Colloids and Surfaces B: Biointerfaces 130 (2015) 272–277
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Ketoprofen-loaded pomegranate seed oil nanoemulsion stabilized by pullulan: Selective antiglioma formulation for intravenous administration Luana M. Ferreira a , Verônica F. Cervi a , Mailine Gehrcke a , Elita F. da Silveira b , Juliana H. Azambuja b , Elizandra Braganhol b , Marcel H.M. Sari c , Vanessa A. Zborowski c , Cristina W. Nogueira c , Letícia Cruz a,∗ a
Programa de Pós-graduac¸ão em Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Programa de Pós-Graduac¸ão em Bioquímica e Bioprospecc¸ão, Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil c Programa de Pós-graduac¸ão em Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 22 March 2015 Accepted 9 April 2015 Available online 18 April 2015 Keywords: Nanoemulsion Pullulan Ketoprofen Pomegranate seed oil Antitumor effect
a b s t r a c t This study aimed to prepare pomegranate seed oil nanoemulsions containing ketoprofen using pullulan as a polymeric stabilizer, and to evaluate antitumor activity against in vitro glioma cells. Formulations were prepared by the spontaneous emulsification method and different concentrations of pullulan were tested. Nanoemulsions presented adequate droplet size, polydispersity index, zeta potential, pH, ketoprofen content and encapsulation efficiency. Nanoemulsions were able to delay the photodegradation profile of ketoprofen under UVC radiation, regardless of the concentration of pullulan. In vitro release study indicates that nanoemulsions were able to release approximately 95.0% of ketoprofen in 5 h. Free ketoprofen and formulations were considered hemocompatible at 1 g/mL, in a hemolysis study, for intravenous administration. In addition, a formulation containing the highest concentration of pullulan was tested against C6 cell line and demonstrated significant activity, and did not reduce fibroblasts viability. Thus, pullulan can be considered an interesting excipient to prepare nanostructured systems and nanoemulsion formulations can be considered promising alternatives for the treatment of glioma. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, many studies have been performed in order to elucidate possible mechanisms through which non-steroidal antiinflammatory drugs (NSAID) are able to reduce tumors, since there are reports which indicate that chronic inflammation may contribute to the cancer progress [1,2]. Recently, some studies have suggested that an inflammatory microenvironment may facilitate carcinogenesis and tumor angiogenesis, furthermore, scientific literature describes a higher expression of cyclooxygenase (COX) in tumor tissues [3] which leads to the hypothesis that the use of NSAIDs may contribute to control tumor progression, thus, increasing the efficacy of cancer therapy. Ketoprofen (KP) is a potent
∗ Corresponding author at: Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil. Tel.: +55 55 32209373; fax: +55 55 32208149. E-mail address: [email protected] (L. Cruz). http://dx.doi.org/10.1016/j.colsurfb.2015.04.023 0927-7765/© 2015 Elsevier B.V. All rights reserved.
inhibitor of the COX enzyme, the main source of prostaglandins, one of the mediators of the inflammatory process [4,5]. This drug was also reported as an inhibitor of cell growth in in vitro and in vivo models of glioma tumors [6]. Among the several types of tumor that affect the central nervous system (CNS), the most common and the most malignant is the glioblastoma multiform (GBM) which is composed mainly by astrocyte cells. These tumors are considered to be chemoresistents and, due to their high proliferation rate and infiltrative growth pattern, it can be quite difficult to achieve an effective treatment. In addition, the blood-brain barrier (BBB) hampers the drug penetration into the CNS, restricting the glioma therapy [7]. An alternative method for crossing the BBB is the use of nanocarrier systems, which can promote an increase of therapeutic efficacy and reduce the side effects of conventional chemotherapy [8]. In this context, nanoemulsions (NE) are very attractive colloidal systems due to their advantages when compared to the conventional emulsions. Because of their small droplet size, NE can be used to incorporate lipophilic drugs improving the contact
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surface with biological membranes and thus, favoring the absorption. An increase of the drug availability at the target area could enable the reduction of the drug dose, therefore reducing side effects [9]. In addition to the biological advantages, NE are also more stable against sedimentation and creaming than conventional emulsions [10]. NE are comprised basically of three components: water, oil and surfactants, and their physicochemical properties may influence the behavior of the system [11]. Many vegetable oils such as linseed oil [12], grape seed oil and almond kernel oil [13], olive and coconut oil [14] among others [15] have been studied to prepare NE. In this context, pomegranate seed oil (PSO) has attracted interest due to its high content of fatty acids, estrogens and polyphenols compounds which provide antioxidant, anti-inflammatory and antitumor properties [16,17]. Moreover, PSO has also been used in the food and cosmetic industry due to its nutraceutical and skin repair properties, respectively [18–20]. Moreover, NE are kinetically stabilized by the addition of surfactants which trigger a reduction in the interfacial tension of the systems and form a film with steric and electrostatic properties around the internal phase globules [21,22]. Traditionally, emulsifiers with low molecular weight have been largely used as stabilizer agents [11]. However, these surfactants are frequently associated with incompatibilities with others raw materials such as preservatives, in addition to problems of instability of formulations. In order to circumvent these aspects, polymeric emulsifiers have been applied in the preparation of nanostructured systems [22,23], e.g. pectins, gums, chitosan, pullulan, dextran and alginate [24]. Pullulan (Fig. 1) is an extracellular polysaccharide obtained by the fermentation of the fungus Aureobasidium pullulans and presents a linear structure composed of maltotriose repeating units connected by ␣-1,6 bonds [25]. This water-soluble polymer has non-toxic, non-carcinogenic and non-immunogenic properties, explaining the crescent interest for its application in the pharmaceutical and biomedical industry. Hydrophobically modified pullulan derivatives have been used in preparing nanostructured systems such as liposomes and magnetic nanoparticles [26,27]. Besides, it has been used in nanoparticles for the treatment of hepatocellular carcinoma providing the bind between the particles and hepatic asialoglycoprotein receptors, lowering hemolytic potential and increasing blood circulation [28–30]. On the other hand, no reports could be found reporting the use of this biopolymer as a stabilizer agent for NE or the use of these formulations for the treatment of glioblastoma multiform. Taking into account the aforementioned information, this work was devoted to prepare NE using pullulan as a stabilizer agent in replacement of the traditional high HLB surfactants (polysorbates and polaxamers) with the aim of proposing alternative formulations to be used in situations where such surfactants should be avoided (incompatibilities, lack of stability, etc.). In order to study
Fig. 1. Pullulan chemical structure.
