Nanoencapsulation of gallic acid and evaluation of its cytotoxicity and antioxidant activity

Materials Science and Engineering C 60 (2016) 126–134

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Nanoencapsulation of gallic acid and evaluation of its cytotoxicity and antioxidant activity Aline de Cristo Soares Alves, Rubiana Mara Mainardes, Najeh Maissar Khalil Department of Pharmacy, Universidade Estadual do Centro-Oeste, Rua Simeão Camargo Varela de Sá 03, 85040-080, Guarapuava, PR, Brazil

a r t i c l e

i n f o

Article history: Received 17 August 2015 Received in revised form 15 October 2015 Accepted 5 November 2015 Available online 11 November 2015 Keywords: Nanoparticles Gallic acid Hemolysis Antioxidant

a b s t r a c t Gallic acid is an important polyphenol compound presenting various biological activities. The objective of this study was to prepare, characterize and evaluate poly(lactic-co-glycolic acid) (PLGA) nanoparticles coated or not with polysorbate 80 (PS80) containing gallic acid. Nanoparticles coated or not with PS80 were produced by emulsion solvent evaporation method and presented a mean size of around 225 nm, gallic acid encapsulation efficiency of around 26% and zeta potential of −22 mV. Nanoparticle formulations were stable during storage, except nanoparticles coated with PS80 stored at room temperature. In vitro release profile demonstrated a quite sustained gallic acid release from nanoparticles and PS80-coating decreased drug release. Cytotoxicity over red blood cells was assessed and gallic acid-loaded PLGA nanoparticles at all analyzed concentrations demonstrated lack of hemolysis, while PS80-nanoparticles containing gallic acid were cytotoxic only in higher concentrations. Antioxidant potential of nanoparticles containing gallic acid was assessed and PLGA uncoated nanoparticles presented greater efficacy than PS80-coated PLGA nanoparticles. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Central Nervous System (CNS) disorders affect a large percentage of the world population. Neurodegenerative diseases such as Alzheimer's, Parkinson's, Amyotrophic Lateral Sclerosis and Multiple Sclerosis result in debilitating conditions, and studies have shown the influence of oxidative stress in the emergence and worsening of these diseases [1,2]. The therapeutic efficacy of treatment of these diseases is limited due to the blood–brain barrier (BBB) regulating drug entry into the brain [3]. Recent and rapid advancement in nanotechnology is an opportunity to circumvent these challenges. Pharmaceutical nanotechnology uses nanoparticles as colloidal drug carriers for biological applications. These carriers can present variable structural arrangement and physicochemical characteristics, making possible its application in prolonged/controlled drug release. Polymeric nanoparticles are often used to improve drug solubility, bioavailability, half-life, efficacy, specificity and tolerability, also protect the drug from premature degradation and depending on surface modification they can be targeted to a selected tissue [4,5]. Moreover, nanoparticles have been used as drug carrier systems capable of overcoming BBB [6], mainly nanoparticle surfaces coated with polysorbate 80 (PS80) [7,8]. Biodegradable polymeric nanoparticles are easily eliminated from the body and they are preferred than nonbiodegradable nanoparticles. Poly(lactic-co-glycolic acid) (PLGA) is one of the most used synthetic polymer for nanoencapsulation due to its

E-mail address: [email protected] (N.M. Khalil).

http://dx.doi.org/10.1016/j.msec.2015.11.014 0928-4931/© 2015 Elsevier B.V. All rights reserved.

