticles due to its well-established mucoadhesive characteristics [6,7]. This co-polymer is comprised of ethyl acrylate, methyl methacrylate, and a methacrylic acid ester content of about 4.5–6.8% quaternary ammonium groups [8]. Ammonium groups are present in the form of salts and are responsible for the permeability of the polymer and for providing a positive surface to the polymer. This positive surface is important for the interaction with negatively charged drugs, cell surfaces, and target tissues, maximizing cellular uptake of the polymer-drug complex [6,8–10]. Although this polymer is not biodegradable, it has adequate biocompatibility for its role in increasing the interaction with the
Abbreviations: DMY, Dihydromyricetin; DMY-F, Free DMY; NC-E, Eudragit RS100® nanocapsules; NC-DMY, Nanocapsules containing DMY; FRAP, Ferric Reducing Antioxidant Power; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tretazolium bromide ⁎ Corresponding author at: Laboratory of Nanotechnology, Franciscan University, Rua dos Andradas 1614, Santa Maria, RS, 97010-032, Brazil. E-mail addresses: [email protected] (A.J.F. Dalcin), [email protected] (B.S. Vizzotto), [email protected] (G.V. Bochi), [email protected] (N.S. Guarda), [email protected] (K. Nascimento), [email protected] (M.R. Sagrillo), [email protected] (R.N. Moresco), [email protected] (A.P. Schuch), [email protected] (A.F. Ourique), [email protected] (P. Gomes). https://doi.org/10.1016/j.colsurfb.2018.10.066 Received 26 July 2018; Received in revised form 11 October 2018; Accepted 24 October 2018 Available online 27 October 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved.
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and conditions were the following: Shimadzu HPLC system (Kyoto, Japan) was used and equipped with an LC-20ATpump, an SPD-M20 A photodiode array (PDA) detector, a CBM-20 A system controller, a C18 Phenomenex (4 × 3.0 mm) pre-column and RP-C18 Phenomenex column (150 mm × 4.0 mm, 5 μm particle size, 100 Å pore diameter). The mobile phase was acetonitrile–water (20:80 v/v) at pH 4.0 (adjusted with acetic acid) at an isocratic flow rate (0.6 mL min−1). Each run lasted 10.0 min at room temperature, and the retention time was 5.6 min. The injection volume was 20 μL. Detection was conducted at 290 nm. The encapsulation efficiency was determined by methodology provided by Santos et al. [19], using ultrafiltration/centrifugation devices (Amicon®10 kDa, Millipore) at 7000 × g for 10 min, followed by determination by HPLC according to the method previously described by Dalcin et al. [17].
mucosa [3,6,11] Eudragit RS100® has been widely employed in the production of oral and topical formulations, and several authors report its good biocompatibility in different applications [7,12–22]. Its use has been accepted by regulatory agencies of the United States, Brazil, Europe, and Japan for oral and topical administration [6,22,23]. Eudragit RS100® can also be used in the development of thermo-sensitive drugs [24], and represents an adequate vehicle for drug dispersion [16]. Nanoencapsulation containing Eudragit RS100® has been reported as an alternative to increase the aqueous solubility of Dihydromyricetin (DMY) [17], a flavonoid extracted mainly from stems and leaves of Ampelopsis grosedentata, a vine from southern China [25]. DMY is known for its different pharmacological properties, including: antioxidant [26,27], anti-inflammatory [25,28], antitumor [25,29], photoprotective [30], depigmenting [31], antimicrobial [17,25,32], hypoglycemic [25,33], hepatoprotective, [25,34,35], neuroprotective [36] among others [25]. Although one of the major advantages of nanomedicine is to provide drugs targeted to diseased organs, the control and prediction of nanocapsules distribution in the human body requires further investigation [37,38]. The relationship between the structure of nanoparticles and behavior in biologically complex environments, such as the human body, is far from being completely understood, making it difficult to predict its biodistribution, as well as possible undesirable cellular interactions and adverse effects [39,40].Therefore, many studies have intended to evaluate and better understand the cytotoxic and genotoxic effects of nanoparticles in in vitro models [39,40]. In this study, we investigated if the nanoencapsulation of the DMY flavonoid produced with the Eudragit RS100® polymer was able to protect against the genotoxicity and cytotoxicity induced by cationic nanocapsules. Additionally, we evaluated the effects of the nanoencapsulation on the formation of the protein corona effect and antioxidant activity of DMY flavonoid. In this study, we evaluated the influence of the nanoencapsulation of the flavonoid DMY on the cytotoxic and genotoxic effects provided by cationic nanocapsules produced with the Eudragit RS100® polymer. Additionally, we evaluated the impact of the nanoencapsulation on the formation of the protein corona effect and antioxidant activity of DMY flavonoid.
