Physico-chemical Characterization and Biosafety Evaluation of Atorvastatin Nanocapsules Co-encapsulated with Ginger Oil or Grape Seed Oil

Cardiovascular diseases are one of the major causes of deaths around the world. The leading cause is related to high cholesterol levels; therefore, controlling these levels has been a successful strategy. Among the drugs used for this purpose, atorvastatin (ATV) has great efficacy; however, some side effects reduce patient adhesion. In this context, the development of ATV polymeric nanocapsules co-encapsulated with ginger (NCAtG) or grape seed (NCAtU) oil can reduce ATV toxicity and increase its efficacy. The objectives of this work were to evaluate the safety and efficacy of these nanoformulations in different experimental models. The formulations had nanometric sizes and suitable physio-chemical parameters. The biosafety was evaluated in terms of hemoglobin measurement, liberation of erythrocyte LDH, and blood coagulation cascade by the extrinsic (PTT) and intrinsic (aPTT) pathways after exposed to the nanoformulations were just high concentrations caused alterations in these parameters. Also, there is no cytotoxicity in the 3T3 cell lines and no alterations in the comet assay. The in vivo assays in Caenorhabditis elegans showed no alterations, in the survival, brood size, and length. And finally, the formulations demonstrated significative effect about the reduction of the lipidic levels of the nematodes, with high lipid levels.


Introduction
Atorvastatin (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl 4(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid) (ATV) is a drug widely employed in the treatment of hypercholesterolemia and atherosclerosis.Its application is based on its ability to inhibit the enzyme HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase), converting the subtract HMG-CoA in mevalonic acid, as consequence reducing the hepatic production of cholesterol [1].Despite being a safe drug, in some cases, ATV may cause muscular myalgia and alter glucose homeostasis, thus leading to diabetes risk [2,3].These factors have been associated as reasons for reduced treatment adherence in patients [4,5].Another alternative therapeutic method to hypercholesterolemia treatment is the use of natural compounds [6,7].Vegetal oils extracted from ginger and grape seed are used due to their pharmacological properties in reducing lipid levels.Some authors suggest that the efficacy of these oils is based on the high levels of phenolic compounds with antioxidant properties [8][9][10][11][12].
To reduce side effects and increase the drug efficacy, the development of formulations based in nanotechnology in the pharmaceutical area has been an important approach for drugs available in the industry.It occurs because the nanometric scale formulations improve pharmacological effects of the active constituents in comparison to their natural structure in a normal scale [13,14].However, due its smaller size (10 to 1000 nm) and the possibility of environmental contamination or human risk to health, the safety of nanomaterials or new nanoformulations needs to be investigated in different organisms [15][16][17].In the last years, the methods for evaluating the safety and efficacy of these new technologies have been improved in order to reduce the use of vertebrates such as rodents, particularly due to ethical concerns [18].
Several studies involving safety assessment of nanoparticles are performed in vitro [19][20][21][22] and in vivo [23][24][25][26] using alternative animal models, such as the invertebrate Caenorhabditis elegans.However, to the best of our knowledge, studies evaluating statin nanostructures are just a few [27][28][29].In relation to ATV nanoformulations, there are studies using chitosan as polymer and lipid nanocapsules to load the drug, but as far we know, this is the only work with ATV-PCL nanoparticles.However, it is known that mouse chitosan decreases the mineral absorption and bone mineral content in rodents, in addition to reprotoxicity effects demonstrated in Danio rerio [30].In addition, the pharmacological effects of the lipid nanoformulation in decreasing the lipid levels were not demonstrated [31].
Taking into consideration, the need of studies involving the biosafety and efficacy evaluation of nanoparticles containing statins and the use of ingredients with biological potential, we have developed polymeric nanocapsules loaded with ATV and ginger oil or grape seed oil.The nanocapsule demonstrated advantages of in vitro and in vivo assays to assess toxicity and lipid reducing activity.Our hypothesis was that the ATV nanoencapsulation associated with ginger or grape seed oils would be biocompatible, had a controlled release, safe to living cells and organisms, besides do not altered ATV efficacy against a high cholesterol diet in C. elegans.