great biocompatibility and biodegradability, with approval by US Food and Drug Administration (FDA) for human use [4,9]. Gallic acid (GA, 3,4,5-trihydroxylbenzoic acid) is an important polyphenol compound present in various fruits, nuts, green tea and red wine with various biologic activities. GA is a potent antioxidant, with the ability to scavenge the DPPH (2,2-Diphenyl-1-picrylhydrazyl), radical more effectively than vitamin E [10,11,12]. Further studies have reported on its neuroprotective capacity [13,14,15], and anticancer [16,17,18], antiinflammatory [19,20], cardiovascular protective [21,22], hepatoprotective [23,24] and gastroprotective activities [12]. However, the therapeutic application of GA is limited by its pharmacokinetic drawbacks. Studies involving oral administration of GA in animals [25,26] showed low bioavailability of this phenolic compound. Although GA absorption is fast, the maximum plasmatic drug concentration (Cmax) reached is low. Besides poor absorption, fast metabolism results in rapid drug elimination. The major metabolites of GA are 4-Omethylgallic acid and pyrogallol, subsequently metabolized in other metabolites excreted in the urine. These metabolites possess inferior antioxidant potential compared to GA [27]. Since therapeutic efficacy is related to drug bioavailability, alternatives tools are necessary to overcome the pharmacokinetics limitations of GA, a molecule with great biological potential applications. The study of Bhattacharyya and coworkers [25] shows the improved antioxidant potential of GA due to its enhanced bioavailability by the formation of a complex of GA with phospholipids. Also, improving the brain distribution of GA is important to effectively improve its neuroprotective effects. A recent work of our group showed the improved neuroprotective effect of resveratrol (polyphenolyc compound presenting low oral bioavailability) when

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loaded in PS80-coated nanoparticles in an animal model of Parkinson's disease [28]. Thereby, the present study proposes the development of PLGA nanoparticles or PS80-coated PLGA nanoparticles containing GA, its physicochemical characterization, physical stability and evaluation of its antioxidant and hemolytic activity. 2. Materials and methods 2.1. Materials 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), polyvinyl alcohol (PVA, MW 31 KDa, 88% hydrolyzed), poly(lactide-co-glycolide) (PLGA, lactide-to-glycolide ratio, 50:50, MW 30-60 KDa), potassium bromide, potassium persulfate, and polysorbate 80 (PS80) were purchased from Sigma-Aldrich® (St. Louis, MO, US). Analytical grade ethyl acetate, dichloromethane, and dimethyl sulfoxide were obtained from Química Moderna® (Brazil). Acetic acid, gallic acid (GA - 99%), and sucrose were purchased from Vetec® Química Fina (Brazil). Sodium chloride, sodium phosphate dibasic, and sodium phosphate monobasic were purchased from Biotec® (Brazil). HPLC grade acetonitrile was purchased from MTedia® (Brazil). Water was purified in a Milli-Q Plus system (Millipore®) with a resistivity of 18.2 MΩ·cm−1. 2.2. Instrumentation and HPLC method conditions HPLC analysis was performed using a Waters 2695 Alliance HPLC system (Milford, MA, US) equipped with a photodiode array wavelength detector (PDA) (Waters 2998), a column compartment with a temperature controller, an on-line degasser, a quaternary pump and an autosampler. Analysis was achieved using a reverse phase C18 column (Xterra Waters®) with a 5 μm particle size, 4 mm internal diameter, and 125 mm length (LiChrospher® 100). Data acquisition, analysis, and reporting were performed using Empower Chromatography Software (Milford, MA, US). HPLC assay was performed using isocratic conditions. Mobile phase consisted of acetonitrile, pure water and acetic acid aqueous solution at 0.4% (v/v) (50:30:20,v/v/v). All the samples (diluted in acetonitrile: water, 40:60, v/v) were filtered through a 0.22 μm membrane prior to injection and ultrasonically degassed prior to use. Fifty microliters of the sample was injected at a flow rate of 0.9 mL/min. Detection was performed at a wavelength of 271 nm. Time analysis was of 2.5 min with a controlled temperature in the column and sample of 30 °C and 25 °C, respectively. Validation of this HPLC method was previously performed through the following parameters: linearity, limit of detection, limit of quantitation, accuracy, robustness, precision, and specificity. The concentration range varied from 10.0 to 80.0 μg/mL. Linearity was 0.9997, and the detection limit was 23.82 ng/mL. 2.3. Preparation of PLGA and PLGA coated PS80 nanoparticles containing GA PLGA nanoparticles containing GA (NP-PLGA-GA) or PS80-coated PLGA nanoparticles containing GA (NP-PLGA/PS80-GA) were prepared using a single emulsion solvent evaporation technique. Briefly, 50 mg of PLGA and 10 mg of GA were dissolved in 1.0 mL of dichloromethane, 0.7 mL of ethyl acetate and 0.3 mL of dimethyl sulfoxide, constituting the organic phase. The aqueous phase, 6 mL of aqueous solution PVA at 0.5% (w/v) was added to the organic phase and emulsified by sonication over 5 min, to produce an oil-in-water emulsion, followed by solvent evaporation under reduced pressure (600 mm Hg, 37 °C, 30 min). Separation of non-incorporated drug was performed by ultracentrifugation at 15,500 rpm for 30 min, at 4 °C and then the collected nanoparticles were washed in pure water using previously described ultracentrifugation. Supernatants were stored for posterior analysis.