2.2. Effect of the protein corona This experimental protocol was conducted in order to predict the possibility of the formation of the protein corona around the nanoparticles when in contact with biological fluids. RPMI (Embriolife, Brazil) and DMEM (Himedia, India) culture media were supplemented with 10% fetal bovine serum and added to the formulations of NC-DMY, NC-E, and free DMY (F-DMY, the non-nanoencapsulated form) at concentrations of 1 μM and 150 μM. Subsequently, they were evaluated for the characterization of particle size, polydispersity index, zeta potential, and pH. The formulations were characterized before contact with the culture medium (after the preparation of the nanocapsule suspensions), after initial contact with the medium (without incubation), 24, and 72 h after incubation at 37C in 5% CO2. The F-DMY samples were analyzed only for pH values. 2.3. Blood collection and cell culture Peripheral blood mononuclear cells (PBMC) were used to assess the cell viability by MTT and comet assays. The peripheral blood samples were obtained of healthy volunteers in Santa Maria, RS, Brazil. The study was approved by the Institucional Ethics Committee (Number: 654264). Samples were obtained by venipuncture using heparin-like Vacutainer tubes. The separation of PBMC by density gradient was done (Histopaque -1077) by centrifugation, and 2000 cells were plated per well (in 96-well plates) for each treatment. Both MTT and comet assay were conducted after treatment with NCE, NC-DMY, and F-DHM. The concentrations used were 1 μM, 10 μM, 50 μM, 100 μM, and 150 μM, and the incubation period with cells was 24 h. In addition, control samples used for the tests consisted of a negative control (C−) only containing culture medium and PBMC, and a positive control (C+) containing culture medium, PBMC and 100 mM hydrogen peroxide (H2O2).
2. Material and methods 2.1. Preparation and physicochemical characterization of nanoparticle suspensions Nanocapsule suspensions containing DMY (NC-DMY) were prepared by interfacial deposition of a pre-formed polymer, according to the method described previously [17]. Eudragit RS100® (Evonik, Germany) was solubilized in acetone (Synth, Brazil), DMY (Sigma-Aldrich, USA) and medium-chain triglycerides (Alpha Quimica, Brazil). Subsequently, this organic phase was added to an aqueous phase containing polysorbate 80 (Synth, Brazil). Magnetic stirring was maintained and the organic solvent was eliminated by evaporation under reduced pressure. Eudragit RS100® nanocapsule suspensions (NC-E) were similarly prepared, omitting the presence of DMY. After the preparation of the nanocapsule suspensions, the average diameter and polydispersity index (1:500 v/v in ultrapure water) were determined by dynamic light scattering (Zetasizer® Nano-ZS model ZEN 3600, Malvern Instruments, UK), and the zeta potential (500 v/v in NaCl 10 mM solution) by electrophoretic mobility (Zetasizer® Nano-ZS model ZEN 3600, Malvern Instruments, UK). In addition, the pH was measured directly from the formulations using a previously calibrated potentiometer (Digimed® DM – 20, Brazil). The DMY content was determined (n = 3) by High Conductance Liquid Chromatography (HPLC) using a method previously validated by validated by our research team25. The chromatographic instruments
2.3.1. Cell viability assay Cell viability was evaluated [44] using RPMI culture medium. In short: a solution of, the 3-(4,5-Dimethyl-2-thiazolyl)-2-5-diphenyl-2Htretazolium bromide (MTT) (Sigma-Aldrich, USA) was dissolved in phosphate buffer (pH 7.4), added to the cells, and incubated for 24 h at 37C in 5% CO2 protected from light. The analyses were conducted in triplicate in a spectrophotometer at 570 nm, and the results were expressed as percentage of viable cells in relation to control samples. Cytotoxicity was defined as [45]: Non-cytotoxic > 90% of cell viability; Slightly cytotoxic = 60–90% of cell viability; Moderately cytotoxic = 30–59% of cell viability; Severely cytotoxic ≤ 30%. 2.3.2. Comet assay The evaluation of genotoxicity was carried out by Comet assay following the protocol previously described [46]. After the incubation, on a glass slide covered previously with a 1.5% agarose layer, the PBMC 799
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were deposited and suspended in low-melting point agarose (Low Melting). The material was immersed in a lysis solution in order to remove the membranes and cytoplasms. The slides were incubated in an alkaline electrophoresis buffer and subjected to electrophoresis for 30 min at 25 V and 300 mA. In order to analyze the genetic material, neutralization, fixation, and coloring processes were carried out. Each sample was evaluated by optic microscopy, and the cells were classified according to the shape of the nuclei into four damage classes, varying from 0 (no damage) to 4 (maximum damage), including the classification of cellular apoptosis. The calculation of the damage index was conducted through the sum of the damages 1, 2, 3, 4 and apoptosis, divided by 100.