Preparation of Atorvastatin Nanocapsules
The nanocapsules loaded with atorvastatin and vegetal oils (NCAtG and NCAtU) were prepared using the pre-formed polymer precipitation method [15].The technique consists, in the preparation of two phases, an organic and an aqueous.After the compound's solubilization, the organic phase was poured on the aqueous phase with the aid of a magnetic stirrer through a narrow funnel.After that, using a rotary evaporator, the organic solvent and aqueous part were evaporated to a final volume of 10 mL until to a concentration of 1000 µg/mL.Formulations without the ATV were prepared and named NCGe and NCUv.Also, an empty nanosphere just with the polymer poli-epsilon caprolactone (PCL) (NEPCL) was made to evaluate the polymer toxicity (Table 1).

Particle Size Measurements
The nanoparticles were evaluated by particle size, polydispersity (SPAN) (Eq. 1) and polydispersity index (PDI) by laser diffractometry (LD) (Mastersizer 2000, Malvern®) and photon correlation spectroscopy (PCS) techniques (Nano-Brook® 90Plus Zeta-Brookhaven Instruments) to provide a confirmation of the size.According Mora-Huertas et al. (2010), the LD measures the diffusion coefficient of particles dispersed in suspension being more accurate for microparticles, and the PCS measures the speed of random movement of particles (Brownian) being more specific to nanoparticles.Equation 1 Determination of SPAN, where the values 10, 50, and 90% of the distribution (d10%, d50%, and d90%, respectively) indicate the percentage of particles with a diameter equal to or less than the determined value.

Zeta Potential Determination
The surface charge of the nanoparticles (zeta potential) was measured in the equipment NanoBrook® 90Plus Zeta-Brookhaven Instruments.This analysis determines the electrical potential at the particle boundary (called the shear plane).Through this assay, the stability of the formulation can be measured and predict instabilities of then.This determination was carried out after 200 times (v / v) dilution of the nanocapsule suspensions in 1 mmol L −1 NaCl solution.

pH Determination
The pH of the formulation was measured by a previously calibrated Hanna® potentiometer.

Content of Formulations
The content of NCAtG and NCAtU was realized with formulations made within 48 h.Therefore, the formulations were previously treated with acetonitrile, which promoted the polymer dissolution and subsequent release of the active component to outside of the nanostructure.The determination of ATV in the formulations was performed using HPLC as methodology previously described [32].

Encapsulation Rate
The encapsulation rate for NCAtG and NCAtU was made by the ultrafiltration-centrifugation technique.Both nanocapsules have a theoretical concentration of 10 mg/mL.To execute, the technique was pipetted 400 µL of NCAtG or NCAtU in Eppendorf Ultrafree® microtubes.After 5 min of centrifugation at 5000 rpm, the volume passed through the filter was collected and analyzed using HPLC [33].The percentage of encapsulated ATV was calculated through the difference between the total concentration in the formulations and the free ATV content present in the filtered, divided by the total concentration multiplied by 100 (Eq.2).
Equation 2 Measurement of the encapsulation efficiency of NCAtG and NCAtU, where CT is the total drug concentration in the sample and CL is the free drug concentration.

Parameters for the Study of In Vitro Release
To the diffusive process be guaranteed throughout the release study, it must be conducted in a sink condition.In this experiment, the medium used for the dissolution of atorvastatin was composed of distilled water + Tween 20 (10%) + HCL (1%).The choice of a medium with a more acidic pH is justified by the better solubilization of the drug, since atorvastatin has an acid-base dissociation constant (pKa) of 4.33.The dialysis bags were composed of cellulose acetate membranes (12,000-14,000 Da) (Sigma Aldrich®).Previously, the bags were hydrated for 12 h and then filled with 3 mL of the free ATV solution and still with the NCAtG or NCAtU formulations.We used 150 mL of release medium in each test, under continuous agitation at 37 °C.

In Vitro Release Test of Formulations
In this assay, aliquots of 1 mL of the liberation medium were collected in times of 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, and 8 h.The medium was replenished in all collections.Subsequently to the final experiment time, the samples were filtered (0.45 µm) (Millipore®), and ATV content released in the medium was measured through methodology previously validated using HPLC.The experiment was carried out in triplicate.

In Vitro Nanocapsule Biocompatibility Assessment
All trials were carried out with prior approval by the Research Ethics Committee (CEP) of UNIPAMPA under N. 13454119.1.0000.5323.