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Considering the formulation NP-PLGA/PS80-GA, the PS80 coating was performed after washing of NP-PLGA-GA nanoparticles. PS80 aqueous solution 1% (v/v) was added to resultant nanoparticles and stirring at 300 rpm for 30 min at 37 °C. Nanoparticle dispersion was centrifuged at 15,500 rpm for 30 min, at 4 °C. Both nanoparticle formulations were dispersed in a sucrose solution 5% (w/v), in order to avoid aggregation of nanoparticles during storage in temperature of −20 °C. When necessary for some analysis, nanoparticles were freeze dried for 24 h to obtain lyophilized nanoparticles. Empty nanoparticles of PLGA (NP-PLGA) and PLGA coated PS80 (NPPLGA/PS80) were prepared as described by procedures only omitting GA in the organic phase. 2.4. Physicochemical characterization 2.4.1. Nanoparticles size, zeta potential and morphological analysis Nanoparticle size distribution, mean particle size and polydispersity index (PI) were assessed by Dynamic Light Scattering (DLS) (BIC 90 Plus-Brookhaven Instruments Corp., US). Zeta potential was evaluated using a Zetasizer Nano S90 (Malvern Instruments, UK). Morphology of the nanoparticles was investigated by scanning electron microscopy equipped with a field emission gun (FE-SEM) (MIRA3 LM, Tescan Orsay Holding, Czech Republic) at an accelerating voltage of 5.0 kV. 2.4.2. Determination of drug encapsulation efficiency GA encapsulation efficiency was determined indirectly [29]. An aliquot of supernatants resultants of ultracentrifugation was appropriately diluted (1:20, v/v) in water: acetonitrile (60:40, v/v) and analyzed by HPLC method validated previously. The amount of GA loaded into nanoparticles was expressed as encapsulation efficiency (EE %), according to Eq. (1): EE% ¼ AI −AA =AI  100

ð1Þ

where AI is the initial amount of GA, and AA is the analytical amount determined by HPLC method. 2.4.3. X-ray diffraction, Fourier-transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analysis X-ray diffraction (XRD) patterns of the GA, NP-PLGA, NP-PLGA-GA, NP-PLGA/PS80 and NP-PLGA/PS80-GA were evaluated by X-ray diffractometer (D2 Phaser, Bruker, Germany). Fourier-transform infrared spectroscopy (FT-IR) of GA, NP-PLGA, NP-PLGA-GA, NP-PLGA/PS80 and NP-PLGA/PS80-GA and physical mixture of drug and polymer were determined by Nicolet IR200 FT-IR Spectrometer (Thermo Scientific, US). Thermograms of GA, NP-PLGA, NP-PLGA-GA, NP-PLGA/PS80 and NPPLGA/PS80-GA were obtained by SDT Q600 Simultaneous DSC-TGA (TA Instruments, US). 2.5. In vitro drug release assay The in vitro release profile of GA from nanoparticles was determined by dissolution followed by ultracentrifugation. In vitro release assay of GA from NP-PLGA and NP-PLGA/PS80 was assessed suspending a volume of nanoparticles containing approximately 1.0 mg of GA into 6 mL of release medium (PS80 aqueous solution of 1% (w/v)) and maintained in an orbital shaker incubator at 37 °C at 150 rpm. At specified time intervals, samples were subjected to ultracentrifugation at 15,500 rpm, at 4 °C for 15 min. Supernatants were directly assayed by HPLC method previously validated to quantify the GA content. Precipitates were redispersed in PS80 solution and incubated again. Analyses were performed for five days and during this period sink conditions were maintained. GA water solubility is 10 mg/mL [30], thus the GA concentration used for this experiment was 1.6% of saturation concentration. The kinetic analyses of the release data were performed using

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various mathematics models, such as zero order, first order, second order, Higuchi and Korsmeyer–Peppas [31,32].

