pharmaceutics Article In Vitro Cytotoxicity of Oleanolic/Ursolic Acids-Loaded in PLGA Nanoparticles in Different Cell Lines Amélia M. Silva 1,2, * , Helen L. Alvarado 2,3 , Guadalupe Abrego 2,4 , Carlos Martins-Gomes 1,2 , Maria L. Garduño-Ramirez 5 , María L. García 3 , Ana C. Calpena 3 and Eliana B. Souto 6,7, * 1 Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os Montes e Alto Douro (UTAD), Quinta de Prados, 5001-801 Vila Real, Portugal 2 Department of Biology and Environment, UTAD, Quinta de Prados, 5001-801 Vila Real, Portugal 3 Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy, University of Barcelona, Ave. Joan XXIII s/n, 08028 Barcelona, Spain 4 Department of Chemical and Instrumental Analysis, Faculty of Chemistry and Pharmacy, University of El Salvador, Final 25 Ave. Norte, 3026 San Salvador, El Salvador 5 Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Av. Universidad No. 1001, Col Chamilpa, 62209 Cuernavaca, Mexico 6 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, 3000-548 Coimbra, Portugal 7 CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal * Correspondence: amsilva@utad.pt (A.M.S.); ebsouto@ebsouto.pt (E.B.S.); Tel.: +351-259-350-106 (A.M.S.); +351-239-488-400 (E.B.S.)  Received: 10 June 2019; Accepted: 22 July 2019; Published: 24 July 2019  Abstract: Oleanolic (OA) and ursolic (UA) acids are recognized triterpenoids with anti-cancer properties, showing cell-specific activity that can be enhanced when loaded into polymeric nanoparticles. The cytotoxic activity of OA and UA was assessed by Alamar Blue assay in three different cell lines, i.e., HepG2 (Human hepatoma cell line), Caco-2 (Human epithelial colorectal adenocarcinoma cell line) and Y-79 (Human retinoblastoma cell line). The natural and synthetic mixtures of these compounds were tested as free and loaded in polymeric nanoparticles in a concentration range from 2 to 32 µmol/L. The highest tested concentrations of the free triterpene mixtures produced statistically significant cell viability reduction in HepG2 and Caco-2 cells, compared to the control (untreated cells). When loaded in the developed PLGA nanoparticles, no differences were recorded for the tested concentrations in the same cell lines. However, in the Y-79 cell line, a decrease on cell viability was observed when testing the lowest concentration of both free triterpene mixtures, and after their loading into PLGA nanoparticles. Keywords: oleanolic acid; ursolic acid; cytotoxicity; PLGA; polymeric nanoparticles; retinoblastoma cell line 1. Introduction Pentacyclic triterpenes are known for their multiple pharmacological properties [1–3], which are attributed to their resemblance to biogenesis to steroids in the cyclization of squalene. As pentacyclic triterpenes, they exist in a range of medicinal plants, becoming an interesting source of new active ingredients for pharmaceutical applications [3]. In the present work, selected pentacyclic triterpenes have oleanane and ursane based structures, i.e., the oleanolic acid (OA) (of chemical name: 3β-3-hydroxyolean-12-en-28-oic acid) and its isomer, ursolic acid (UA) (of chemical name: 3β-3-hydroxyurs-12-en-28-oic acid). Both structures are shown in Figure 1. These compounds are Pharmaceutics 2019, 11, 362; doi:10.3390/pharmaceutics11080362 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2019, 11, 362 2 of 11 Pharmaceutics 2019, 11, x FOR PEER REVIEW 2 of 11 found a setinofa medicinal set of medicinal herbs herbs and fruits and fruits [2–5].[2–5]. WhileWhile having having interesting interesting biological biological activities activities (anti- (anti-inflammatory, anticancer, hepatoprotective, antioxidant, antifungal, antimicrobial inflammatory, anticancer, hepatoprotective, antioxidant, antifungal, antimicrobial activity), their activity), their bioavailability bioavailability is greatly is greatly hindered hindered bybytheir theirpoor poorwater water solubility. Due to totheir theirsimilar similarchemical chemical structure, structure,thethebiological biologicalactivity activityand andtherapeutic therapeuticinterest interestofofOA OAand andUAUAare arerather rathersimilar, similar,which which is is highly related to their dose-dependent cytotoxicity profile [6]. Previous studies have highly related to their dose-dependent cytotoxicity profile [6]. Previous studies have shown that these shown that these triterpenes triterpenesexhibited exhibitedcytotoxicity cytotoxicityininseveral severaltypes typesofofcancer cancercell lines cell [6–8]. lines Besides [6–8]. Besidestargeting targeting tumor tumor cells by induction of apoptosis, oleanolic and ursolic acids also modulate the cells by induction of apoptosis, oleanolic and ursolic acids also modulate the tumor environment tumor environment exhibiting exhibitingantiangiogenic antiangiogenicand and anti-inflammatory anti-inflammatory activities, togethertogetherwithwithantioxidant antioxidanteffects effects andand cell cell differentiation differentiation (e.g., (e.g., [3]). [3]). AsAsthethe efficacy efficacy of of each each single single compound compound might might differ differ withwith respect respect to to the the identified bioactivities, the combination of these identified bioactivities, the combination of these substances substances in a single dosage form might a single dosage form might have have synergistic synergistic effects inin effects thethe treatment treatment of of cancer [9].[9]. cancer Figure The 1. 