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the application of the proposed formulations, Ketoprofen, a nonsteroidal anti-inflammatory drug, was incorporated in the NE and this formulation was tested against C6 cells. 2. Materials and methods 2.1. Materials Ketoprofen (99% purity) was obtained from Henrifarma (São Paulo, Brazil). PSO was purchased from ViaFarma (São Paulo, Brazil), Span 80® (sorbitan monooleate) was acquired from Sigma Aldrich (São Paulo, Brazil) and Tween 80® (polysorbate 80) purchased from Delaware (Porto Alegre, Brazil). Pullulan was kindly donated by Hayashibara. HPLC-grade methanol was acquired from Tedia (Rio de Janeiro, Brazil). Ultrapure water was obtained from Mili-Q® Plus apparatus. All other solvents and reagents were analytical grade and used as received. 2.2. Analytical procedures The experiments were performed on a LC-10A HPLC system (Shimadzu, Japan) equipped with a LC-20AT pump, an UV-VIS SPD-M20A detector, a CBM-20A system controller and a SIL-20A HT valve sample automatic injector. Separation was achieved at room temperature using a Gemini C18 Phenomenex column ˚ coupled to a C18 guard column. (150 mm × 4.60 mm, 5 m; 110 A) The isocratic mobile phase consisted of methanol and water pH 3.0 (70:30, v/v) at 1 mL/min flow rate. KP was detected at 254 nm. According to the ICH guidelines, the method was validated for the determination of KP in NE. The method was found to be linear (r = 0.9985), specific, accurate (100.96–102.62%) and precise (relative standard deviation ≤ 1.71%) in the concentration range of 3.0–15.0 g/mL. 2.3. Preparation and characterization of nanoemulsions PSO nanoemulsions (n = 3) containing pullulan were prepared by the spontaneous emulsification solvent diffusion method. Briefly, PSO (1.5 w/v%), Span 80® (0.077 g) and KP (0.01 g) were solubilized in acetone (50 mL) and mixed with a pullulan aqueous phase (50 mL) at three different concentrations: 0.5 w/v% (NE 0.5 KP), 1.0 w/v% (NE 1.0 KP) and 1.5 w/v% (NE 1.5 KP) and magnetically stirred for 10 min. Afterwards, the organic solvent was eliminated by evaporation under reduced pressure to achieve a 10 mL final volume and 1 mg/mL KP concentration. For comparison purposes blank NE were prepared simultaneously (NE 0.5 B, NE 1.0 B and NE 1.5B). Photon correlation spectroscopy was used to determine the mean droplet size and polydispersity indexes (PDI) (n = 3) at 25 ◦ C (Zetasizer Nanoseries, Malvern Instruments, UK) after diluting the samples in ultrapure water (1:500). Zeta potential analyses (ZP) were performed using the same instrument after dilution of the samples in 10 mM NaCl (1:500). pH values of NE were determined by directly immersing the electrode of a calibrated potentiometer (Model pH 21, Hanna Instruments, Brazil) in the formulations. Measurements were performed at room temperature (25 ± 2 ◦ C) in triplicate. The total drug content in NE (n = 3) was measured by diluting an aliquot of the sample in 10 mL ethanol followed by sonication for 30 min. This procedure ensures the complete extraction of ketoprofen. Subsequently, samples were filtered through a 45 m membrane and injected into the HPLC system according to the method previously described. The encapsulation efficiency was determined by ultrafiltration/centrifugation technique. An aliquot of the samples was placed in a 10,000 MW centrifugal device (Amicon® Ultra, Millipore) and free drug was separated at 2200 × g
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for 10 min. The ultrafiltrate was diluted 30 times and analyzed by HPLC method. The difference between the total and the free concentration of KP, determined in the NE and in the ultrafiltrate, respectively, was calculated as the encapsulation efficiency (EE%). The morphology of NE was evaluated by scanning electron microscopy. NE were previously lyophilized using trehalose as cryoprotectant and the samples were gold sputtered on a Desk II Cold Sputter (Denton Vacuum, USA) and subsequently analyzed using an accelerating voltage of 10 kV (Scanning microscope JSM-6360, Jeol, Japan). 2.4. Photostability study For the photostability study, 700 L of a methanolic drug solution (1 mg/mL) and KP-loaded NE (1 mg/mL) were placed individually in plastic cuvettes with covers disposed at a fixed distance and subsequently exposed to ultraviolet radiation (Phillips TUV lamp–UVC long life, 30 W) during 4 h in a mirrored chamber (1 m × 25 cm × 25 cm). At predetermined intervals of time (0, 1, 2, 3 and 4 h), an aliquot of 90 L was withdrawn and diluted in ethanol to determine the drug concentration in each sample by HPLC. To discard the influence of other factors, cuvettes containing the samples, wrapped in aluminum foil, were also evaluated (dark controls). The experiment was performed in triplicate. The degradation kinetics of KP was determined by fitting the data to zero order (C = C0 − kt), first order (ln C = ln C0 − kt) and second order equations (1/C = 1/C0 + kt). Correlation coefficients (r) indicated the reaction order. 2.5. In vitro release study In order to determine the release profiles of KP from NE the dialysis bag diffusion technique was used. For this, an aliquot of each formulation was placed in the dialysis bag (MWCO 10,000, Spectra Por 7), which was subsequently immersed in 150 mL phosphate buffer pH 6.8 at 37 ◦ C under continuous stirring at 50 rpm to maintain sink conditions. Aliquots of 1 mL were withdrawn in predetermined intervals, during 5 h and replaced by the same volume of fresh medium. The percentage of drug released was determined using the HPLC conditions previously mentioned. For comparison purposes, a methanolic drug solution at 1 mg/mL KP (MS KP) was studied as control. The experiment was carried out in triplicate (n = 3) and followed sink conditions. Mathematical behavior as well as drug release mechanisms were evaluated by fitting the data to first order (ln C = ln C0 − kt) and to the Korsmeyer–Peppas model (ft = a·tn ), respectively. In these models, C is the concentration of the drug released at time t, C0 is the initial concentration of the drug, k is the kinetic rate constant, a is a constant incorporating structural and geometric characteristics of the carrier, and n is the release exponent, indicative of the mechanism of drug release [31]. For spherical systems, n = 0.43 implies Fickian diffusion, while n ≥ 0.85 is related to case II transport. Values between the both limits indicate anomalous transport [32]. The