3. Results and discussion 3.1. Obtaining of PLGA or PS80 coated PLGA nanoparticles containing GA

2.6. Physical stability study Physical stability of NP-PLGA-GA and NP-PLGA/PS80-GA was evaluated under −20 °C (samples dispersed in sucrose aqueous solution 5%) or room temperature of 25 ± 2 °C (dispersed in water), both protect from light. Stability of the formulations was analyzed in terms of mean size, PI and zeta potential in the periods of 0, 1, 2, 3, 4, 8 and 12 weeks, in triplicate. 2.7. Biologic characterization 2.7.1. Evaluation of cytotoxicity over erythrocytes Blood was collected from healthy volunteers according to the experimental protocol approved by the Institutional Human Ethics Committee of the Universidade Estadual do Centro-Oeste, Brazil (Registration no. 501.330/2013), into tubes with heparin. Heparinized blood was centrifuged for 5 min at 2500 rpm. Supernatant was removed and erythrocytes were washed three times with phosphate buffer sodium (PBS) (10 mM, pH 7.4 and 0.85% NaCl) containing glucose (1.08 mg/mL), penicillin G (0.3 mg/mL) and streptomycin (0.5 mg/mL). Erythrocytes were diluted in PBS until a 2.0% hematocrit was reached. Thereafter, erythrocyte suspensions were incubated with GA, NP-PLGA-GA or NP-PLGA/ PS80-GA in concentrations of 80, 40 or 20 μg/mL at 37 °C. In determined times (24, 48 or 72 h), an aliquot of each sample was withdrawn and centrifuged for 5 min at 2500 rpm. The amount of hemoglobin released from erythrocytes was measured by absorbance of the resultant supernatants at 540 nm, representing the extension of hemolysis. Hemolysis percentage was calculated according to Eq. (2): %Hemolysis : Abs =Abc  100

ð2Þ

where, Abs is the sample absorbance and Abc, the absorbance of sample treated with deionized water, representing 100% of hemolysis. 2.7.2. Determination of antioxidant activity in vitro Antioxidant activity of GA, NP-PLGA-GA and NP-PLGA/PS80-GA was assessed by colorimetric measure of radical cation 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), as described elsewhere [33], with some modifications. Radical ABTS•+ was generated by incubation of ABTS (7 mM) with potassium persulfate (140 mM) into pure water in the dark at room temperature for 12 h before use. ABTS•+ solution was diluted to an absorbance of 0.8 at 734 nm in a 50 mM phosphate buffer pH 7.4. Samples of GA, NP-PLGA-AG and NP-PLGA/PS80-AG with GA concentrations of 15.62, 7.81 or 3.90 μg/mL were incubated in PB (10 mM, pH 7.4) under stirring of 150 rpm at 37 °C. In determined times (1, 2, 4, 8, 24 and 48 h), samples aliquots were mixed to ABTS• + solution and absorbance measured at 734 nm. All determinations were performed in triplicate. The results were expressed as inhibition percentage, according to Eq. (3): %Inhibition ¼ Ac −As =Ac  100

ð3Þ

where, Ac is the control absorbance and As, the sample absorbance. 2.8. Statistical analysis Results are presented as mean ± SD. The significance of differences between means was evaluated by variance analysis (p ≤ 0.05) with Tukey posttest.