1. Figure chemical The structure chemical ofof structure ursolic acid ursolic (UA) acid and (UA) oleanolic and acid oleanolic (OA). acid (OA). Cytotoxicity Cytotoxicity assays assays are arewidely widelyusedusedby theby the pharmaceutical pharmaceutical industry toindustry screen theto screenof biosafety the biosafety of compounds, and also to evaluate the compounds, and also to evaluate the potential anti-tumoral effect of chemotherapeutic drugs. Among potential anti-tumoral effect ofthechemotherapeutic methodologies commonly drugs. used Among to evaluate the cellmethodologies viability, the authorscommonly used to the can highlight evaluate MTT (3- cell viability, the authors can highlight the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) [10,11], SRB (sulforhodamine B), [6,8], tetrazolium trypan bluebromide) [12], WST-8 [10,11], SRB (sulforhodamine B), [6,8], trypan blue [12], WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, tetrazolium, monosodium salt) assay [13], clonogenic assay [11], Alamar blue [14–18],monosodium among others salt) assay [19]. [13], clonogenic assay [11], Alamar blue [14–18], among others [19]. InInthethelast lastdecades, decades,the thesuccess successofofchemotherapy chemotherapyhas hassignificantly significantlyincreased increasedbybysite sitespecific specific targeting of anti-cancer drugs when loaded into nanoparticles [20]. targeting of anti-cancer drugs when loaded into nanoparticles [20]. These submicron-sized carriers These submicron-sized carriers offer offeradvantages advantages over overstandard standard dosage dosage forms, forms, withwiththe thepossibility possibility totoovercome overcome adverse adverse side sidereactions reactions and and systemic systemic toxicity. Tumor-targeted toxicity. Tumor-targeted drugdrug delivery increases delivery intra-tumoral increases drug concentration intra-tumoral while drug concentration lowering the chemotherapeutic concentration in healthy while lowering the chemotherapeutic concentration in healthy tissues [21]. tissues [21]. The Theloading loadingofofnaturalnaturalproducts productsininnanoparticles nanoparticlesfor foranticancer anticancertherapy, therapy,asaswellwellasasforforthethe treatment treatmentofofother otherdiseases, diseases,is isbeing being widely widely explored exploredwith with thetheaimaim toto protect protectsensitive sensitive compounds compounds from from degradation, degradation, to increase theirtheir to increase solubility and thus solubility and their thus bioavailability their bioavailability[22,23].[22,23]. Nanoparticles have Nanoparticles been used for targeting and delivery [24], to reduce inflammation or even have been used for targeting and delivery [24], to reduce inflammation or even facilitate the action of facilitate the action of other drugs other[25,26]. drugs The evaluation [25,26]. of the cytotoxicity/safety The evaluation of new drug-loaded of the cytotoxicity/safety of newnanoparticle drug-loadedformulations, nanoparticle and the comparison of drug effects after being encapsulated becomes formulations, and the comparison of drug effects after being encapsulated becomes therefore therefore of crucial relevance.of Incrucial this work, the cytotoxic relevance. In this profile work, the of natural cytotoxic(NM) and of profile synthetic natural(SM) (NM)mixtures of oleanolic and synthetic acid (OA)of (SM) mixtures and ursolic acid (UA) before and after their loading into poly (d,l-lactide-co-glycolide) oleanolic acid (OA) and ursolic acid (UA) before and after their loading into poly (D,L-lactide-co- acid (PLGA) nanoparticles (PLGA-NPs) have been reported. The Alamar blue glycolide) acid (PLGA) nanoparticles (PLGA-NPs) have been reported. The Alamar blue assay assay was carried out using three was different carriedcelloutlines, usingi.e., human three hepatoma different (HepG2), cell lines, humanhepatoma i.e., human epithelial(HepG2), colorectalhuman adenocarcinoma epithelial (Caco-2) colorectal and human retinoblastoma adenocarcinoma (Caco-2) (Y-79). and human The characterization retinoblastoma (Y-79). of the developed nanoparticle The characterization of formulations the developed was reported in a previous work [25], in which the stability nanoparticle formulations was reported in a previous work [25], in which the stability of NPs over 6 months wasofstudied NPs over at 6 two temperatures ◦ (4 C ◦ months was studied at and two 25 C).Our results temperatures (4 °Cshowed that, from and 25 °C).Our month results 0 to month showed 6: (i) that, from Only0ato month month 6: (i) Only a slight increase of Z-ave, from 220 nm to 230 nm (SM-NPs) and from 210 nm to 230 nm (NM-NPs), without changing polydispersity index (PI) at 4 °C (=0.08) for SM-NPs and with a Pharmaceutics 2019, 11, 362 3 of 11 slight increase of Z-ave, from 220 nm to 230 nm (SM-NPs) and from 210 nm to 230 nm (NM-NPs), without changing polydispersity index (PI) at 4 ◦ C (=0.08) for SM-NPs and with a slight increase, from 0.098 to 0.111, for NM-NPs, was observed; (ii) ZP decreased approximately 2–3 mV, (initial ZP was −27 mV and −25 mV, SM-NPs and NM-NPs, respectively); and (iii) release kinetics occurred over a period of 75 h for both formulations (i.e., 75% released). The reproducibility of NPs characteristics has also been reported [25–27]. Thus, this study aimed to compare the safety/cytotoxicity of OA/UA mixtures (of natural source obtained from Plumeria obtusa leaves extraction [26] and of synthetic source) with the corresponding encapsulated mixtures (SM-OA/UA NPs and NM-OA/UA NPs). 