Evaluation of the Incidence of Hemolysis
The hemolysis test was performed in order to evaluate if the NCAtG or NCAtU was able to cause blood incompatibility, as the formation of clots and thrombosis caused by the aggregation of proteins present in the human blood, for example [19,22].To these assay samples of total human blood, collected with sodium citrate were incorporated together with each ATV nanoformulation in concentrations of 10, 50, and 100 µg/mL at 37 °C.The commercial control of hemoglobin and dosages with PBS (pH 7.2) were used as control and negative control.The samples were then incubated for 2 h with mild magnetic stirring.Posteriorly, a microplate reader (Spectramax M5®) was used to determine absorbances in 540 nm.All analyzes were performed in triplicate.

Erythrocyte Membrane Integrity Evaluation
The erythrocyte membrane integrity was realized by collecting samples of total human blood with sodium citrate and measuring the increase in the liberation of the enzyme lactate dehydrogenase (LDH) erythrocyte.This enzyme was quantified through a colorimetric reaction in a microplate reader (Spectramax M5®) at 340 nm.In this case, we use PBS (pH 7.2) as negative control and Triton X100 (1%) as a 100% LDH release control (positive control).The formulations NCAtG and NCAtU were evaluated in concentrations of 10, 50, and 100 µg/mL in the whole blood samples.The results were determined by the difference between the positive control (100% hemolysis) and the formulations added to blood samples.

Blood Clotting Assessment
The activated partial thromboplastin time (aPTT) and prothrombin time (PTT) tests were performed in tubes containing sodium citrate as an anticoagulant.Whole blood samples previously incubated with NCAtG and NCAtU in concentrations of 10, 50, and 100 µg/mL for 30 min at 37 °C were gently shaken for analysis.Samples of citrated whole blood containing an equal amount of PBS (pH 7.2) were incubated and used as sample blank.Clot formation times were measured using a digital coagulometer (Humaclot Jr, In Vitro Diagnóstica, Brazil).

Comet Test
For the comet test, the cells were seeded in 12-well plates (Nest Biotech Co., Ltd., China) at a density of 500,000 cells per well.After 24 h, the medium, consisting of 1% fetal bovine serum, was aspirated, and the cells were incubated with nanocapsules and the free ATV solution at 10, 50, and 100 µM at 37 °C.As a positive control, 1% DMSO (dimethyl sulfoxide) was used.And as a negative control, the cell culture medium was used.After 24 h of incubation time, cells were harvested by trypsinization (0.05% trypsin/EDTA).The cell suspensions were centrifuged (400 × g, 5 min, 4 °C), and the cell pellets obtained were resuspended in low melting point agarose (0.75%, 150 µL) (Sigma-Aldrich (St.Louis, USA), and 60 µL aliquots were distributed in two slides coated with 1% normal melting point agarose.The samples were incubated in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, distilled water, 10% DMSO and 1% Triton X100) at 4 °C for 24 h in the dark; the slides were then incubated with electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13) for 20 min at 4 °C before electrophoresis, which was performed for 20 min at 25 V and 300 mA.After that, the slides were neutralized with 0.4 M Tris-HCL for 15 min in the dark; DNA was fixed by immersing the slides in 70% ethanol for 15 and allowed to dry at rest during the night.For microscopic analysis, the dried slides were stained with gel red dye (20 µg/mL), and DNA migration was observed in at least 100 cells at × 400 magnification using a fluorescence microscope (Olympus, Japan) equipped with a 510-550 nm excitation filter attached to a camera.The images were evaluated using the Comet Score™ software obtained from the public domain (http:// www.trite kcorp.com/ produ cts_ comet score.php).The percentage of DNA in the tail of the comet (% of DNA in the tail) was the parameter evaluated to describe the formation of comets.

Cytotoxicity Assay
Simultaneously with the comet assay, a number of samples identical to that of the comet were prepared, lysed, and immediately fixed and stained without electrophoresis to assess cytotoxicity using the low molecular weight DNA diffusion assay.

Caenorhabditis Elegans as an In Vivo Model to Safety and Efficacy Evaluation
Strain Maintenance For all tests, we have used C. elegans N2 Bristol (wild-type) strain.The worms were obtained from the Caenorhabditis Genetics Center (CGC), Minnesota University-USA.Worms were maintained in nematode growth medium (NGM) and fed with Escherichia Coli OP50 (E. coli OP50) at 21 °C in a humidity-controlled condition.Synchronized populations at the first larval (L1) stage were obtained by embryos isolated from pregnant hermaphrodites, using lysis solution (1% NaOCl; 0.25 M NaOH) [34].