Emulsion solvent evaporation technique is most commonly used to produce PLGA nanoparticles. GA has a relatively high theoretical solubility (10 mg/mL, log P = − 0.53 ± 0.05) [30], thus, the nanoencapsulation technique recommended for hydrophilic drugs is the multiple emulsification (water-in-oil-in-water) followed by solvent evaporation. Nanoparticles containing GA were initially developed by this technique, but the greater entrapment efficiency reached was 13.9 ± 1.3% (n = 3). Nanoprecipitation method also was tested and the results also showed low entrapment efficiency of 13.1 ± 6.6% (n = 3). Therefore, nanoparticles obtained by simple emulsification (oil-in-water) solvent evaporation technique were optimized, since entrapment efficiencies greater than 20% were obtained. The chosen surfactant was PVA. In high PVA concentrations, interfacial tension is decreased resulting in lower particles concluding that PVA concentration is inversely proportional to nanoparticles size, as noticed by Mainardes & Evangelista [34]. However, higher PVA concentrations increase the viscosity of aqueous external phase resulting in higher particles. Thereby, the chosen concentration should be adequate to decrease the superficial tension without changing the medium viscosity [35]. In this study, PVA concentration determined with more favorable results was of 0.5% (w/v). GA has great solubility in dimethyl sulfoxide and PLGA in dichloromethane, and thus, both solvents were essential in formulations. Presence of ethyl acetate in formulations assisted in the reduction of nanoparticle size. Sonication time of 5.0 min resulted in nanoparticles with suitable size and PI.

3.2. Nanoparticle size, zeta potential and morphology NP-PLGA-GA and NP-PLGA/PS80-GA had a mean size of 223 ± 12 nm (n = 10) and 228 ± 11 nm (n = 10), respectively. PI is a parameter determining particle dispersion assessing how the size of each nanoparticle turned away from the mean size. Therefore, a lower PI means a narrow size distribution. A good condition is achieved when PI is lower or equal to 0.1, which shows that particles present homogeneity in size [36]. The PI of NP-PLGA-GA and NP-PLGA/PS80-GA was 0.06 ± 0.02 (n = 10) and 0.05 ± 0.02 (n = 10), respectively, confirming the ideal size distribution. In Fig. 1(A) we represented the size distribution of one produced batch of NP-PLGA-GA with a mean size of 240 nm, PI of 0.033 and size distribution of 100% in the range from 228 to 231 nm. Nagpal, Singh & Mishra [37] developed chitosan nanoparticles containing GA with a mean size of 166 ± 5 nm, however with a high PI value of 0.24 ± 0.08. Thus, nanoparticles developed in this study demonstrate the effectiveness and reproducibility of the method to produce formulations with a mean size, distribution and PI that are satisfactory to the GA carrier. Zeta potential of NP-PLGA-GA and NP-PLGA/PS80-GA was −22.18 ± 1.57 mV (n = 3) and −22.50 ± 1.57 mV (n = 3), respectively, confirming the PLGA and PS80 characteristic of providing a negative potential to the nanoparticle surface. A zeta potential value approaching −30 mV or + 30 mV is essential to avoid aggregation of nanoparticles in suspension [38]. FE-SEM images of nanoparticles revealed their regular spherical shape and range of size (Fig. 1B and 1C). Morphological analysis of both formulations proved the presence of nanoparticles with diameter next to 200 nm. Sizes were similar to those determined by DLS technique and consistent with emulsification solvent evaporation technique producing nanospheres.

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Fig. 1. Size distribution profile of PLGA nanoparticles containing GA (A); SEM images of PLGA nanoparticles containing GA (B) and PS80-coated PLGA nanoparticles containing GA (C).

3.2.1. Determination of drug encapsulation efficiency Encapsulation efficiency obtained by GA in NP-PLGA and NP-PLGA/ PS80 nanoparticles was 27 ± 4% (n = 10) and 25 ± 5% (n = 10), respectively. Both formulations presented low entrapment efficiency, however consistent with GA aqueous solubility. GA has hydrophilic characteristics constituting a challenge in its encapsulation because of rapid drug portioning to external aqueous phase.

3.2.2. X-ray diffraction (XRD) XRD patterns of GA, NP-PLGA, NP-PLGA-GA, NP-PLGA/PS80 and NPPLGA/PS80-GA are shown in Fig. 2. GA XRD pattern (Fig. 2A) showed several distinct characteristic peaks at diffraction angles of 11.86°, 16.3° and 18.83°, confirming the crystalline form of GA. These characteristics were not observed in XRD of GA loaded in coated (Fig. 2E) or uncoated (Fig. 2C) PS80 nanoparticles, evidencing entire drug molecularly dispersion in polymeric matrix in their amorphous state. Empty nanoparticles also presented in amorphous form (Fig. 2B and 2D). This change in the crystalline character of GA to amorphous state is favorable, whereas amorphous forms are more absorbed than crystalline increasing its bioavailability [39].