2. Materials and Methods 2.1. Chemicals Resomer® RG756 S [poly (d,l-lactide-co-glycolide) acid (PLGA)] of 98,000 Da of molecular weight (MW) and 75/25 molar ratio of lactide/glycolide was a kind gift from Boehringer (Ingelheim, Germany). The selected triterpenoids, Oleanolic Acid (OA) (≥97%) and Ursolic Acid (UA) (≥90%) were purchased from Sigma-Aldrich (Madrid, Spain). Lutrol® F68 (Poloxamer 188, P188) was offered by BASF (Barcelona, Spain). The reagents and consumables for the cell culture were purchased from Gibco (Alfagene, Invitrogen, Carcavelos, Portugal). MilliQ water was obtained by a MilliQ® Plus System lab supplied. All other reagents were of analytical or high-performance liquid chromatography (HPLC) grade obtained from Fisher Scientific (Leicestershire, UK). 2.2. Production of Natural (NM) and Synthetic (SM) Mixtures of Pentacyclic Triterpenes The leaves of Plumeria obtusa L. var. sericifolia (C. Wright ex Griseb.) Woodson (Apocynaceae), collected in Calakmul, Campeche, Mexico [28] were used as a source of natural mixture of oleanolic and ursolic acids (NM-OA/UA). Briefly, the methanol extract of leaves was processed in a column chromatography under reduced pressure, and eluted with mixtures of hexane/ethyl acetate of increasing polarity. A 50:50 hexane/ethyl acetate chromatography elution system was used to obtain a POLHC-1OA fraction, as a white amorphous powder of 250–260 ◦ C of melting point. The retention factor (Rf) was 0.63. The natural mixture (NM-OA/UA) was obtained with a purity of 58% and analyzed by HPLC to determine the ratio between each triterpene. A synthetic mixture (SM-OA/UA), using the two triterpenes acquired from Sigma-Aldrich, was further prepared, in the same proportion of oleanolic and ursolic acids as found in NM-OA/UA (1:9 w/w). 2.3. Preparation of Nanoparticles PLGA-NPs loaded with NM-OA/UA or SM-OA/UA were prepared applying the solvent displacement production procedure [29]. Briefly, 90 mg PLGA was dissolved in 5 mL of acetone containing 1.0 mg/mL of mixture (NM or SM). This organic solution was poured into 10 mL of an aqueous solution, adjusted to pH 5.5, of 10 mg/mL poloxamer 188 (P 188), under moderate stirring. Acetone was then evaporated and PLGA-NPs dispersions were then concentrated under reduced pressure, as described [26]. 2.4. Cell Culture Y-79 (Human retinoblastoma cell line) and Caco-2 (Human epithelial colorectal adenocarcinoma cell line) were purchased from Cell Lines Service CLS (Eppelheim, Germany). HepG2 (Human hepatoma cell line) was purchased from ATCC (LGC Standards S.L.U., Andorra, Spain). The adherent cell lines (Caco-2 and HepG2) were maintained in DMEM (Dulbecco’s Modified Eagle Medium; Gibco, Alfagene, Portugal), whereas Y-79 cell line was maintained in RPMI-1640 (Roswell Park Memorial Institute medium). Both media were supplemented with 10% (v/v) FBS (fetal bovine serum), 2 mM l-glutamine, and antibiotics (100 U/mL penicillin and 100 µg/mL of streptomycin), in an incubator with atmosphere of 5% CO2 in air at 37 ◦ C, as previously described [14,17]. Pharmaceutics 2019, 11, 362 4 of 11 2.5. Cytotoxicity Assay For the cytotoxicity assays, the adherent cells (Caco-2 and HepG2) were handled as described in [17] and the non-adherent Y-79 cells, that grow in suspension forming aggregates, were handled as described in [14] and then seeded into previously poly-l-Lysine coated 96-well plates. Briefly, the three cell lines were counted and, after appropriate dilution in culture media, were seeded at 5 × 104 cells/mL (Caco-2 and HepG2) or 1 × 105 cells/mL (Y-79) density, in 96-well plates (100 µL/well). The stock solutions of NM-OA/UA and SM-OA/UA were prepared in dimethyl sulfoxide (DMSO) at a concentration of 1.0 µg/µL (2.2 mM). For each mixture and formulation (NM-OA/UA, SM-OA/UA, NM-OA/UA NPs, SM-OA/UA NPs and blank-NPs), five concentrations (2, 4, 8, 16 and 32 µmol/L) were tested, by diluting the required volume of NM or SM with FBS-free culture media to achieve the final concentrations, or the appropriate volume of NPs that contain the required volume of OA/UA, and added to cells 24 h after seeding (100 µL/well). The cell viability was evaluated, after 24 h or 48 h of cells exposure to above mentioned solutions, by applying the Alamar blue assay (Alfagene, Invitrogen, Carcavelos, Portugal). The sample solutions were replaced by a 10% (v/v) Alamar blue solution, diluted in FBS-free culture medium, adding 100 µL to each well. Absorbance was read at 570 nm (reduced form; resorufin) and 620 nm (oxidized form; resazurin). The data were analyzed by calculating the percentage of Alamar blue reduction (according to the manufactures recommendation) and expressed as a percentage of the control (untreated cells), as previously reported [30]. 2.6. Statistical Analysis The data presented are the mean ± standard deviation of four determinations, following analysis of variance (ANOVA) in the GraphPad Prism software (version 5.01). The Tukey multiple comparison test was used to compare the significance of the difference between the groups, a p-value < 0.05 was accepted as significant. 3. Results and Discussion 3.1. Characterization of Nanoparticles Submicron-sized PLGA-NPs were produced by a solvent displacement procedure. A mean particle size of 213.55 ± 1.60 nm was obtained for the NM-OA/UA NPs, with a polydispersity index of 0.090 ± 0.038 and zeta potential of −27.12 ± 0.27 mV. For SM-OA/UA NPs, a mean particle size of 217.98 ± 2.74 nm, polydispersity index of 0.073 ± 0.011 and zeta potential of −26.85 ± 0.49 mV were recorded. The encapsulation efficiency reached 79.01 ± 4.22% for NM-OA/UA NPs and 78.25 ± 3.01% for SM-OA/UA NPs. Both formulations exhibited a Newtonian behavior and high long-term physical stability, without presenting phenomena of destabilization over a period of 6 months. The production of these NPs has been optimized using a 23 factorial design, as reported in Alvarado et al. [25], and the used methodology provides particles with reproducible characteristics. The NPs presented a spherical-shape with identical sizes (~200 nm) for both mixtures, as reported [25]. In a previous in vitro study, these NPs showed to have identical release kinetics, being the release from NM-OA/UA NPs slightly higher up to 40 h of assay, and attaining nearly maximal and identical % release values from 40 h to 72 h (about 75% [25]). This formulation exhibited enhanced OA/UA corneal permeation, showing an identical cumulative permeated amount for both mixtures over time [25]. With this in mind, aiming to use these drugs for ocular application or oral administration, the cytotoxicity of blank NPs, individual mixtures and the loaded NPs were evaluated. 