Table 1 Physicochemical characterization of nanoparticles suspensions. Parameter
NC-DMY
NC-E
Particle size (nm) Polydispersity index Zeta Potential (mV) pH Value Drug content (%) Encapsulation efficiency
146.9 ± 0.7 0.107 ± 0.01 +10.3 ± 0.7 4.0 ± 0.02 100.2 ± 0.5 80.88% ± 2.88
140.7 ± 0.4 0.097 ± 0.01 +10.2 ± 0.1 4.7 ± 0.12 – –
efficiency was about 80%, drug content was 100%, and pH was slightly acid due to the characteristics of the DMY.
2.3.3. DNA interaction The assay to evaluate DNA interaction was conducted according to the methodology previously described [47]. All samples (NC-DMY, NCE, and F-DMY) were tested at concentrations of 1 μM, 10 μM, 50 μM, 100 μM, 150 μM, 500 μM, 750 μM, and 1000 μM, and incubated overnight with 300 ng/μL plasmidial DNA pCMUT48 at room temperature, protected from light. After incubation, the samples were electrophoresed in 0.8% agarose gel in 0.5X TBE buffer. In order to determine the interaction of the formulations with the DNA and possible changes in the electrophoretic migration pattern, the samples were evaluated by densitometry analysis (Amersham Imager 600 GE®, United Kingdom).
3.2. Protein corona effect The results of the protein corona effect are presented in Fig. 1. In the initial contact of the nanocapsules with both media, there was a significant increase of the diameter for NC-DMY in the lowest concentration (1 μM). However, this increase in diameter was not found in the highest concentration evaluated (150 μM). For NC-E, a significant increase was only found in the RPMI culture medium, and in the lowest concentration (1 μM) after initial contact, as well as after 24 h of incubation with the medium. Further incubation times did not provide significant increase in particle size. The stability of the nanocapsules was also evaluated by monitoring the polydispersity changes of the formulations evaluated in the culture medium (Fig. 1B). After contact with the culture medium, the results demonstrated a significant increase in all incubation times both for RPMI and DMEM medium for all samples at the lowest concentration of 1 μM, for both NC-DMY and NC-E. However, no significant differences were demonstrated for the highest concentration of 150 μM. Fig. 1C shows that after contact with the culture medium, all samples presented a change in charge from the positive zeta potential into negative, regardless of time and culture medium evaluated. The initial pH values for the NC-DMY, NC-E, and F-DMY formulations were 4.03 ± 0.02, 4.70 ± 0.02, and 4.45 ± 0.02, respectively (data not shown). The culture media have pHs of 7.42 ± 0.02, and 7.82 ± 0.02 for RPMI and DMEM, respectively. After contact with the media it was possible to verify an increase in the pH values for all samples, independent of the medium and incubation time. The pH values increased and were similar to the pH values of the individualized medium.