fit of the experimental data to the models was performed using the Scientist 2.0 software (Micromath, USA). 2.6. In vitro hemolysis study Hemolysis induced by NE or free KP incubation was evaluated spectrophotometrically. Mice were used according to the guidelines established by the Committee on Care and Use of Experimental Animal Resources, from the Federal University of Santa Maria, Brazil (protocol number 041/2014). Fresh mice blood (n = 6) was collected by cardiac puncture, and centrifuged for 10 min, at a rotation of 3400 rpm, to sort out the plasma and erythrocytes fractions. The plasma was discarded and erythrocytes fraction was washed three times with 0.9% saline solution and centrifuged with the same conditions aforementioned and suspended in the same solution. NE and free KP at different concentrations (50, 25, 10, 5, 1 g/mL) were incubated with 10% erythrocytes suspension during 1 h at 37 ◦ C. After the incubation period, all samples were centrifuged at 1200 rpm for 5 min and 500 L supernatant was mixed with 500 L sodium lauryl sulfate to measure free hemoglobin content at 540 nm (UV-Vis 1601 PC Spectrophotometer, Shimadzu, Japan). Water and 0.9% saline solution were used for positive and negative controls of hemolysis, respectively. 2.7. Cell viability assay in glioblastoma cells (C6) and fibroblasts cell line (3T3) The rat malignant glioma (C6) and fibroblasts cell line (3T3) were obtained from the American Type Culture Collection (ATCC). The cells were grown and maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 0.1% fungizone and 100 U/L penicillin/streptomycin and supplemented with 5% or 10% of fetal bovine serum (FBS) and kept at 37 ◦ C in a humidified atmosphere with 5% CO2 . After the cells reached 90% confluency, they were subjected to trypsinization and counted in a Neubauer chamber, after seeding 10,000 cells per well in 96-well plates. These plates were kept at 37 ◦ C in a humidified atmosphere of 5% CO2 for 24 h and were further treated with free KP or NE (NE 1.5 KP and NE 1.5 B) at 50 and 100 M concentrations. Cells treated with DMSO or NE without drugs were used as control. After 72 h of treatment, cell viability was determined by the MTT (3(4, 5-dimethyl)-2,5diphenyl tetrazolium bromide) method as previously described, which evaluates the mitochondrial functionality [6]. 3. Results and discussion 3.1. Preparation and characterization of nanoemulsions After preparation, the NE obtained by spontaneous emulsification had a milky appearance, characteristic of colloidal systems. Table 1 shows the results of the physicochemical characterization. All formulations presented nanometric size, in the range of 190–257 nm. Besides, there is a tendency in droplet size increase
Table 1 Physicochemical characteristics of nanoemulsions (n = 3). Samples
Drug content (%)
Mean diameter (nm)
NE 0.5 B NE 1.0 B NE 1.5 B NE 0.5 KP NE 1.0 KP NE 1.5 KP
– – – 94.86 ± 2.96 95.02 ± 2.43 102.04 ± 3.71
198 190 200 226 191 257
± ± ± ± ± ±
15 7 22 46 6 70
PDI 0.11 0.08 0.10 0.17 0.09 0.20
± ± ± ± ± ±
0.02 0.04 0.02 0.06 0.03 0.11
Zeta potential (mV)
pH
−35.92 −37.40 −36.69 −17.72 −18.18 −20.81
7.52 6.88 6.83 4.48 4.36 4.28
± denotes standard deviation of a triplicate. a Significant level by comparing ketoprofen-loaded nanoemulsions and the formulation without drug (blank nanoemulsion).
± ± ± ± ± ±
1.00a 1.86a 1.25a 1.73 1.27 2.32
± ± ± ± ± ±
0.08a 0.14a 0.03a 0.16 0.04 0.05
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Fig. 3. Remaining concentration of KP from nanoemulsions and methanolic solution after UVC radiation exposure. The asterisks denote significant level between both formulations and methanolic solution of KP (p < 0.05) by two-way ANOVA analysis, followed by the Tukey’s test.
Table 2 Correlation coefficients of photodegradation kinetic of ketoprofen-loaded nanoemulsions.
Fig. 2. Scanning electron microscopy images of ketoprofen-loaded pomegranate seed oil nanoemulsions stabilized by pullulan (a) and trehalose (b).
with the augmentation in the pullulan concentration, probably due to the increase of aqueous phase viscosity (data not shown), which hindered the solvent diffusion. Polydispersity index values were lower than 0.2 for all formulations, indicating a homogeneous distribution of the systems. Zeta potential values were negative for all formulations, which can be explained by the anionic nature of pullulan. Moreover, the results showed that the presence of KP provides a significant decrease in modulus values of zeta potential, probably indicating that at least part of the drug can be located at the oil/water interface of NE. Furthermore, the presence of the drug caused a reduction in pH values (around 4.0), probably because of the acid nature of KP. Drug content was close to the theoretical value (1 mg/mL) and encapsulation efficiency was higher than 90% for all formulations. Regarding morphology, in Fig. 2a it is possible to note the presence of the nanodroplets at the cryoprotectant surface. Moreover, a thin layer can be visualized around of the nanodroplets, probably due to the presence of the pullulan at the droplet interface. Fig. 2b shows the absence of such structures at the cryoprotectant surface. 3.2. Photostability study NE were evaluated for their ability to protect KP against photodegradation in a drastic condition. For this, a kinetic study was carried out with the NE containing 1 mg/mL KP and with a methanolic solution of the drug (MS KP) at the same concentration. In 75 min of exposure to UVC radiation, only 55% of drug remained
Degradation order
NE 0.5 KP
NE 1.0 KP
NE 1.5 KP
Zero First Second
0.976 0.960 0.936
0.965 0.949 0.927
0.974 0.956 0.930
in the methanolic solution (Fig. 3). On the other hand, NE photoprotected the drug and after 4 h, KP content in NE 0.5 KP, NE 1.0 KP and NE 1.5 KP was approximately 64, 59 and 57%, respectively. No significant difference was observed among the formulations (p > 0.05), indicating that pullulan concentration did not influence the photodegradation profile. Regarding the dark controls, KP concentrations were closed to 100%, discarding the influence of the chamber temperature on drug degradation process. The photoprotection conferred to KP by NE can be attributed to the drug confinement in the oil droplets in addition to the presence of fatty acids, estrogens and polyphenols in PSO composition, which are antioxidants compounds. Moreover, due to the colloidal droplets, NE can scatter the light and improve the photostability. Aiming to elucidate the kinetics of KP photodegradation from the NE, experimental data were fitted to zero, first and second order equations. By comparing the correlation coefficients, all the formulations showed a better adjustment to the zero order equation (Table 2). Thus it can be suggested that the degradation rate is not dependent on the concentration of the drug.