3.2. Cytotoxicity Assay Alamar blue was selected for the evaluation of the pentacyclic triterpenoids cytotoxicity, as it is a quantitative measure of cell proliferation and metabolic activity, useful in the cytotoxicity screening of tested compounds/formulations and can be used as a baseline for further in vivo studies. Alamar blue is a reliable, simple, non-radioactive, sensitive fluorometric/colorimetric assay commonly used to Pharmaceutics 2019, 11, 362 5 of 11 Pharmaceutics 2019, 11, x FOR PEER REVIEW 5 of 11 to detect detect thethe metabolic metabolic activity activity of cells. of cells. ThisThis assay assay combines combines additional additional advantages, advantages, as itas is it is ready ready to to use, use, extremely extremely stable,stable, minimallyminimally toxic toxic to the to cells,theandcells, and for allows allows for the continuous the continuous monitoring monitoring of cultures.of cultures. It may also be useful to predict if formulations cause cellular It may also be useful to predict if formulations cause cellular damage which can result in the loss ofdamage which can result in the metabolic the loss of the cellmetabolic cell function. function. Indeed, Indeed, the oxidation-reduction the oxidation-reduction (REDOX) indicator(REDOX) indicator is incorporated in theis incorporated cell which, when in the cell which, under whenenvironment a reducing under a reducing environment active of a metabolically of a metabolically cell, is reduced.activeAlamar cell, is reduced. blue Alamar becomes blue becomes therefore fluorescent therefore and changesfluorescent its colorandfromchanges blue toitspink. colorThefrom blue to of reduction pink. The Alamar reduction blue can be ofmediated Alamar blue can be mediated by mitochondrial by mitochondrial enzymes or cytosolicenzymes or cytosolic and microsomal and microsomal enzymes [14,31,32]. enzymes [14,31,32]. The cytotoxic activity of free and encapsulated mixtures The cytotoxic activity of free and encapsulated mixtures of OA/UA was evaluated on three human cell of OA/UA was evaluated on three lines, human HepG2, cell lines, Caco-2 HepG2, Caco-2 and Y-79. and Y-79. Figure 22 shows Figure shows the the cell cell viability viability in in HepG2 HepG2 cell cell line line after after 24 24 and and 48 48 hh of of exposure exposure to to different different concentrations of both mixtures of OA/UA and of the mixtures loaded concentrations of both mixtures of OA/UA and of the mixtures loaded into PLGA nanoparticles. Afterinto PLGA nanoparticles. After 24 hhofofexposure, 24 exposure, at at concentrations concentrations up up to 16toµmol/L, 16 µmol/L, the mixture the pure pure mixture of triterpenoid of triterpenoid acids (NM- acids (NM-OA/UA; OA/UA; Figure Figure 2A) 2A)produce did not did not produce significantsignificant changes changeson the cell on viability the cell viability when compared when compared to the to the control control (untreated cells). SM-OA/UA (Figure 2B) at the highest concentration (untreated cells). SM-OA/UA (Figure 2B) at the highest concentration (32 µmol/L) produced a decrease (32 µmol/L) produced a decrease of of approximately approximately 35% on the cell 35%viability on the cell (cell viability viability (cell viability is 65.23% is 65.23%The of control). of nanoparticles control). The loading the mixtures showed no changes (Figure 2B,D). After 48 h of exposure, both pure mixturesboth nanoparticles loading the mixtures showed no changes (Figure 2B,D). After 48 h of exposure, (i.e., pure mixtures non-loaded (i.e., non-loaded mixtures) showed a mixtures) showeddecrease fairly significant a fairly onsignificant decreasei.e., the cell viability, on at the32cell viability, µmol/L cell i.e., at 32reached viability µmol/Lvalues cell viability of 62.89%reached values of 62.89% for NM-OA/UA (Figurefor2A)NM-OA/UA and 17.52% (Figure 2A) and(Figure for SM-OA/UA 17.52%2C),for SM-OA/UA (Figure 2C), compared to the unexposed control. However, compared to the unexposed control. However, no significant negative changes on the cell viability no significant negative changes were on thewhen recorded cell viability loadingwere recordedinwhen the mixtures loading the NPs, the mixturesthat demonstrating in the NPsNPs, demonstrating reduce drug toxicity that at NPs reduce drug toxicity the highest tested concentration. at the highest tested concentration. Figure 2. The effect Figure effect of OA/UA on HepG2 (human hepatoma) cells viability. viability. HepG2 cells were exposed to the the natural mixture of oleanolic and ursolic acids (NM-OA/UA) (A), natural mixture of oleanolic and ursolic acids (NM-OA/UA) (A), NM-OA/UA NM-OA/UA nanoparticles nanoparticles (NPs) (NPs) (B), (B), the the synthetic synthetic mixture mixture of of oleanolic oleanolic acid acid and and ursolic ursolic acids acids (SM-OA/UA) (SM-OA/UA) (C), (C), SM-OA/UA