3.2.3. Fourier-transform infrared spectroscopy FTIR spectra of GA, physical mixture of GA and PLGA, NP-PLGA, NPPLGA-GA, NP-PLGA/PS80 and NP-PLGA/PS80-GA are presented in Fig. 3. Characteristics bands of GA (Fig. 3A) appear at 3409 cm− 1 due to O–H stretching and at 1715 cm− 1 for the CO stretching of carboxylic acid. The bands at 1540 and 1470 cm− 1 are due to CC aromatic ring stretching vibration, at 1029 cm − 1 related to C–H aromatic ring stretching and at 868 cm− 1 referring to the C–H aromatic ring outside stretching with tetra-replacement in 1,3,4,5, which agreed with previously studies [40]. FTIR of physical mixture of PLGA and GA (Fig. 3B) showed the peak absorption at 3490 cm−1 of the O–H stretching vibration presents in NP-PLGA and GA spectrum. Absorption peak at 1530 cm− 1 refers to the CC stretching vibration of GA phenyl. In absorption band 881 cm−1, refer to the C–H stretching of AG. NP-PLGA spectra (Fig. 3C) and NP-PLGA/PS80 spectra (Fig. 3E) presented the peak of hydroxyl group of the PLGA at 3477 cm−1, the CH, CH2 and CH3 stretching vibrations of simples bindings occur from 2950 to 3004 cm−1, at 1760 cm−1 refers the CO stretching vibrations and from 1097 to 1179 cm−1 the C–O stretching. FTIR of NP-PLGA-GA (Fig. 3D) and NP-PLGA/PS80-GA (Fig. 3F) showed no significant changes

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Fig. 2. X-ray diffractogram of GA (A); PLGA nanoparticles (B); PLGA nanoparticles containing GA (C); PS80-coated PLGA nanoparticles (D) and PS80-coated PLGA nanoparticles containing GA (E).

between the spectra due to superposition of PLGA bands on GA bands, suggesting nanoencapsulation process did not alter chemical structure of GA.

3.2.4. Differential scanning calorimetry and thermogravimetric analysis Results of DSC and TG of samples are presented in Fig. 4. GA showed an endothermic peak next to 100 °C (Fig. 4A) which is probably due to water loss (ΔH = 294.3 J/g), indicating its crystalline nature. The second peak at 266.78 °C corresponding to its melting point (ΔH = 437.8 J/g). TG curve confirms weight loss refers to water loss (10.8%) and its melting point (52.1%) occurs in same temperature range as DSC curve. DSC curve of NP-PLGA (Fig. 4B) showed an endothermic peak at 296.97 °C (ΔH = 173.4 J/g). When encapsulated in the PLGA nanoparticles, GA original peaks disappeared (Fig. 4C) with presence only melting point peak at 303.65 °C (ΔH = 287.4 J/g), indicating the formation of an amorphous inclusion complex. Rosa et al. [41] described same profile of GA protection for polymeric matrix when loaded in chitosan, ciclodextrin and xantan microparticles. TG curves indicated weight loss in melting point of NP-PLGA (93.6%) (Fig. 4B) and NP-PLGA-GA (100%) (Fig. 4C) in the same temperature range as DSC curve. DSC curve of NP-PLGA/PS80 (Fig. 4D) showed melting point at 311.29 °C (ΔH = 300.7 J/g), a higher temperature than NP-PLGA indicating the presence of PS80 in the nanoparticle surface. NP-PLGA/ PS80-GA curve (Fig. 4E) presented a melting point peak at 365.77 °C (Δ H = 213.5 J/g). Comparing the curves, it is suggested that PS80 is an additional protection for thermal stability of GA loaded in PLGA nanoparticles. In TG curve, weight loss of NP-PLGA/PS80 (Fig. 4D) and NP-PLGA/PS80-GA (Fig. 4E) was 95% and 93.6%, respectively, in same temperature as DSC curve. These results corroborate with XRD data suggesting GA dispersion into polymeric matrix. Further, both formulations were thermally more stable than free drug. 3.3. In vitro drug release assay The release profile of GA from NP-PLGA and NP-PLGA/PS80 is represented in Fig. 5. Within 72 h, 20 and 12% of GA was released from NPPLGA and NP-PLGA/PS80, respectively, indicating a very sustained drug release. GA entrapped in NP-PLGA/PS80 was released slower than when entrapped in NP-PLGA probably due to PS80 presence in surface nanoparticles constituting an additional barrier to GA release. This characteristic can be advantageous to brain release, since it is ideal to release the drug only after reaching the CNS. Mathematical modeling demonstrated a release profile of GA into nanoparticles followed by a biphasic model for both formulations. This model determines the drug release process from nanoparticles which occurs at two different rates [31]. The release constant of NP-PLGA-GA was of 0.0005 h− 1 in fast phase and 0.232 h− 1 in slow phase, while NP-PLGA/PS80-GA presented release constant in fast phase of 0.0004 h−1 and 0.164 h−1 in slow phase. The first stage was initially fast, called burst effect, probably related to the desorption of surfaceadsorbed drug whereas the second stage was slow reflecting the release of drug within the polymer matrix. It is known that nanoparticles developed by emulsification solvent evaporation presented a burst release in the first hours [42]. A higher GA initial release could in vivo allow reaching a higher drug concentration in plasma faster, while the slow posterior release helps to maintain the drug concentration for a long period. The data obtained from in vitro GA release assay were also fitted in the Korsmeyer–Peppas model. The found values of release exponent (n) for NP-PLGA-GA and NP-PLGA/PS80-GA were 0.13 and 0.29, respectively. For spherical forms, n value is less than 0.43 indicating that the drug release mechanism occurs by Fickian diffusion [32]. 3.4. Physical stability