C. Elegans Exposure to Nanoparticles and Mortality Assessment
A survival curve at the concentrations of 10-100 µg/mL of the four formulations (NCAtG, NCGe, NCAtU, and NCUv) and free ATV was made in order to obtain a suitable safe concentration range for worms, without toxicity.Therefore, the safety concentration range chosen to assays was between 10 and 30 µg/mL.Worms in the L1 stage were exposed to chronic treatment for 30 min in liquid medium under constant agitation.Samples were then centrifuged at 7000 rpm for 3 min.Posteriorly, the treated worms were placed on the plates seeded with the E. coli, inactivated by UV light (this procedure was made in the length/area and brood size assay too).After 24 h, the surviving animals were counted, and the percentage in relation to the untreated control group was used to determine the survival rate.Nanospheres containing PCL polymer were also developed and concomitantly evaluated to assess if the polymer could cause toxicity to worms, as previously reported [23,35].The number of live animals per treatment was normalized to percentage of control.
Brood Size Worms exposed to nanoparticles (NCAtG, NCGe, NCAtU, and NCUv) and free ATV were evaluated throughout their reproductive period (3 days).To that end, one worm at L4 stage was transferred to a new NGM/E.coli plate and allowed to lay eggs.The number of larvae was counted daily, and the P0 worm was transferred to a new plate.Assays were performed in triplicates in three independent experiments.The number of live animals per treatment was normalized to percentage of control.

Determination of the Body Size and Area
The body size and area of the treated worms were measured 24 h after treatment.Images were obtained in an EVOS® Floid® Cell Imaging Station microscope.The size of 5 worms per treatment was measured using the software Image J®, and the average was used for statistical analysis.Three independent experiments were performed.The number of live animals per treatment was normalized to percentage of control.

Exposure Protocol to Induce High Triglyceride Levels
In order to induce increases in triacylglyceride (TAG) levels in the nematodes and to evaluate the effectiveness of the nanoparticles in reducing their levels, a protocol adapted from Purwakusumah et al. (2017) was performed [36].For this, the NGM medium was enriched with 10 times more cholesterol than recommended.After synchronization, eggs were placed in this cholesterol-enriched medium or in regular NGM (as control).When the worms reached the L4 larval stage, they were transferred to plates with normal NGM medium seeded with E. coli OP50 (previously inactivated by UV light), treated with the nanoparticles or ATV for 24 h.Posteriorly, the worms were collected from plates and transferred to microtubes, washed twice with M9 medium to remove excess bacteria.Finally, we added lysis solution (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA), and the worms were frozen and thawed three times to break the cuticles of the nematodes with the aid of an ultrasonicator (3 cycles of 10 s/amplitude 60).The samples were centrifuged at 10,000 rpm for 3 min at 4 °C; the supernatant was transferred to 96-well microplates, and colorimetric reaction was made using a TAG assay kit (LABTEST®).Readings from the curve and samples were obtained in Spectramax M5® at 500 nm.Protein determination was used to normalize the data and followed the Bradford method, using albumin as a standard.

Statistical Analysis
All assays were performed at least three individual times, and GraphPad Prism 8 software was used to generate charts and statistical analysis.One-way ANOVA was used, and a p < 0.05 was considered statistically significant.Post hoc tests were performed using Tukey's post hoc test.The values expressed in percentage (%) were normalized considering a value of 100% for the day control.In all figures, bars represent mean ± the standard error of the mean.

Average Particle Size and Polydispersity Demonstrated Variability According to the Active Compound Present in the Formulations
The results of the average particle size and polydispersity are shown in Table 2.We observed that the average particle sizes (D 0.5) for the formulations containing ginger NCAtG and NCGe varied between 300 and 364 nm.However, when the grape seed oil in the core of the capsule was used, the formulations NCAtU and NCUv demonstrated smaller particle sizes between 232 and 280 nm.The result is expected because the oils have different constituents, and it may cause variations in the particle sizes [37].NEPCL analysis revealed similar sizes to the nanocapsules.The variation in the sizes of the empty formulations between the ones containing ATV can be associated by the presence of the drug in particles, and it may increase the average size [16].According to Table 2, we also observe that the SPAN and PDI of the formulations are suitable as describe in the study of Schaffazick et al. (2003) [38].