SM-OA/UA NPs NPs (D), (D), for for 24 24 and and 48 48 h, h, as as denoted. denoted. Statistically Statistically significant differences, pp < significant differences, 0.05, are < 0.05, are denoted denoted with with ** or or **, **, in relation to respective control at 24 or 48 h, respectively; # denotes statistical differences in relation to respective control at 24 or 48 h, respectively; # denotes statistical differences for for incubation periods incubationat indicated periods atconcentration. The results The indicated concentration. are the mean results ± the are of n =± 4. S.D.mean S.D. of n = 4. The cell viability in Caco-2 cells is shown in Figure 3, where similar results to the HepG2 cells The cell viability in Caco-2 cells is shown in Figure 3, where similar results to the HepG2 cells are observed. After 24 h of exposure, the pure mixtures of both natural and synthetic triterpenes are observed. After 24 h of exposure, the pure mixtures of both natural and synthetic triterpenes showed no significant changes from the control in concentrations from 2 to 16 µmol/L (NM-OA/UA) showed no significant changes from the control in concentrations from 2 to 16 µmol/L (NM-OA/UA) and from 2 to 8 µmol/L (SM-OA/UA). However, a reduction on cell viability to the concentration of and from 2 to 8 µmol/L (SM-OA/UA). However, a reduction on cell viability to the concentration of 32 µmol/L was observed, where a decrease of approximately 14% and 48% (cell viability of 86.8% and mixtures encapsulated in PLGA-NPs produced no significant changes on the cell viability at all concentrations (Figure 3B,D). Concerning the 48 h exposure (Figure 3, square symbols), Figure 3C shows that SM-OA/UA produced a significant reduction on the cell viability, starting from the concentration of 8 µmol/L and reaching a value of 10.41% (~90% reduction) at the concentration of 32 Pharmaceutics 2019, 11, 362 (Figure 3A) only started producing a significant reduction on the cell viability µmol/L. NM-OA/UA 6 of 11 at 16 µmol/L, reaching 36.5% at 32 µmol/L (reduction of 63.5% on the cell viability in comparison to the control). As in the case of the HepG2 cells, OA/UA mixtures loaded into NPs produced no 32 µmol/L was observed, where a decrease of approximately 14% and 48% (cell viability of 86.8% significant changes on the cell viability compared to control cells, showing again that NPs decreased and 52.30%) for NM-OA/UA (Figure 3A) and SM-OA/UA (Figure 3C) respectively, was observed. the toxicity of the compounds. The mixtures encapsulated in PLGA-NPs produced no significant changes on the cell viability at all The obtained data demonstrate that these terpenoids have cytotoxic activity against HepG2 and concentrations (Figure 3B,D). Concerning the 48 h exposure (Figure 3, square symbols), Figure 3C shows Caco-2 cells, which may translate to anti-tumoral activity. The toxicity of these compounds was that SM-OA/UA produced a significant reduction on the cell viability, starting from the concentration significantly reduced or abolished when loaded into PLGA nanoparticles. This was attributed to a of 8 µmol/L and reaching a value of 10.41% (~90% reduction) at the concentration of 32 µmol/L. controlled release leading to concentrations onto the cells, that were not enough to exhibit cytotoxic NM-OA/UA (Figure 3A) only started producing a significant reduction on the cell viability at 16 µmol/L, effects but that could have a protective role. These results may demonstrate that the developed reaching 36.5% at 32 µmol/L (reduction of 63.5% on the cell viability in comparison to the control). formulation exhibit some protective effects in the intestine and liver as HepG2 and Caco-2 cells are As in the case of the HepG2 cells, OA/UA mixtures loaded into NPs produced no significant changes in vitro models for these organs, respectively. This might be a promising result for the formulation on the cell viability compared to control cells, showing again that NPs decreased the toxicity of of these drugs in innovative oral dosage forms. the compounds. The effect Figure 3. The effect of of OA/UA OA/UA on on Caco-2 Caco-2 (human (humanepithelial epithelialcolorectal colorectaladenocarcinoma) adenocarcinoma)cells cellsviability. viability. Caco-2 cells were exposed to NM-OA/UA (A), NM-OA/UA NPs (B), SM-OA/UA (C), SM-OA/UA NPs denoted. Statistically significant differences, p < (D), for 24 and 48 h, as denoted. 0.05 are < 0.05 are denoted denoted with with ** or or **, **, in relation relationtotorespective respective control at 24ator24 control 48 or h, respectively; # denotes 48 h, respectively; # statistical differencesdifferences denotes statistical for incubationfor periods incubationat indicated periods atconcentration. The results The indicated concentration. are the mean results are± the of n = S.D.mean 4. of n = 4. ± S.D. The Y-79obtained is a human data demonstrate that retinoblastoma (RB)these terpenoids cell line that has have been cytotoxic activity against firstly established from anHepG2 explant and of Caco-2 a primarycells, which tumor of amay translate donor to anti-tumoral with a family history ofactivity. The toxicity retinoblastoma. of these tumoral The cultured compoundscells was had significantly ultrastructural reduced or abolished characteristics when similar loaded to those seeninto PLGA in the nanoparticles. original tumor, such This was attributedtriple as microtubules, to a controlled release leading to concentrations onto the cells, that were not enough membrane structures, nuclear membrane infoldings, centrioles, large coated vesicles, basal bodies to exhibit cytotoxic effects but that