3.3. In vitro release profile Concerning the in vitro drug release study (Fig. 4), formulations had a similar release profiles in phosphate buffer pH 6.8, regardless of the pullulan concentration (p > 0.05). After 5 h, formulations released 97.82, 94.73 and 95.45% of KP, from NEs prepared with 0.5, 1.0 and 1.5% of pullulan, respectively. The statistical comparison of the release profiles revealed that NE improved KP release in relation to the drug solution, used as a control. Probably, the low drug solubility in the release medium and not the dialysis membrane is a limiting factor for KP release. A high solubility could promote an increase in drug’s concentration circulating in the blood and, consequently, could ameliorate brain permeation. Mathematical modeling of experimental data indicated that the formulations followed a first order kinetics (r > 0.99), suggesting that the release occurred in a single step and it was dependent on the drug concentration.
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Fig. 4. Drug release profiles of KP from methanolic solution and NEs. Asterisk denotes significant level between formulations and methanolic solution of KP (p < 0.05), analyzed by two-way ANOVA, followed by the Tukey’s test.
3.4. In vitro hemolysis study Hemolysis testing is considered a rapid and reliable method to determine the hemocompatibility of biomaterials, especially when the drug is to be administered intravenously [33,34]. Fresh mice blood was used to evaluate the hemolytic property of the formulations. Different concentrations of NEs and free KP were tested, as well as the respective blank NEs (data not shown). Fig. 5 shows the results of percentage of hemolysis for NEs containing drug and free KP. No statistical difference was observed (p > 0.05) among the formulations, regardless of the pullulan concentration. At the highest concentration tested (50 g/mL), KP-loaded NE showed around 70% of hemolysis. This higher hemolytic activity can be explained by the higher surface area of the nanocarriers, which improves the contact area with the erythrocytes, especially if the drug is associated more externally [35,36]. According to the literature, the accepted limits should vary between 1 and 5% for spontaneous hemolysis [37,38]. Therefore, 50 g/mL was not considered suitable for intravenous administration. However, at 1 g/mL the hemolysis was less than 1% for NE 0.5 KP, while free KP and the other NE induced about 5% of hemolysis. The most common intravenous dose of ketoprofen is 1 mg/kg, depending on the therapeutic goal (anti-inflammatory, analgesic or antipyretic). However, no dosage regime has yet been established for ketoprofen in the treatment of glioma tumors. Considering that nanostructured systems have presented site-specific delivery for anticancer drugs, it is possible that the intravenous dose of ketoprofen in nanoemulsion is lower than the dose employed using conventional therapy. Thus, we believe that 1 g/mL is a good reference for the hemolysis test in this case.
Fig. 5. Percentage of hemolysis at concentration range of 1–50 g/mL for free KP and NE, analyzed by One-way ANOVA. Values of p > 0.05 was considered not significant. Each line represents the mean with S.E.M. of triplicate.
According to the literature, surfactant molecules can solubilize the lipids and proteins and destabilize the membrane of the erythrocytes [36]. In this case, our formulations employing pullulan instead of conventional surfactants can be an alternative for intravenous dosage forms. 3.5. Cell viability assay in glioblastoma cells (C6) and fibroblasts cell line (3T3) In order to determine the antitumor activity of formulation, free KP, NE 1.5 KP and blank formulation were incubated with C6 cells at two different concentrations (50 and 100 M). After 72 h of incubation, NE 1.5 KP presented 40% inhibition of cell growth (Fig. 6b), which can be considered a promising result regarding that GBM possess high proliferation rate and degree of invasiveness [6,39]. Probably, the small droplet size of NE provides a high contact area with the cell membrane which increases the input of KP. Regarding the literature, the incorporation of antineoplasic agents in nanocarriers can improve antitumor activity [40]. It can be noted that NE 1.5 B did not present antitumor activity, indicating that the effect is due to the presence of KP. Moreover, the formulations tested did not exhibit cytotoxicity action against fibroblasts (Fig. 6a), when used as a non-transformed cells model. In spite of this promising result, the blood–brain barrier represents an obstacle to great part of the antitumor agents. However, the use of nanocarriers is a promising approach to improve the treatment efficiency of brain tumors. Recently, our group found that the encapsulation of KP in poly--caprolactone nanocapsules inhibited glioma growth in vivo, showing, therefore, the ability of these nanostructures to cross the blood-brain barrier. In this work,
Fig. 6. Antiproliferative effect of free KP, NE 1.5 KP and NE 1.5 B by MTT assays. (a) Fibroblasts cell line and (b) glioma cell line (C6). Each column represents the mean with S.E.M. of triplicate. Asterisks denote the significance level compared to the control group (p < 0.05), analyzed by One-Way ANOVA, followed by the Tukey’s test.