2.4. Determination of the antioxidant capacity For the determination of the antioxidant capacity of the formulations, the Ferric Reducing Antioxidant Power (FRAP) assay was used according to the methodology previously described [49], adapted to the automated system BS 380 (Mindray®, China). In short, NC-DMY, NC-E, and F-DMY were evaluated at different concentrations (1, 10, 50, 100 and 150 μM), diluted in distilled water. The FRAP reagent was prepared from the combination of acetate buffer with 2,4,6-Tris(2-Pyridyl)-striazine (TPTZ) (Sigma-Aldrich, USA) solution and an aqueous solution of ferric chloride (Sigma-Aldrich, USA). At acid pH, the Fe3+ complex is reduced to Fe2+ and forms an intense blue solution. The FRAP solution was used as the reference reagent, and the absorbance was measured at 605 nm. For the calibration curve, ferrous sulphate was used as standard at concentrations ranging from 0 to 1000 μmol/L. Distilled water was used as the negative control. The assay was conducted in triplicate (n = 3), and the results were expressed in μmol/L. 2.5. Statistical analysis
3.3. Cell viability assay The results of the characterization were expressed as a mean ± standard deviation. For the analysis of the protein corona effect, evaluation of cytotoxicity, genotoxicity, and determination of antioxidant capacity, data were expressed as mean ± standard deviation and analyzed by One-Way ANOVA, followed by the Dunett´s test. Values with p ≤ 0.001 were considered statistically significant. For determination of the antioxidant capacity, the t-test was conducted to compare NC-DMY and F-DMY. Values with p ≤ 0.05 were considered statistically significant. All graphs and statistical analyzes were done on GraphPad Prism® software version 4.0 (GraphPad, USA). 3. Results
The MTT cell viability assay demonstrated that the different concentrations of NC-DMY did not show significant difference as compared to the negative control (Fig. 2). This indicates that the nanocapsules containing the DMY drug were not cytotoxic. NC-E showed a significant decrease in viability for the highest concentrations of 100 and 150 μM. NC-E at the concentration of 100 μM demonstrated mild cytotoxicity (62%), and at the concentration of 150 μM (59%), moderate cytotoxicity. F-DMY showed no reduction in cell viability at the concentrations evaluated; however, there was increased cell viability at the highest concentrations (50, 100 and 150 μM).
3.1. Physicochemical characterization of nanocapsule suspensions
3.4. Comet assay
The results of the physicochemical characterization of NC-DMY and NC-E are described in Table 1. The mean particle diameters were around 140 nm for both formulations. The samples presented low polydispersity index, which demonstrates good homogeneity of the systems, as well as positive zeta potential around + 10 mV due to the presence of the Eudragit RS 100® cationic polymer. The encapsulation
The levels of DNA damage induced by the formulations are depicted in Fig. 3. NC-DMY and F-DMY did not show significant difference as compared to the negative control. This indicates that there was no damage to the cellular DNA and, according to this test, that the formulations of NC-DMY and F-DMY were not genotoxic. For the NC-E, concentrations of 50, 100 and 150 μM caused 800
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Fig. 1. Results of protein corona effect for the formulations NC-DMY and NC-E characterized in particle size (A), polydispersity index (B), zeta potential (C), before and after contact with RPMI and DMEM culture medium in the different incubation periods. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by variance One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
Surprisingly, the results found for NC-DMY and NC-E indicate a high interaction with DNA in concentrations higher than 10 μM (Fig. 4B and C), demonstrating the occurrence of DNA sequestration by these formulations at the concentrations of 50, 100, 150, 500, 750, and 1000 μM (DNA retained on the upper part of the gel).
significant DNA damage to the cells in a concentration-dependent manner. This result is consonant to the MTT viability test, where for the same concentrations there was a decrease in cell viability, indicating a possible toxic effect of the NC-E in the highest concentrations.
3.4.1. Interaction with DNA We evaluated 8 concentrations, 3 of them higher than those evaluated in previous tests, in order to verify its interaction with DNA. The analysis of a plasmid DNA sample interaction with NC-DMY, NC-E and F-DMY can be seen in Fig. 4, and these results indicate that F-DMY does not interact with DNA.
3.5. Determination of the antioxidant capacity The antioxidant capacity was evaluated for all formulations at the same concentrations used for the assessment of cytotoxicity and genotoxicity (Fig. 5). 801
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Fig. 2. Cell viability assay by MTT with 24 h incubation. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.