and annulate could [33]. lamellae haveThea protective incidencerole. These resultsremains of retinoblastoma may demonstrate that the at constant worldwide developed one case formulation per 15,000–20,000 live births, which corresponds to approximately 9000 new cases every yearcells exhibit some protective effects in the intestine and liver as HepG2 and Caco-2 are [34,35]. in vitro models for these organs, respectively. This might be a promising result Retinoblastoma is the most common malignant eye tumor diagnosed in childhood and can occur as for the formulation of these drugs heritable in innovative (usually oral bilateral) anddosage forms. (unilateral) forms in infants [36]. In health conditions, non-heritable Y-79 is a human retinoblastoma (RB) cell line that has been firstly established from an explant of a primary tumor of a donor with a family history of retinoblastoma. The cultured tumoral cells had ultrastructural characteristics similar to those seen in the original tumor, such as microtubules, triple membrane structures, nuclear membrane infoldings, centrioles, large coated vesicles, basal bodies and annulate lamellae [33]. The incidence of retinoblastoma remains constant worldwide at one case per 15,000–20,000 live births, which corresponds to approximately 9000 new cases every year [34,35]. Pharmaceutics 2019, 11, 362 7 of 11 Pharmaceutics 2019, 11, x FOR PEER REVIEW 7 of 11 Retinoblastoma is the most common malignant eye tumor diagnosed in childhood and can occur as heritable retinoblasts(usually sufferbilateral) division andinto non-heritable new cells and(unilateral) forms fill the retina, in infants dividing and[36]. In healthinto developing conditions, mature retinoblasts retinal cells. suffer division This occurs intoearly during new stages cells and fill thedevelopment of human retina, dividing and in the developing womb. into mature Under pathological retinal cells.instead conditions, This occurs during early of maturing stages cells into special of human development that detect light, somein the womb. Under retinoblasts pathological continue to divide conditions, instead of maturing into special cells that detect light, some retinoblasts and grow out of control, forming the retinoblastoma. The retinoblastoma gene (RBl) is a molecular continue to divide and grow marker of out of control, forming retinoblastoma tumors. theThisretinoblastoma. gene is located The retinoblastoma in chromosome gene (RBl) is a molecular 13q14. marker of retinoblastoma Figure 4 shows the resultstumors. of This gene the cell is located viability in chromosome using Y-79 cell line.13q14. Further, 24 h after applying Figure 4 showstothe the formulations theresults cells,ofnon-loaded the cell viability usingproduced mixtures Y-79 cell line. Further,reductions significant 24 h after applying the on the cell formulations viability. From tothe thefirst cells,concentration non-loaded mixtures of 2 µmol/Lproduced significant to the highest reductions tested on the cell concentrations viability. (32 µmol/L), From theviability the cell first concentration values reached of 2 15.16% µmol/Land to the highest 15.43% for tested NM-OA/UA concentrations (32 µmol/L), (Figure 4A) the cell and SM-OA/UA viability values (Figure 4C), reached 15.16% respectively, and 15.43% (a reduction forviability on cell NM-OA/UA (Figure 4A) and of approximately SM-OA/UA 85%). While NPs(Figure 4C), decreased respectively, the toxicity of (a reduction triterpenoid on compounds cell viabilityinof HepG2 approximately and Caco-285%).cell While NPs lines, indecreased the Y-79, the the toxicity loaded of triterpenoid mixtures compounds still produced in HepG2reduction a significant and Caco-2 on cell lines, the cell in the Y-79, viability. Fromthe loaded to 4 µmol/L mixtures still the highest produced a significant(32 tested concentration reduction µmol/L), onthe thevalues cell viability. From of the cell 4 µmol/L viability to the ca. reached highest ~65.62%tested forconcentration NM-OA/UA (32 (Figure the NPsµmol/L), 4B)values of the for and 36.06% cellSM-OA/UA viability reached ca. ~65.62% NPs (Figure 4D). for NM-OA/UA After NPs (Figure 48 h of exposure, 4B) and a significant 36.06% decreasefor onSM-OA/UA NPs (Figure the cell viability, from the4D). After concentration, lowest 48 h of exposure, a significant reached 32 µmol/L decrease valuesonof the cell cell viability, from viability of the lowest 18.84% concentration, for NM-OA/UA, 12.24%reached 32 µmol/L 28.97% for SM-OA/UA, values of forthe cell viability NM-OA/UA NPsofand 18.84% for 21.01% NM-OA/UA, for SM-OA/UA 12.24% NPs. for SM-OA/UA, 28.97% for NM-OA/UA NPs and 21.01% for SM-OA/UA NPs. Figure 4. The effect of OA/UA on Y-79, human retinoblastoma cells viability. Figure viability. Y-79 Y-79 cells cells were were exposed exposed to NM-OA/UA (A), (A), NM-OA/UA NM-OA/UA NPs NPs(B), (B),SM-OA/UA SM-OA/UA(C), (C),SM-OA/UA SM-OA/UANPs NPs(D),(D),forfor 2424 andand48 48 h, as h, as denoted. Statistically significant differences, p < 0.05 are denoted with * or **, in relation denoted. Statistically significant differences, p < 0.05 are denoted with * or **, in relation to respective at 24 control at 24 or or 48 48 h, h, respectively; respectively; ## denotes denotes statistical statistical differences differences for for incubation incubation periods periods atat indicated indicated concentration. The results are the mean ± concentration. ± S.D. of n ==4. 