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it was demonstrated that the nanostructures increased cell membrane permeability, which is an indicative of cell death by necrosis [6]. 4. Conclusion In conclusion, for the first time it was found that it is feasible to prepare pomegranate seed oil nanoemulsions containing ketoprofen, using pullulan as a new polymeric emulsifier. Nanoemulsions improved drug photostability and have the ability to ameliorate drug solubility. Moreover, these formulations can be considered suitable for intravenous administration and showed significant activity against glioma cells in vitro. Acknowledgements The authors thank C.B. da Silva for Zetasizer access. L.M.F. thanks CNPq/Brasília/Brazil for granting a master fellowship. References [1] A. Bernardi, R.L. Frozza, J.B. Hoppe, C. Salbego, A.R. Pohlmann, A.M.O. Battastini, S.S. Guterres, Int. J. Nanomed. 8 (2013) 711–729. [2] Y. Dagistan, I. Karaca, E.R. Bozkurt, A. Bilir, J. Coll. Physicians Surg. Pak. 22 (2012) 690–693. [3] Y.Q. Liu, Y. Lu, J. Wang, L. Xie, T.J. Li, Y. He, Q.L. Peng, X. Qin, S. Li, Br. J. Clin. Pharmacol. 78 (2014) 58–68. [4] M.H.F. Sakeena, M.F. Yam, S.M. Elrashid, A.S. Munavvar, M.N. Azmin, J. Oleo Sci. 59 (2010) 667–671. [5] G. Pinardi, F. Sierralta, H.F. Miranda, Inflammation 25 (2001) 233–239. [6] E.F. da Silveira, J.M. Chassot, F.C. Teixeira, J.H. Azambuja, G. Debom, F.T. Beira, F.A.B. Del Pino, A. Lourenco, A.P. Horn, L. Cruz, R.M. Spanevello, E. Braganhol, Investig. N. Drugs 31 (2013) 1424–1435. [7] A. Behin, K. Hoang-Xuan, A.F. Carpentier, J.Y. Delattre, Lancet 361 (2003) 323–331. [8] G. Mattheolabakis, B. Rigas, P.P. Constantinides, Nanomedicine 7 (2012) 1577–1590. [9] T.G. Mason, J.N. Wilking, K. Meleson, C.B. Chang, S.M. Graves, J. Phys. Condens. Matter 18 (2006) R635–R666. [10] T. Tadros, R. Izquierdo, J. Esquena, C. Solans, Adv. Colloid Interface Sci. 108 (2004) 303–318. [11] K. Bouchemal, S. Briancon, E. Perrier, H. Fessi, Int. J. Pharm. 280 (2004) 241–251.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Ketoprofen-loaded pomegranate seed oil nanoemulsion stabilized by pullulan: Selective antiglioma formulation for intravenous administration Luana M. Ferreira a , Verônica F. Cervi a , Mailine Gehrcke a , Elita F. da Silveira b , Juliana H. Azambuja b , Elizandra Braganhol b , Marcel H.M. Sari c , Vanessa A. Zborowski c , Cristina W. Nogueira c , Letícia Cruz a,∗ a
Programa de Pós-graduac¸ão em Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Programa de Pós-Graduac¸ão em Bioquímica e Bioprospecc¸ão, Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas, RS, Brazil c Programa de Pós-graduac¸ão em Bioquímica Toxicológica, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 18 November 2014 Received in revised form 22 March 2015 Accepted 9 April 2015 Available online 18 April 2015 Keywords: Nanoemulsion Pullulan Ketoprofen Pomegranate seed oil Antitumor effect
a b s t r a c t This study aimed to prepare pomegranate seed oil nanoemulsions containing ketoprofen using pullulan as a polymeric stabilizer, and to evaluate antitumor activity against in vitro glioma cells. Formulations were prepared by the spontaneous emulsification method and different concentrations of pullulan were tested. Nanoemulsions presented adequate droplet size, polydispersity index, zeta potential, pH, ketoprofen content and encapsulation efficiency. Nanoemulsions were able to delay the photodegradation profile of ketoprofen under UVC radiation, regardless of the concentration of pullulan. In vitro release study indicates that nanoemulsions were able to release approximately 95.0% of ketoprofen in 5 h. Free ketoprofen and formulations were considered hemocompatible at 1 g/mL, in a hemolysis study, for intravenous administration. In addition, a formulation containing the highest concentration of pullulan was tested against C6 cell line and demonstrated significant activity, and did not reduce fibroblasts viability. Thus, pullulan can be considered an interesting excipient to prepare nanostructured systems and nanoemulsion formulations can be considered promising alternatives for the treatment of glioma. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, many studies have been performed in order to elucidate possible mechanisms through which non-steroidal antiinflammatory drugs (NSAID) are able to reduce tumors, since there are reports which indicate that chronic inflammation may contribute to the cancer progress [1,2]. Recently, some studies have suggested that an inflammatory microenvironment may facilitate carcinogenesis and tumor angiogenesis, furthermore, scientific literature describes a higher expression of cyclooxygenase (COX) in tumor tissues [3] which leads to the hypothesis that the use of NSAIDs may contribute to control tumor progression, thus, increasing the efficacy of cancer therapy. Ketoprofen (KP) is a potent
∗ Corresponding author at: Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil. Tel.: +55 55 32209373; fax: +55 55 32208149. E-mail address: [email protected] (L. Cruz). http://dx.doi.org/10.1016/j.colsurfb.2015.04.023 0927-7765/© 2015 Elsevier B.V. All rights reserved.
inhibitor of the COX enzyme, the main source of prostaglandins, one of the mediators of the inflammatory process [4,5]. This drug was also reported as an inhibitor of cell growth in in vitro and in vivo models of glioma tumors [6]. Among the several types of tumor that affect the central nervous system (CNS), the most common and the most malignant is the glioblastoma multiform (GBM) which is composed mainly by astrocyte cells. These tumors are considered to be chemoresistents and, due to their high proliferation rate and infiltrative growth pattern, it can be quite difficult to achieve an effective treatment. In addition, the blood-brain barrier (BBB) hampers the drug penetration into the CNS, restricting the glioma therapy [7]. An alternative method for crossing the BBB is the use of nanocarrier systems, which can promote an increase of therapeutic efficacy and reduce the side effects of conventional chemotherapy [8]. In this context, nanoemulsions (NE) are very attractive colloidal systems due to their advantages when compared to the conventional emulsions. Because of their small droplet size, NE can be used to incorporate lipophilic drugs improving the contact
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surface with biological membranes and thus, favoring the absorption. An increase of the drug availability at the target area could enable the reduction of the drug dose, therefore reducing side effects [9]. In addition to the biological advantages, NE are also more stable against sedimentation and creaming than conventional emulsions [10]. NE are comprised basically of three components: water, oil and surfactants, and their physicochemical properties may influence the behavior of the system [11]. Many vegetable oils such as linseed oil [12], grape seed oil and almond kernel oil [13], olive and coconut oil [14] among others [15] have been studied to prepare NE. In this context, pomegranate seed oil (PSO) has attracted interest due to its high content of fatty acids, estrogens and polyphenols compounds which provide antioxidant, anti-inflammatory and antitumor properties [16,17]. Moreover, PSO has also been used in the food and cosmetic industry due to its nutraceutical and skin repair properties, respectively [18–20]. Moreover, NE are kinetically stabilized by the addition of surfactants which trigger a reduction in the interfacial tension of the systems and form a film with steric and electrostatic properties around the internal phase globules [21,22]. Traditionally, emulsifiers with low molecular weight have been largely used as stabilizer agents [11]. However, these surfactants are frequently associated with incompatibilities with others raw materials such as preservatives, in addition to problems of instability of formulations. In order to circumvent these aspects, polymeric emulsifiers have been applied in the preparation of nanostructured systems [22,23], e.g. pectins, gums, chitosan, pullulan, dextran and alginate [24]. Pullulan (Fig. 1) is an extracellular polysaccharide obtained by the fermentation of the fungus Aureobasidium pullulans and presents a linear structure composed of maltotriose repeating units connected by ␣-1,6 bonds [25]. This water-soluble polymer has non-toxic, non-carcinogenic and non-immunogenic properties, explaining the crescent interest for its application in the pharmaceutical and biomedical industry. Hydrophobically modified pullulan derivatives have been used in preparing nanostructured systems such as liposomes and magnetic nanoparticles [26,27]. Besides, it has been used in nanoparticles for the treatment of hepatocellular carcinoma providing the bind between the particles and hepatic asialoglycoprotein receptors, lowering hemolytic potential and increasing blood circulation [28–30]. On the other hand, no reports could be found reporting the use of this biopolymer as a stabilizer agent for NE or the use of these formulations for the treatment of glioblastoma multiform. Taking into account the aforementioned information, this work was devoted to prepare NE using pullulan as a stabilizer agent in replacement of the traditional high HLB surfactants (polysorbates and polaxamers) with the aim of proposing alternative formulations to be used in situations where such surfactants should be avoided (incompatibilities, lack of stability, etc.). In order to study
Fig. 1. Pullulan chemical structure.