Zeta Potential Demonstrated Suitable Values
The analysis of the distribution of electrical charges around the surface of the nanoparticles, called the Zeta Potential, is an important parameter for investigating the morphological and stability aspects of the particles [16].As we can see in Table 2, the zeta potential of all nanoformulations remained adequate [39,40].The presence of negative charges was also expected due to the presence of a more electronegative polymer, as well as the structural formula of ATV.The presence of little variation between formulations containing ATV may be related to the fact that different oils promote different superficial charges [41].

The Nanoformulations Reduce the Acidity ATV
pH measurements are important when evaluating possible routes of administration of formulations.In addition, sudden changes in pH can be related to system instability and also provide information about their compositions.In this study, the pH results for NCAtG and NCAtU were 5.79 ± 0.4 and 5.60 ± 0.3, respectively.These values indicating formulations with an acid character are usual in other studies that used PCL as a coating polymer [42,43].In addition, both ginger oil and grape seed oil have phenolic compounds in their compositions and hydroxyls that can be ionized in their chemical structures [44].Another factor to consider is the characteristic of greater acidity related to ATV that has hydroxyl and carbonyl groups.This becomes more evident when dealing with formulations without ATV.In this case, NCGe and NCUv showed a slightly higher pH, being 6.4 ± 0.4 and 6.3 ± 0.2, respectively.

The Mostly Content of ATV is Encapsulated in the Nanostructure
The dosing of nanoformulations demonstrated suitable according to other authors [45,46].In Table 3, we observe that the dosing values of the formulations are 90.29 ± 1.22 to NCAtG and 90.68 ± 1.34 to NCAtU.The values show that the nanometric size did not affect the content of the formulations and then are suitable for the use in biological tests.
The rate of encapsulation of the nanoformulations is adequate with 98.24 ± 0.96 for NCAtG and 97.37 ± 1.5 for NCAtU showing that the drug is mostly in the nanostructure [38].

In Vitro Assays Release Demonstrated that ATV Nanoformulations Have a Controlled Release
The in vitro dissolution/release profiles of free ATV and NCAtG and NCAtU are described in Fig. 1.Our results demonstrated that in the first 15 min of dialysis, free ATV and the formulations showed 80% of dissolution/release of drug and approximately 70% for nanocapsules.After this initial period, NCAtG and NCAtU demonstrated a gradual and slight increase in the release over up to 8 h of testing.In this period, the release was 81% of the ATV for both formulations.These results collaborate with results found in other studies [29,47].Probably, this greater release in the first minutes of analysis is due to the portion of ATV adsorbed in the polymeric wall and therefore more available to dissolve in the short term than the ATV dispersed in the oily nucleus.On the other hand, the sustained profile of the release of ATV present in the oily core is desired for their future use in therapies of cholesterol disorders.

Hemolysis Incidence Was Higher for NCAtU in Relation to NCAtG
To evaluate a possible incidence of hemolysis caused by nanoparticles, free hemoglobin was measured after exposure of whole blood samples with the formulations NCAtG, NCAtU (Fig. 2A).We can observe that, in view of the hemoglobin pattern, the particles caused significant damage when the concentrations were relatively high (50 and 100 µg/mL).More specifically, we can say that the levels of free hemoglobin were higher for NCAtU at concentrations of 50 and 100 µg/mL and for NCAtG only at the highest concentration.

NCAtU Promotes Alteration in the Erythrocyte Integrity Through LDH Release
To reaffirm the hemolysis results obtained by the measurement of free hemoglobin, the erythrocyte integrity was evaluated by quantifying the released LDH enzyme.In this way, the NCAtG and NCAtU formulations were again added in concentrations of 10, 50, and 100 µg/mL in whole blood (Fig. 2B).We can observe that, when compared to the negative control (PBS) and positive control of hemolysis (TRITON X100), the NCAtG nanoparticles did not show significant damage to the erythrocyte membrane.In this case, the percentage of LDH released did not differ from that observed for the addition of the inert substance PBS.However, NCAtU demonstrated, from the lowest concentration, to cause alterations in the erythrocyte integrity, whose values are similar to those obtained by the inducer of erythrocyte lesion TRITON X100.
The results in Fig. 2 demonstrated that the NCAtG in these concentrations is safer in relation to NCAtU.But, in the present study, we wanted to use higher concentrations of the formulations to show that even in higher doses, it is still relatively safe.So, we can say that both formulations are safe in a concentration until 50 µg/mL and higher dosages can be used but with precaution.Our results can be compared to Gehrcke et al. (2017), where the authors also find that low concentrations of nanoparticles loaded with PCL and rose hip oil have a better hemocompatibility [48].Natrajan et al. (2015) find in their study evaluating chitosan-alginate nanocapsules loaded with turmeric oil and lemongrass oil any hemolysis event when compared to the positive control [49].