4. The effect effect of of blank-NPs blank-NPs on on the the cell cell viability viability of of all all the the cell cell lines lines was was tested tested at the two time-points and showed to be statistically statistically different from the control. from the control. However, the observed observed decrease was only approximately 15% of of the the control control (cell (cell viability viability ~85–90%, ~85–90%, at at highest highest concentrations). concentrations). Figure 5 shows the results obtained obtained for Y-79 Y-79 cells cells exposure exposure to to blank-NPs blank-NPs as as these these cells cells showed showed thethe most most notorious notorious effect of the encapsulated triterpene mixtures, showing a cell viability of 82 effect the encapsulated triterpene mixtures, showing a cell viability of 82 ± 7% of the control, ± 7% of the control, after after 48 h exposure 48 h exposure to 32toµmol/L 32 µmol/L (equivalent (equivalent of blank of blank NPs). NPs). Ath48exposure At 48 h exposure to µmol/L to 32 32 µmol/L equivalent equivalent of of blank blank NPs, NPs, thethe viability viability ofof Caco-2and Caco-2 andHepG2 HepG2cells cellswas was88% 88%andand95%, 95%,respectively respectively(data (data not not shown). shown). These small reductions on the cell viability is considered non-toxic [19], making these NPs safe for drug delivery. Pharmaceutics 2019, 11, 362 8 of 11 These small reductions on the cell viability is considered non-toxic [19], making these NPs safe for drug delivery. Pharmaceutics 2019, 11, x FOR PEER REVIEW 8 of 11 Figure5.5.The Figure Theeffect effectofofblank blankNPs NPs ononY-79 cellcell Y-79 viability. Y-79Y-79 viability. cellscells werewere exposed to blank exposed NPs, NPs, to blank for 24for and24 48 h, as and 48 denoted. Statistically h, as denoted. significant Statistically differences, significant p < 0.05, differences, are denoted p < 0.05, withwith are denoted * or ***, orin **,relation to in relation respective control to respective at 24ator control 2448 orh,48respectively. The The h, respectively. results are the results are mean ± S.D. the mean ± S.D. = n4.= 4. of nof These Thesedecreases decreaseson onthethecell cellviability, viability,especially especiallypronounced pronouncedininY-79 Y-79cell cellline, line,are arepossible possiblebecause because these these compounds exhibit potential anticancer effects both in vitro and in vivo.They compounds exhibit potential anticancer effects both in vitro and in vivo. Theyinhibited inhibited proliferation proliferationand andcaused causedapoptosis apoptosisininthe thecells cellsofofnumerous numerouscancers, cancers,including includingmelanoma, melanoma,pancreatic pancreatic cancer, cancer, breast cancer, lung cancer, colon cancer, cervical cancer [37–41]. However, most of breast cancer, lung cancer, colon cancer, cervical cancer [37–41]. However, most oftheir their mechanisms of action are still unclear. Further, UA and OA have mechanisms of action are still unclear. Further, UA and OA have been described as having cytostaticbeen described as having cytostatic activity activityby byinducing inducingcell cellcycle cyclearrest arrestininG0/G1 G0/G1phase phase[42], [42],bybyinducing inducingapoptosis apoptosisthrough throughgenerating generating reactive oxygen species (ROS) and caspase signaling pathways reactive oxygen species (ROS) and caspase signaling pathways [3]. Thymus plant extracts, [3]. Thymus plant extracts, rich in UAinand rich UA OA triterpenoids (~12% w/w; with UA:OA of ~2:1) were also reported and OA triterpenoids (~12% w/w; with UA:OA of ~2:1) were also reported to exhibit high anti- to exhibit high anti-proliferative effects througheffects proliferative cell cycle througharrest and cell apoptosis cycle arrest and [18].apoptosis From these data, [18]. From it can thesebedata, seen it that candifferent be seen cell that lines different cell lines exhibit different sensitivity both to the unloaded and to the loaded Table exhibit different sensitivity both to the unloaded and to the loaded OA/UA mixtures (see OA/UA 1). The OA/UA mixtures mixtures (see Table 1). produced The OA/UA a more pronounced mixtures producedcytotoxic a more effect than the corresponding pronounced cytotoxic effect NPs. thanThis the might result from the release kinetics of these systems. Indeed, corresponding NPs. This might result from the release kinetics of these systems. Indeed, Alvarado et Alvarado et al. [25] reported that NM-AO/UA NPs and al. [25] reported thatSM-AO/UA NM-AO/UA NPsNPs showed approximately and SM-AO/UA NPs50% and 75% showed release, respectively, approximately 50% andat75% 24 h,release, and both presented 75% respectively, at 24release h, andatboth 48 h.presented As the UA75% and release OA content at 48inh.NM-AO/UA As the UA and and SM-AO/UA OA content isin immediately accessible to cells, it is expected that their action is more NM-AO/UA and SM-AO/UA is immediately accessible to cells, it is expected that their action is more pronounced, as it is demonstrated by the significantly pronounced, as it lower IC50 (Table 1), is demonstrated bycompared to the encapsulated the significantly lower IC50 ones. (TableThe 1), synthetic compared mixture to the (SM-AO/UA) seems to be slightly more effective in reducing encapsulated ones. The synthetic mixture (SM-AO/UA) seems to be slightly more effective the cell viability than the natural one. in This may result from the higher purity of purchased compounds (named reducing the cell viability than the natural one. This may result from the higher purity of purchased as synthetic; see material) in comparison compoundsto(named the ones asobtained synthetic; byseeextraction, material)asinreferred comparison in Alvarado to the ones et al.obtained [26]. Theby lower purity of extraction, as NM-OA/UA, in comparison to