(4 ± 2C) [17]. Our group also verified the efficacy of the anti-biofilm effect of dihydromyricetin-loaded nanocapsules on urinary catheter infected by Pseudomonas aeruginosa. NC-DMY was more effective when compared with free DMY, demonstrating that NC-DMY can potentially be used as an innovative approach to urinary catheter biofilm treatment or prevention. [17] However, few in vitro and in vivo studies are devoted to the toxicity of nanoparticulate systems, in particular to the nanocapsules produced with the Eudragit RS100® polymer. In addition, most of these studies used only one assay (usually cell viability by MTT) to assess the toxicity of nanoparticles. Regarding the results of the protein corona assay, the initial increase in diameter can be explained by the fact that the analysis was conducted after the nanocapsules were in immediate contact with the medium, what may not have granted sufficient time for the nanoparticles to stabilize in the solutions containing the culture medium. The significant increase in particle diameter after the initial period was verified only at the 24-hour incubation for NC-E at the concentration of 1 μM for the RPMI medium. The other concentrations of NC-DMY or NC-E did not demonstrate increase in the mean diameter of the particles, regardless of the incubation time and culture medium evaluated. Similar results were found [49] who evaluated the diameter of white nanocapsules produced with Eudragit RL100® and PMMA (poly-methylmethacrylate), and verified that there was no increase in particle diameter after contact with the RPMI medium. The increased polydispersity probably occurred due to the heterogeneity of the media, and to the fact that the graphical profile of the size distribution of the culture media alone shows more than one peak,
As expected, the F-DMY formulations demonstrated high dose-dependent antioxidant capacity, and these results corroborate previous studies demonstrating the antioxidant activity of F-DMY [31,34,65–68]. NC-E did not demonstrate significant antioxidant capacity when compared to the drug in the free or nanostructured form. The values found ranged from 13 to 390 μM/L FRAP, demonstrating that the constituents of the formulation and the structure of the nanocapsule do not have antioxidant potential. As verified for F-DMY, NC-DMY demonstrated an important antioxidant capacity and in a dose-dependent manner, reaching values higher than 5000 FRAP μmol/L. Results from the FRAP assay demonstrated that DMY inserted into polymeric nanocapsules maintained its antioxidant capacity when compared to F-DMY. 4. Discussion The present study reported at first time that the nanoencapsulation of DMY was able to protect the cytotoxicity and genotoxicity induced by cationic nanocapsules produced with the cationic polymer Eudragit RS100® in PBMCs. The results of the physicochemical characterization of the nanocapsule suspensions corroborate with results previously found by us [17], as well as by other authors who used the same nanostructure with other drugs [6,7,18–20,50–52]. Studies of the stability of NC-DMY and NC-E for 90 days under different storage conditions (refrigeration, room temperature and climatic chamber) were previously conducted and demonstrated that the best condition for suspension storage without losing physical and chemical characteristics was under refrigeration
Fig. 3. Comet assay with 24 h of incubation. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗p < 0.05, ∗∗p < 0.01, and ∗∗∗ p < 0.001. 802
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Fig. 4. Results of interaction with DNA in electrophoresis in agarose gel 0.8% for F-DHM (A), NC-DMY (B) and NC-E (C).
dissociation, as well as changes in the environment, and may subsequently change their behavior. [54] The pH change of the formulations upon contact with the medium allows us to ensure that even being acidic, when in contact with the biological medium they did not cause imbalance in pH values. This can be explained by the characteristic of the constituents of the culture media, which have buffering systems in their composition in order to maintain pH regulation and to establish adequate culture conditions. [55] The results of the corona protein effect show that the nanocapsules were stable when in contact with the culture media. In addition, it was possible to evaluate that there was no particle agglomeration by the average particle diameter measurement when in contact with culture media; or if the formation of the protein corona occurred, this was not sufficient to provide an increase in particle size. This indicates the absence of protein corona formation for the nanoparticles evaluated in the RPMI and DMEM culture media. The results of the cell viability assay by MTT for F-DMY demonstrated no reduction in viability and corroborate with the findings [56,57] who