The Nanoformulations Not Able to Interfere Negatively the Coagulation Cascade
The early activation of the blood clotting cascade can cause the formation of coagulative thrombi capable of negatively influencing blood circulation in its different vascular beds [18,19].In order to evaluate whether the NCAtG and NCAtU formulations could interfere with the extrinsic (prothrombin time) or intrinsic (activated partial thromboplastin time) blood coagulation pathways, concentrations of 10, 50, and 100 µg/mL of the formulations were added to citrate plasma samples.After incubation, it was demonstrated that the prothrombin time (Fig. 3A) and the activated partial thromboplastin time (Fig. 3B) did not promote changes compatible with a greater or lesser variation in the time of blood clot formation.These results were different from what occurred in the hemolysis and erythrocyte LDH release tests.In these cases, the highest concentrations of NCAtG and NCAtU ended up significantly increasing those parameters.But, as mentioned earlier, the study wanted to stipulate a safe range of dosage.

The Nanoformulations Caused No DNA Damage in Genotoxicity Assays
The comet test is a relatively simple, fast, and low-cost technique to detect and evaluate pre-mutagenic lesions.In this study, different nanoparticle formulations, in addition to free ATV, were evaluated as possible DNA damage in cells of the 3T3 lineage (Fig. 4A).
After electrophoresis, possible DNA damage was assessed.We can verify that any formulation was able to generate damage to the DNA of cells in relation to positive control and their tested concentrations (10, 50, and 100 µg/ mL).
Simultaneously with the comet assay, the cytotoxicity of the formulations containing or not the ATV was also determined (Fig. 4B).For that, the cells after the treatments were initially lysed and then fixed and stained without electrophoresis in order to determine possible low molecular weight DNA diffusion effects.In this case, no formulation in its different concentrations was able to significantly alter cell integrity.
The results are promising because the comet assay in polymeric nanoparticles is important to confirm the biosafety of this formulation.According to Vandghanooni and Eskandan et al. (2011), different constituents of the formulations may cause different cell damages or none [50].Furthermore, the results found in the present work collaborate with the funds by Dandekar et al. (2011).In this case, the authors found no damage in nanoparticles loaded with curcumin [51].This is a bioactive compound widely used in cardiovascular diseases due to its antioxidant properties.Other authors too did not find any significant damage in study with various nanoparticles with different modified surfaces and polymers [52].

Nanocapsules Do Not Affect the Survival Rate in C. Elegans
Initially, it was performed to evaluate the dose response of the nanocapsules using survival rate as a toxicity end point.
Figure 5A shows that the different formulations added to NGM medium did not cause significant alterations in the survival of the nematodes.On the other hand, the Fig. 5B shows that empty PCL nanospheres caused a reduction in worm survival rate at higher concentrations.Considering that PCL-loaded ATV nanocapsules did not present this toxicity, at the same PCL concentrations in the nanospheres, we can assume that these oils generate a protective effect against the decrease in the survival rate caused by the polymer only.Despite the fact that the nanocapsules did not cause mortality in the worms, the finding with nanospheres is troubling.According to Charao et al. (2015), formulations containing PCL caused similar toxicity [23].In another study, the PCL polymer is toxic to the worms, but in this case, the exposure to the PCL was in 48 h and caused even more damage than our study.The authors suggest in this case that the toxicity is progressive according to the time [35].In this way, taking into consideration PCL toxicity to the nematodes, we proceeded our experiments using the concentration of 10 µg/mL., with melatonin-loaded lipid-core nanocapsules, demonstrated that the nanocapsules just with the PCL caused a decrease in the body size in the higher concentration [23].Therefore, we can state that under these experimental conditions, the nanocapsules are not toxic and do not affect C. elegans growth.