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the application of the proposed formulations, Ketoprofen, a nonsteroidal anti-inflammatory drug, was incorporated in the NE and this formulation was tested against C6 cells. 2. Materials and methods 2.1. Materials Ketoprofen (99% purity) was obtained from Henrifarma (São Paulo, Brazil). PSO was purchased from ViaFarma (São Paulo, Brazil), Span 80® (sorbitan monooleate) was acquired from Sigma Aldrich (São Paulo, Brazil) and Tween 80® (polysorbate 80) purchased from Delaware (Porto Alegre, Brazil). Pullulan was kindly donated by Hayashibara. HPLC-grade methanol was acquired from Tedia (Rio de Janeiro, Brazil). Ultrapure water was obtained from Mili-Q® Plus apparatus. All other solvents and reagents were analytical grade and used as received. 2.2. Analytical procedures The experiments were performed on a LC-10A HPLC system (Shimadzu, Japan) equipped with a LC-20AT pump, an UV-VIS SPD-M20A detector, a CBM-20A system controller and a SIL-20A HT valve sample automatic injector. Separation was achieved at room temperature using a Gemini C18 Phenomenex column ˚ coupled to a C18 guard column. (150 mm × 4.60 mm, 5 m; 110 A) The isocratic mobile phase consisted of methanol and water pH 3.0 (70:30, v/v) at 1 mL/min flow rate. KP was detected at 254 nm. According to the ICH guidelines, the method was validated for the determination of KP in NE. The method was found to be linear (r = 0.9985), specific, accurate (100.96–102.62%) and precise (relative standard deviation ≤ 1.71%) in the concentration range of 3.0–15.0 g/mL. 2.3. Preparation and characterization of nanoemulsions PSO nanoemulsions (n = 3) containing pullulan were prepared by the spontaneous emulsification solvent diffusion method. Briefly, PSO (1.5 w/v%), Span 80® (0.077 g) and KP (0.01 g) were solubilized in acetone (50 mL) and mixed with a pullulan aqueous phase (50 mL) at three different concentrations: 0.5 w/v% (NE 0.5 KP), 1.0 w/v% (NE 1.0 KP) and 1.5 w/v% (NE 1.5 KP) and magnetically stirred for 10 min. Afterwards, the organic solvent was eliminated by evaporation under reduced pressure to achieve a 10 mL final volume and 1 mg/mL KP concentration. For comparison purposes blank NE were prepared simultaneously (NE 0.5 B, NE 1.0 B and NE 1.5B). Photon correlation spectroscopy was used to determine the mean droplet size and polydispersity indexes (PDI) (n = 3) at 25 ◦ C (Zetasizer Nanoseries, Malvern Instruments, UK) after diluting the samples in ultrapure water (1:500). Zeta potential analyses (ZP) were performed using the same instrument after dilution of the samples in 10 mM NaCl (1:500). pH values of NE were determined by directly immersing the electrode of a calibrated potentiometer (Model pH 21, Hanna Instruments, Brazil) in the formulations. Measurements were performed at room temperature (25 ± 2 ◦ C) in triplicate. The total drug content in NE (n = 3) was measured by diluting an aliquot of the sample in 10 mL ethanol followed by sonication for 30 min. This procedure ensures the complete extraction of ketoprofen. Subsequently, samples were filtered through a 45 m membrane and injected into the HPLC system according to the method previously described. The encapsulation efficiency was determined by ultrafiltration/centrifugation technique. An aliquot of the samples was placed in a 10,000 MW centrifugal device (Amicon® Ultra, Millipore) and free drug was separated at 2200 × g
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for 10 min. The ultrafiltrate was diluted 30 times and analyzed by HPLC method. The difference between the total and the free concentration of KP, determined in the NE and in the ultrafiltrate, respectively, was calculated as the encapsulation efficiency (EE%). The morphology of NE was evaluated by scanning electron microscopy. NE were previously lyophilized using trehalose as cryoprotectant and the samples were gold sputtered on a Desk II Cold Sputter (Denton Vacuum, USA) and subsequently analyzed using an accelerating voltage of 10 kV (Scanning microscope JSM-6360, Jeol, Japan). 2.4. Photostability study For the photostability study, 700 L of a methanolic drug solution (1 mg/mL) and KP-loaded NE (1 mg/mL) were placed individually in plastic cuvettes with covers disposed at a fixed distance and subsequently exposed to ultraviolet radiation (Phillips TUV lamp–UVC long life, 30 W) during 4 h in a mirrored chamber (1 m × 25 cm × 25 cm). At predetermined intervals of time (0, 1, 2, 3 and 4 h), an aliquot of 90 L was withdrawn and diluted in ethanol to determine the drug concentration in each sample by HPLC. To discard the influence of other factors, cuvettes containing the samples, wrapped in aluminum foil, were also evaluated (dark controls). The experiment was performed in triplicate. The degradation kinetics of KP was determined by fitting the data to zero order (C = C0 − kt), first order (ln C = ln C0 − kt) and second order equations (1/C = 1/C0 + kt). Correlation coefficients (r) indicated the reaction order. 