evaluated it in other cell lines, such as human liver cancer cells (HepG2), and human umbilical vein endothelial cells (HUVECs), respectively. However, our results demonstrated increased F-DMY viability (above the concentration of 10μM), suggesting a proliferative effect. Ferreira et al. [58], attributed this effect to the stimulation of the immune system by the flavonoid, which has antioxidant activity and can provide immunostimulatory effects modulating gene expression, which may be related with increased cell metabolism.59 It is important to mention that NC-DMY showed no changes in cell viability. Therefore, the results of the present study demonstrated that NC-DMY and F-DMY did not induce cytotoxicity. However, for NC-E, a decrease in cell viability was noted in a dose-dependent manner with moderate
consequent to the various nutrients contained in the culture media. In addition, this changed the profiles of particle size distribution of the formulations from unimodal to bimodal or multimodal (supplementary information - S1) when inserted into the culture media. Increases in the polydispersity index values were also found [7] when evaluating nanocapsules containing carvedilol in the porcine sublingual mucosal medium. This increase in polydispersity values indicated an increase in heterogeneity of the size distribution [7]. It is important to consider that the culture media consist of various nutrients which provide diversity in sample composition and results in increased heterogeneity and consequently, increase in the polydispersity index. The results found suggested that the most affected physicochemical parameter by the interaction of nanocapsules with the components of the cellular medium was the charge. As of today, there are no reports in the literature evaluating the stability of nanocapsules containing Eudragit RS 100® in contact with culture media RPMI and DMEM. Nevertheless, researchers [7] noted a similar behavior in porcine sublingual mucosal medium, as well as verified this charge changes for their formulations. The results were attributed to the high capacity of the nanoencapsulated drug to interact with mucin molecules when compared to the drug in the free form, suggesting that the Eudragit RS100® polymer can interact with negative mucin molecules by electrostatic attraction. We believe that the same effect found can be justified in our experiment. Graça et al., [53] also noted changes in positive to negative charge when evaluating nanocapsules produced with Eudragit RS100® and poly methyl-methacrylate (PMMA) on immediate dilution in the RPMI medium. This was justified as a probable consequence of the dynamic formation of a protein corona, which may be coated by biomolecules present in the biological environment that bind to the surface of nanocapsules. In contrast, the protein corona composition varies over time due to continuous protein association and
Fig. 5. Result of antioxidant capacity by FRAP. *Data are expressed as mean ± standard deviation (SD). Analyses were conducted by One-Way ANOVA, followed by the Dunnett’s test. Values with p < 0.05 were considered statistically significant.∗p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. 803
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[25–27], and the reason for this antioxidant activity is the chemical structure of flavonoid compounds, which can scavenge free radicals, chelate metals, and donate electrons and hydrogen atoms. Its chemical structure consists of 15 carbons arranged in a configuration of 6C-3C6C, having 6 hydroxyl groups, and the ortho-trihydroxy group (ring B) in DMY mainly conducts antioxidant activity [64]. NC-DMY maintained its antioxidant capacity when compared to FDMY. Therefore, we believe that the incorporation of DMY into a nanostructured carrier did not interfere in its antioxidant capacity. In addition, it can be noticed that NC-DMY suggest potentiation in the antioxidant effect when compared to F-DMY (p = 0.0158 for the concentration of 150 μM, and p = 0.0052 for the concentration of 100 μM). It is important to mention that the encapsulation efficiency values for NC-DMY were 80%. That is, 80% of the DMY is inside the nanocapsule, what suggests that the remaining 20% are adhered to the polymeric membrane. We may therefore imply that nanoencapsulated DMY in addition to providing the effect of the drug sustained release [17], may provide the effect of protection against the toxicity generated by NC-E through an antioxidant effect generated by DMY adhered to the surface of the nanostructure that acts against the generation of free radicals. We suggest that DMY also can be added to nanocapsules of Eudragit RS100® as an ingredient of the nanocapsule to prevent the cytotoxicity induced by Eudragit in order to prepare other drug formulations and also execute its antioxidant effect.