Nanoformulations and the Free ATV Did Not Affect the Brood Size
The maintenance of the reproductive ability of the nematode is an important parameter to be studied due the fact that the lipid metabolisms of the worms interfere directly in the reproduction [53].Some nuclear receptors, such as NHR-8, are important to the maintenance of normal lipid levels in the nematode, and it includes the sterol transport to the gonads and eggs, production of derivative metabolites, and dafachronic acid [54].Figure 7 shows that the nanoformulations and the free ATV did not affect the reproduction.These results are similar to those already observed also for nematode body size and area.In a study, using 6-shogaol from Zingiber officinale was observed that this compound did not affect reproduction and body length of the worms, besides increased life span [55].
Yue Shen et al. ( 2019) reported that trans-Tris methoxy resveratrol present in the grape seed oil has a potential to reduce fat  acids and did not affect progeny size [56].And as mentioned early, the normal levels of steroids and lipids are important to the growth, reproduction, and development of C. elegans.

Nanocapslues Promote a Significant Decrease in TAG Levels Using C. Elegans as Model to Metabolic Alterations
In order to verify the biological efficacy of nanoparticles, we have determined TAG levels and compared it to free drug ATV.It is important to highlight that C. elegans does not synthesize endogenous cholesterol and acquires it from the diet, but the nematode has the mevalonate pathways as mammalian, so the use of ATV in C. elegans is valid [2].For this purpose, we have induced high cholesterol levels by feeding worms with high cholesterol concentration, which modulates TAG synthesis in worms and promotes a model of dyslipidemia like.According to Fig. 8, the group of nematodes that received normal treatment without high cholesterol levels Fig. 5 A C. elegans survival rate following exposure to different nanocapsules after 24 h of chronic treatment in relation to the control.B Worms survival rate following PCL nanospheres treatment after 24 h of chronic treatment in relation to the control.Each bar represents mean ± S.E.M (n = 3).Statistical analysis performed by one-way ANOVA followed by Tukey's post-hoc where * indicates p < 0.05.NCGe, nanocapsule with ginger oil; NCUv, nanocapsule with grape oil; NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil; NEPCL, empty nanosphere only with polymer became known as control without the addition of cholesterol (CS).In turn, the group that received a cholesterol-rich diet and was not treated with any formulation (CT) served as a comparison parameter for an increase or decrease in TG levels between groups.
Figure 8B, the per se group, shows that even without the supplementation with high cholesterol levels, all the treatments do not reduce the TAG levels compared with the control.This result is interesting due the fact that a reduction of the lipid levels in the per se will not be interesting due the fact that it may cause metabolic disorders.
In Fig. 8A, we observe that the formulation and ATV promote a significant decrease in TAG levels compared to the CT group.According to other authors, lower doses of ginger could not cause a reduction in the lipid levels, but in our work, this does not occur [9,44].Rodrigues et al. (2018) in their study using Salvia hispanica L. (chia) seeds oil extracts to reduce lipid levels showed similar to our results, where the extract has a potential to reduce the TAG levels in the worms [57].Another study using liposomes loaded with ethanolic extract of purple pitanga (Eugenia uniflora) also showed that the encapsulation of natural products improves the efficacy in reducing the lipid levels of the nematode [25].Yue et al. (2019) in their study using resveratrol alone also decreased the lipid levels of the nematode [56].These studies collaborate to our studies demonstrating that the nematode is a suitable model to evaluate lipid levels reduction when treated with natural products.Despite the fact that the formulation had similar effects with the free ATV, the controlled release of the formulation and the biological biocompatibility are valid arguments to use this new technology.Due to the possible side effects of ATV, patient adherence to treatment may be compromised.In this way, nanoencapsulation could improve patient care and decrease side effects.