2.5. In vitro release study In order to determine the release profiles of KP from NE the dialysis bag diffusion technique was used. For this, an aliquot of each formulation was placed in the dialysis bag (MWCO 10,000, Spectra Por 7), which was subsequently immersed in 150 mL phosphate buffer pH 6.8 at 37 ◦ C under continuous stirring at 50 rpm to maintain sink conditions. Aliquots of 1 mL were withdrawn in predetermined intervals, during 5 h and replaced by the same volume of fresh medium. The percentage of drug released was determined using the HPLC conditions previously mentioned. For comparison purposes, a methanolic drug solution at 1 mg/mL KP (MS KP) was studied as control. The experiment was carried out in triplicate (n = 3) and followed sink conditions. Mathematical behavior as well as drug release mechanisms were evaluated by fitting the data to first order (ln C = ln C0 − kt) and to the Korsmeyer–Peppas model (ft = a·tn ), respectively. In these models, C is the concentration of the drug released at time t, C0 is the initial concentration of the drug, k is the kinetic rate constant, a is a constant incorporating structural and geometric characteristics of the carrier, and n is the release exponent, indicative of the mechanism of drug release [31]. For spherical systems, n = 0.43 implies Fickian diffusion, while n ≥ 0.85 is related to case II transport. Values between the both limits indicate anomalous transport [32]. The
fit of the experimental data to the models was performed using the Scientist 2.0 software (Micromath, USA). 2.6. In vitro hemolysis study Hemolysis induced by NE or free KP incubation was evaluated spectrophotometrically. Mice were used according to the guidelines established by the Committee on Care and Use of Experimental Animal Resources, from the Federal University of Santa Maria, Brazil (protocol number 041/2014). Fresh mice blood (n = 6) was collected by cardiac puncture, and centrifuged for 10 min, at a rotation of 3400 rpm, to sort out the plasma and erythrocytes fractions. The plasma was discarded and erythrocytes fraction was washed three times with 0.9% saline solution and centrifuged with the same conditions aforementioned and suspended in the same solution. NE and free KP at different concentrations (50, 25, 10, 5, 1 g/mL) were incubated with 10% erythrocytes suspension during 1 h at 37 ◦ C. After the incubation period, all samples were centrifuged at 1200 rpm for 5 min and 500 L supernatant was mixed with 500 L sodium lauryl sulfate to measure free hemoglobin content at 540 nm (UV-Vis 1601 PC Spectrophotometer, Shimadzu, Japan). Water and 0.9% saline solution were used for positive and negative controls of hemolysis, respectively. 2.7. Cell viability assay in glioblastoma cells (C6) and fibroblasts cell line (3T3) The rat malignant glioma (C6) and fibroblasts cell line (3T3) were obtained from the American Type Culture Collection (ATCC). The cells were grown and maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 0.1% fungizone and 100 U/L penicillin/streptomycin and supplemented with 5% or 10% of fetal bovine serum (FBS) and kept at 37 ◦ C in a humidified atmosphere with 5% CO2 . After the cells reached 90% confluency, they were subjected to trypsinization and counted in a Neubauer chamber, after seeding 10,000 cells per well in 96-well plates. These plates were kept at 37 ◦ C in a humidified atmosphere of 5% CO2 for 24 h and were further treated with free KP or NE (NE 1.5 KP and NE 1.5 B) at 50 and 100 M concentrations. Cells treated with DMSO or NE without drugs were used as control. After 72 h of treatment, cell viability was determined by the MTT (3(4, 5-dimethyl)-2,5diphenyl tetrazolium bromide) method as previously described, which evaluates the mitochondrial functionality [6]. 3. Results and discussion 3.1. Preparation and characterization of nanoemulsions After preparation, the NE obtained by spontaneous emulsification had a milky appearance, characteristic of colloidal systems. Table 1 shows the results of the physicochemical characterization. All formulations presented nanometric size, in the range of 190–257 nm. Besides, there is a tendency in droplet size increase
Table 1 Physicochemical characteristics of nanoemulsions (n = 3). Samples
Drug content (%)
Mean diameter (nm)
NE 0.5 B NE 1.0 B NE 1.5 B NE 0.5 KP NE 1.0 KP NE 1.5 KP
– – – 94.86 ± 2.96 95.02 ± 2.43 102.04 ± 3.71
198 190 200 226 191 257
± ± ± ± ± ±
15 7 22 46 6 70
PDI 0.11 0.08 0.10 0.17 0.09 0.20
± ± ± ± ± ±
0.02 0.04 0.02 0.06 0.03 0.11
Zeta potential (mV)
pH
−35.92 −37.40 −36.69 −17.72 −18.18 −20.81
7.52 6.88 6.83 4.48 4.36 4.28
± denotes standard deviation of a triplicate. a Significant level by comparing ketoprofen-loaded nanoemulsions and the formulation without drug (blank nanoemulsion).
± ± ± ± ± ±
1.00a 1.86a 1.25a 1.73 1.27 2.32
± ± ± ± ± ±
0.08a 0.14a 0.03a 0.16 0.04 0.05
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Fig. 3. Remaining concentration of KP from nanoemulsions and methanolic solution after UVC radiation exposure. The asterisks denote significant level between both formulations and methanolic solution of KP (p < 0.05) by two-way ANOVA analysis, followed by the Tukey’s test.
Table 2 Correlation coefficients of photodegradation kinetic of ketoprofen-loaded nanoemulsions.
Fig. 2. Scanning electron microscopy images of ketoprofen-loaded pomegranate seed oil nanoemulsions stabilized by pullulan (a) and trehalose (b).