cytotoxicity at the highest concentration (59%), demonstrating the cytotoxic potential of the cationic nanocapsules produced with the polymer Eudragit RS100® when evaluated in the absence of the drug. This same effect was also found by Eidi et al., [10] where nanocapsules produced with Eudragit RS100® and without the drug showed concentration-dependent cytotoxicity and increased toxicity when compared to nanocapsules containing the drug in macrophage cells (NR8383). Gargouri et al., [47] also reported that nanocapsules without the DNA were more cytotoxic than nanocapsules containing DNA in pharyngeal cancer cells (FaDu) and in human breast adenocarcinoma cells (MDA-MB 231 and MCF-7). Contri et al. [52], evaluated the viability of human fibroblasts and keratinocytes against white nanocapsules produced with Eudragit RS100®, and reported a decrease in cell viability in a concentration-dependent manner of treatment, although this cytotoxicity was not significant. In contrast, the mild toxicity noted supports the hypothesis of a nonspecific cell response to stress associated to the exposure to nanocapsules at very high concentrations [60]. The results of the comet assay for NC-E were different from those found in the literature [10,52,53]. However, these authors used other cell lines, other concentrations, and other methodologies for the assessment of genotoxicity. We chose the PBMC cell line because it is the most sensitive human cell line to evaluate the toxicity of various chemical compounds, and has been applied for decades as cytotoxic and genotoxic biomarkers because it is abundant in the blood circulation. In addition, when exposed to any mutagenic agent they are able to reflect recent damage [57]. However, in assay conducted by Eidi et al. [10], on cellular apoptosis, the conclusion was that Eudragit RS100® nanocapsules can induce cell death, although the type of cell death (necrosis or apoptosis) involved should be better investigated. It should also be investigated whether cell death induced by nanocapsules is consequent to their internalization by cells or their interaction with cell membranes. Nevertheless, it is important to mention that Louro et al. [54] report that such primary lesions usually arise soon after exposure to genotoxic agents and may be easily repaired by the DNA repair machinery. As for the DNA interaction assays, we verified the disappearance of the bands for NC-E and NC-DMY in the highest concentrations. This same behavior was observed by Du et al. [62] where bovine serum albumin was synthesized with the cationic polymers tetraethylenepentamine (TEPA) and polyethyleneimine (PEI) and used as a potential gene transporter. The results demonstrated that the DNA bands did not appear on the gel in the highest concentrations tested, which meant that the formulation evaluated had the capacity to sequestrate DNA due to the agitation effect of the α-helical structure and to the positive potential of the complexes. Furthermore, the complexes demonstrated high transfection efficiency of DNA, proving to be a potential gene transporter. Lu and Liu [63] also verified this gel retargeting effect by cationic nanocapsules produced with PEG-PCL-PEI. The authors justify that this binding affinity of copolymers with the DNA occurs consequent to the electrostatic interaction between negatively charged phosphates along nucleic acid and cationic copolymers segments. Therefore, we believe that this same behavior described [64] occurred in our experiment. The disappearance of the DNA bands may indicate the use of NC-DMY and NC-E formulations as potential gene carriers. We strongly suggest, therefore, that this same effect of sequestration/retardation for NC-DMY and NC-E can be justified by the electrostatic interaction of the DNA with the quaternary ammonium groups present in the Eudragit RS100® polymer. It is important to point out that the highest concentrations of NC-E induced a decrease in cell viability and increased DNA damage. Furthermore, no cytogenotoxicity was demonstrated for the NC-DMY, since it did not decrease cell viability, as well as did not induce DNA damage. Therefore, our results demonstrate the preliminary safety of these nanoparticles. DMY, as well as most flavonoids, possess antioxidant properties
5. Conclusions For the first time, different in vitro biological tests were conducted in order to evaluate the cytotoxicity and genotoxicity of cationic nanocapsules produced with Eudragit RS100® and containing DMY and NC-E in peripheral blood mononuclear cells, as well as their interaction with the DNA molecule. Our work is the first to show that the nanoencapsulation of the flavonoid Dihydromyricetin (DMY) may decrease the cytotoxicity provided by the cationic polymer Eudragit RS100® due to its potential antioxidant effect. The results of the protein corona demonstrated that there was no increase in particle diameter, suggesting absence of protein corona, or if it occurred, no interference in the mean particle diameter. NC-E at the highest concentrations demonstrated moderate cytotoxicity and increased DNA damage as compared to control, suggesting toxic potential. The results demonstrate that DMY, when nanoencapsulated, maintained its antioxidant capacity and prevented the cytogenotoxic effect of the cationic polymer Eudragit RS100®, reinforcing that its use can be better exploited by nanomedicine for different biological applications. Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. The authors also would like to thank Evonik for supplied Eudragit RS100®. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.10.066. References [1] A.D. Mali, R.S. Bathe, Updated review on nanoparticles as drug delivery systems, Int. J. Anal. Pharm. Biomed. Sci. 8 (9) (2015) 18–34. [2] C.E. Mora-huertas, H. Fessi, A. Elaissari, Polymer-based nanocapsules for drug delivery, Int. J. Pharm. 385 (2010) 113–142. [3] L.A. Frank, R.V. Contri, R.C.R. Beck, A.R. Pohlmann, S.S. Guterres, Improving drug biological effects by encapsulation into polymeric nanocapsules, WIREs Nanomed. Nanobiotechnol. 7 (2015) 623–639. [4] L. Bregoli, D. Movia, J.D. Gavigan-imedio, J. Lysaght, J. Reynolds, A. Prina-mello, Nanomedicine applied to translational oncology: a future perspective on cancer
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