Conclusion
The present work aimed to develop and evaluate the biosafety of nanocapsules containing ATV co-encapsulated with ginger or grape seed oils.It was found that NCAtG and NACtU had homogeneous nanometric populations and adequate physio chemical characteristics.The release profile of ATV remained growing and sustained for up to 8 h.
Through hemolysis tests, release of the erythrocyte LDH enzyme and blood clotting evaluation, we verified that only higher concentrations (50 and 100 µg/mL) can be associated with possible changes in blood parameters, whereas the most evident toxicity was found in in NCAtU exposure.
The formulations did not cause changes in DNA integrity or cytotoxicity in 3T3 cell lines.They also did not change parameters of mortality, body size, and area in vivo in C. elegans.Significant effects on the lipid levels of nematodes with high TAG levels were observed in those animals treated with safe nanocapsule concentration (10 µg/mL).Finally, the use of ATV in nanostructured systems can improve the treatment of lipid disturbs, but the criteria that attest to the biological safety of their applications cannot be ignored.Also, with all these results, we can suppose that despite the fact that the formulation had similar effects with the free ATV, the nanoencapsulation may improve the patients' adherence to treatment and decrease the side effects caused by the drug in long time treatments.Also, one of the future perspectives of the work is to transpose these results to mammalian models to evaluate the safety and efficacy of this formulation, in a model of atherosclerosis to confirm these results.

Fig. 2 A
Fig. 2 A The hemoglobin measurement compared to the treatment with the particles.B Dosage of erythrocyte LDH compared to treatment with nanoparticles.Each bar represents mean ± S.E.M (n = 3) where p < 0.05 was considered "*."Statistical analysis by one-way ANOVA with Tukey's post-hoc.NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil

Fig. 3 A 3 3. 11
Fig. 3 A Prothrombin time test measured after exposure to nanocapsules, comparison made between the PBS as negative control.B Assay for activated partial thromboplastin time measured after exposure to nanocapsules, comparison made between the PBS as negative control.Each bar represents mean ± S.E.M (n = 3).Statistical analysis by one-way ANOVA with Tukey's post-hoc.NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil

Figure
6A and B refers to body length and area of treated worms, respectively.None of the formulations caused significant alterations in both parameters.This lack of toxicity is promising since Campos et al. (2017) have found that loaded and unloaded PCL nanoparticles caused a drastic reduction in worm body size [35].Another study made by Charao et al. (2015)

Fig. 4 A
Fig. 4 A Evaluation of the percentage of cytotoxic damage in cell of line 3 T3, all comparisons were made between the positive control and the other groups.B The comet test performed to assess DNA damage in a 3 T3 cell; all comparisons were made between the positive control and the other groups.Each bar represents mean ± S.E.M (n = 3).Statistical analysis by ANOVA of a road with Tukey's post-hoc; *indicates difference in relation to the control group with damage, where ****p < 0.0001.ATV, atorvastatin; NCGe, nanocapsule with ginger oil; NCUv, nanocapsule with grape oil; NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil

Fig. 6 Fig. 7
Fig. 6 Growth end points following treatment with nanocapsules at 10 µg/ml.A Length of the treated worms in relation to control.B Worms body area in relation to control.Each bar represents mean ± S.E.M (n = 3).Statistical analyis performed by ANOVA of a one-way Tukey's post-hoc.ATV, atorvastatin; NCGe, nanocapsule with ginger oil; NCUv, nanocapsule with grape oil; NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil

Fig. 8 A
Fig. 8 A TAG measurement in C. elegans in relation to the group without cholesterol induction (CS) of the ginger formulations and grape seed formulation.B The per se test of the TAG measurement in relation to the control without cholesterol induction.Each bar represents mean ± S.E.M (n = 3).Statistical analysis performed by one-way ANOVA, where "*" indicates statistical difference between CS and "#" statistical difference between CT.Values of p > 0.05 were considered statistically significant.ATV, atorvastatin; NCGe, nanocapsule with ginger oil; NCUv, nanocapsule with grape oil; NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil

Table 1
Qualitative and quantitative composition of nanocapsule formulations

Table 2
Parameters related to the average particle size, size distribution, and surface charge for nanocapsule formulations DLS laser diffractometry, PCS photon correlation spectroscopy, SPAN polydispersity, PDI polydispersity index, NCGe nanocapsule with ginger oil, NCUv nanocapsule with grape oil, NCAtG nanocapsule loaded with ATV and ginger oil, NCAtU nanocapsule loaded with ATV and grape oil, NEPCL empty nanosphere only with polymer

Table 3
Formulation content data and encapsulation rateNCAtG nanocapsule loaded with ATV and ginger oil, NCAtU nanocapsule loaded with ATV and grape oil Fig. 1 Release profile of the nanoformulations in relation to free ATV.Time in hours (h).NCAtG, nanocapsule loaded with ATV and ginger oil; NCAtU, nanocapsule loaded with ATV and grape oil