with the augmentation in the pullulan concentration, probably due to the increase of aqueous phase viscosity (data not shown), which hindered the solvent diffusion. Polydispersity index values were lower than 0.2 for all formulations, indicating a homogeneous distribution of the systems. Zeta potential values were negative for all formulations, which can be explained by the anionic nature of pullulan. Moreover, the results showed that the presence of KP provides a significant decrease in modulus values of zeta potential, probably indicating that at least part of the drug can be located at the oil/water interface of NE. Furthermore, the presence of the drug caused a reduction in pH values (around 4.0), probably because of the acid nature of KP. Drug content was close to the theoretical value (1 mg/mL) and encapsulation efficiency was higher than 90% for all formulations. Regarding morphology, in Fig. 2a it is possible to note the presence of the nanodroplets at the cryoprotectant surface. Moreover, a thin layer can be visualized around of the nanodroplets, probably due to the presence of the pullulan at the droplet interface. Fig. 2b shows the absence of such structures at the cryoprotectant surface. 3.2. Photostability study NE were evaluated for their ability to protect KP against photodegradation in a drastic condition. For this, a kinetic study was carried out with the NE containing 1 mg/mL KP and with a methanolic solution of the drug (MS KP) at the same concentration. In 75 min of exposure to UVC radiation, only 55% of drug remained
Degradation order
NE 0.5 KP
NE 1.0 KP
NE 1.5 KP
Zero First Second
0.976 0.960 0.936
0.965 0.949 0.927
0.974 0.956 0.930
in the methanolic solution (Fig. 3). On the other hand, NE photoprotected the drug and after 4 h, KP content in NE 0.5 KP, NE 1.0 KP and NE 1.5 KP was approximately 64, 59 and 57%, respectively. No significant difference was observed among the formulations (p > 0.05), indicating that pullulan concentration did not influence the photodegradation profile. Regarding the dark controls, KP concentrations were closed to 100%, discarding the influence of the chamber temperature on drug degradation process. The photoprotection conferred to KP by NE can be attributed to the drug confinement in the oil droplets in addition to the presence of fatty acids, estrogens and polyphenols in PSO composition, which are antioxidants compounds. Moreover, due to the colloidal droplets, NE can scatter the light and improve the photostability. Aiming to elucidate the kinetics of KP photodegradation from the NE, experimental data were fitted to zero, first and second order equations. By comparing the correlation coefficients, all the formulations showed a better adjustment to the zero order equation (Table 2). Thus it can be suggested that the degradation rate is not dependent on the concentration of the drug.
3.3. In vitro release profile Concerning the in vitro drug release study (Fig. 4), formulations had a similar release profiles in phosphate buffer pH 6.8, regardless of the pullulan concentration (p > 0.05). After 5 h, formulations released 97.82, 94.73 and 95.45% of KP, from NEs prepared with 0.5, 1.0 and 1.5% of pullulan, respectively. The statistical comparison of the release profiles revealed that NE improved KP release in relation to the drug solution, used as a control. Probably, the low drug solubility in the release medium and not the dialysis membrane is a limiting factor for KP release. A high solubility could promote an increase in drug’s concentration circulating in the blood and, consequently, could ameliorate brain permeation. Mathematical modeling of experimental data indicated that the formulations followed a first order kinetics (r > 0.99), suggesting that the release occurred in a single step and it was dependent on the drug concentration.
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Fig. 4. Drug release profiles of KP from methanolic solution and NEs. Asterisk denotes significant level between formulations and methanolic solution of KP (p < 0.05), analyzed by two-way ANOVA, followed by the Tukey’s test.
3.4. In vitro hemolysis study Hemolysis testing is considered a rapid and reliable method to determine the hemocompatibility of biomaterials, especially when the drug is to be administered intravenously [33,34]. Fresh mice blood was used to evaluate the hemolytic property of the formulations. Different concentrations of NEs and free KP were tested, as well as the respective blank NEs (data not shown). Fig. 5 shows the results of percentage of hemolysis for NEs containing drug and free KP. No statistical difference was observed (p > 0.05) among the formulations, regardless of the pullulan concentration. At the highest concentration tested (50 g/mL), KP-loaded NE showed around 70% of hemolysis. This higher hemolytic activity can be explained by the higher surface area of the nanocarriers, which improves the contact area with the erythrocytes, especially if the drug is associated more externally [35,36]. According to the literature, the accepted limits should vary between 1 and 5% for spontaneous hemolysis [37,38]. Therefore, 50 g/mL was not considered suitable for intravenous administration. However, at 1 g/mL the hemolysis was less than 1% for NE 0.5 KP, while free KP and the other NE induced about 5% of hemolysis. The most common intravenous dose of ketoprofen is 1 mg/kg, depending on the therapeutic goal (anti-inflammatory, analgesic or antipyretic). However, no dosage regime has yet been established for ketoprofen in the treatment of glioma tumors. Considering that nanostructured systems have presented site-specific delivery for anticancer drugs, it is possible that the intravenous dose of ketoprofen in nanoemulsion is lower than the dose employed using conventional therapy. Thus, we believe that 1 g/mL is a good reference for the hemolysis test in this case.
Fig. 5. Percentage of hemolysis at concentration range of 1–50 g/mL for free KP and NE, analyzed by One-way ANOVA. Values of p > 0.05 was considered not significant. Each line represents the mean with S.E.M. of triplicate.
According to the literature, surfactant molecules can solubilize the lipids and proteins and destabilize the membrane of the erythrocytes [36]. In this case, our formulations employing pullulan instead of conventional surfactants can be an alternative for intravenous dosage forms. 3.5. Cell viability assay in glioblastoma cells (C6) and fibroblasts cell line (3T3) In order to determine the antitumor activity of formulation, free KP, NE 1.5 KP and blank formulation were incubated with C6 cells at two different concentrations (50 and 100 M). After 72 h of incubation, NE 1.5 KP presented 40% inhibition of cell growth (Fig. 6b), which can be considered a promising result regarding that GBM possess high proliferation rate and degree of invasiveness [6,39]. Probably, the small droplet size of NE provides a high contact area with the cell membrane which increases the input of KP. Regarding the literature, the incorporation of antineoplasic agents in nanocarriers can improve antitumor activity [40]. It can be noted that NE 1.5 B did not present antitumor activity, indicating that the effect is due to the presence of KP. Moreover, the formulations tested did not exhibit cytotoxicity action against fibroblasts (Fig. 6a), when used as a non-transformed cells model. In spite of this promising result, the blood–brain barrier represents an obstacle to great part of the antitumor agents. However, the use of nanocarriers is a promising approach to improve the treatment efficiency of brain tumors. Recently, our group found that the encapsulation of KP in poly--caprolactone nanocapsules inhibited glioma growth in vivo, showing, therefore, the ability of these nanostructures to cross the blood-brain barrier. In this work,
Fig. 6. Antiproliferative effect of free KP, NE 1.5 KP and NE 1.5 B by MTT assays. (a) Fibroblasts cell line and (b) glioma cell line (C6). Each column represents the mean with S.E.M. of triplicate. Asterisks denote the significance level compared to the control group (p < 0.05), analyzed by One-Way ANOVA, followed by the Tukey’s test.
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