SM-OA/UA, might also explain the referred in Alvarado et al. [26]. The lower purity of NM-OA/UA, in comparison to SM-OA/UA, might reason why SM-OA/UA produced lower IC50 , forthe also explain HepG2reason andwhy Caco-2 than NM-OA/UA SM-OA/UA produced (Table lower 1).IC Concerning Y-79 cells, it was observed 50, for HepG2 and Caco-2 than NM- that OA/UAthey are very1). (Table sensitive Concerning to these Y-79triterpenoids cells, it was as the IC50 ofthat observed boththey mixtures are very are very low (Table sensitive 1), to these indicating a high toxicity as shown in Figure 4. In these cells, as triterpenoids as the IC50 of both mixtures are very low (Table 1), indicating a high toxicity as shown they are very sensitive to these triterpenoids, in Figure 4. the In encapsulated these cells, as triterpenoids they are very exhibit an anti-proliferative sensitive effect over time. to these triterpenoids, the Most natural encapsulated bioactive compounds triterpenoids exhibit an have a dualistic effect, anti-proliferative depending effect over time. on Most the concentration natural bioactive and mode compounds of action. have Ina this case, by controlling drug release, NPs promote the protection of dualistic effect, depending on the concentration and mode of action. In this case, by controlling drug Caco-2 (colon cells) and HepG2 (hepatic release, cells) which arethe NPs promote notprotection that sensitive to these of Caco-2 compounds (colon cells) and while HepG2 promoting (hepatic efficient cell viability cells) which are not reduction in Y-79 cells. They are also capable of exhibiting anticancer that sensitive to these compounds while promoting efficient cell viability reduction in Y-79 cells. Theyeffects as these are more sensitive toare OA/UA triterpenoids. also capable of exhibiting Cell specific-sensitivity anticancer effects as tothese drugsare and natural more compounds sensitive to OA/UA has triterpenoids. been widely reported. From the experience Cell specific-sensitivity to drugsof theand authors, natural Thymus plant extracts, compounds has been rich in UA and widely OA, induce reported. Fromvery the low toxicity in Caco-2 and HepG2 cells [18] compared to MCF-7 experience of the authors, Thymus plant extracts, rich in UA and OA, induce very low toxicity in (breast cancer cells), BT-474 (breast cancer Caco-2cells) and Raw and HepG2 264.7 cells [18]cells [4], being compared the Raw(breast to MCF-7 264.7 cancer cells ICcells), 50 , forBT-474 24 h exposure, of 24 µg/mL (breast cancer of cells) and extract Raw 264.7(~12% of the cells [4], extract being the is UARawand OA) 264.7 [4].IC50, for 24 h exposure, of 24 µg/mL of extract (~12% of cells the extract is UA and OA) [4]. Pharmaceutics 2019, 11, 362 9 of 11 Table 1. The values of IC50 (µmol/L), (half maximal inhibitory concentration) obtained for HepG2, Caco-2 and Y-79 cells exposed NM-OA/UA, SM-OA/UA, NM-OA/UA NPs and SM-OA/UA NPs. The results are expressed as the mean ± SD, n = 4. IC50 (µmol/L) NM-AO/UA SM-AO/UA NM-AO/UA NPs SM-AO/UA NPs HepG2 24 h >32.0 >32.0 >>32.0 >>32.0 48 h >32.0 24.3 ± 1.8 >>32.0 >>32.0 Caco-2 24 h >32.0 32.4 ± 0.9 >32.0 >32.0 48 h 24.3 ± 0.9 16.3 ± 0.6 >32.0 >32.0 Y-79 24 h 6.4 ± 0.9 6.5 ± 0.9 82.0 ± 10.2 16.6 ± 1.3 48 h 2.6 ± 0.4 2.9 ± 0.3 19.1 ± 2.2 11.1 ± 0.8 Abbreviation: IC50 , half maximal inhibitory concentration. In table, > denotes higher than; >> denotes much higher than. 4. Conclusions In summary, oleanolic (OA) and ursolic (UA) acids have some toxicity at high concentrations (anticancer effect), especially pure (non-loaded) synthetic mixtures which show higher toxicity than the natural mixture in all cell lines. This is attributed to the purity or to the presence of other isomer with lower activity than UA or OA. The potent cytotoxic activity against Y-79 cell line exhibited by these compounds is a promising result to develop new formulations for the treatment of retinoblastoma as prospective anticancer agents, as previous studies also showed them to have a good anti-inflammatory and antioxidant activity. The lower toxicity against Caco-2 cells (colon) and HepG2 (liver) is also very important as it opens new perspectives in oral-delivery formulations with low toxicity, and also ensures low enteric toxicity if inadvertently ingested. Author Contributions: A.M.S., H.L.A., G.A. and C.M.G. contributed for the conceptualization, methodology, validation, formal analysis and investigation. A.M.S., H.L.A., G.A. contributed for the writing—original draft preparation. A.M.S., M.L.G.R., M.L.G., A.C., and E.B.S. contributed for supervision, writing—review and editing, project administration, resources and funding acquisition. Funding: This research was funded by the Ministry of Science and Innovation of Spain under the project with reference MAT2014-59134R and the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, and co-financed by FEDER, under the project references M-ERA-NET/0004/2015 (PAIRED) and UID/AGR/04033/2019 (CITAB), co-financed by FEDER, under the Partnership Agreement PT2020. Acknowledgments: The authors acknowledge Boehringer (Ingelheim, Germany) for the gift of Resomer® RG756 S and BASF (Barcelona, Spain) for the gift of Lutrol® F68 (Poloxamer 188, P188). Conflicts of Interest: The authors declare no conflict of interest. References 1. Jesus, J.A. Antimicrobial activity of oleanolic and ursolic acids: An update. Evid. Based Complement. Altern. Med. 2015, 2015, 14. [CrossRef] [PubMed] 2. Valdes, K. Potential use of nanocarriers with pentacyclic triterpenes in cancer treatments. Nanomedicine 2016, 11, 3139–3156. 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