Fig. 3. FT-IR of GA (A); physical mixture of GA and PLGA (B); PLGA nanoparticles (C); PLGA nanoparticles containing GA (D); PS80-coated PLGA nanoparticles (E) and PS80-coated PLGA nanoparticles containing GA (F).

Fig. 6 represents the results of physical stability of NP-PLGA-GA and NP-PLGA/PS80-GA, considering mean size, PI and zeta potential, over 12 weeks. Mean size of NP-PLGA-GA (Fig. 6A) and NP-PLGA/PS80-GA

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Fig. 4. DSC and TG thermogram of GA (A); PLGA nanoparticles (B); PLGA nanoparticles containing GA (C); PS80-coated PLGA nanoparticles (D) and PS80-coated PLGA nanoparticles containing GA (E).

(Fig. 6B) did not change (p N 0.05) in both storage conditions. Similarly, PI was not changed over 12 weeks (Fig. 6C and 6D). NP-PLGA-GA maintained (p N 0.05) zeta potential after 12 weeks of storage at room temperature or at − 20 °C (Fig 6E) but NP-PLGA/PS80-GA stored at room temperature maintained zeta potential until 8 weeks (Fig 6F). When stored at −20 °C the formulation was stable. 3.5. Biologic characterization

Fig. 5. In vitro release of GA from PLGA nanoparticles and PS80-coated PLGA nanoparticles (n = 3).

3.5.1. Evaluation of cytotoxicity over erythrocytes GA, NP-PLGA and NP-PLGA-GA presented no hemolytic activity in erythrocytes in all tested concentrations (20, 40 or 80 μg/mL) after 72 h (results not shown for NP-PLGA and NP-PLGA-GA). Fig. 7 shows the results of percentage of hemolysis of GA, NP-PLGA/PS80 and NPPLGA/PS80-GA in concentrations of 80, 40 or 20 μg/mL after 24, 48 and 72 h. Blank NP-PLGA/PS80 and NP-PLGA/PS80-GA in concentrations of 20 or 40 μg/mL showed no cytotoxic effect on red blood cells, but in concentration of 80 μg/mL after 48 h induced low hemolysis and significant hemolysis after 72 h. Free GA and NP-PLGA-GA at the same concentration presented no significant hemolysis after 72 h, indicating that PS80

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Fig. 6. Physical stability of nanoparticles over 12 weeks. Mean size of PLGA nanoparticles containing GA (A) and PS80-coated PLGA nanoparticles containing GA (B) (n = 3). Polydispersity index of PLGA nanoparticles containing GA (A) and PS80-coated PLGA nanoparticles containing GA (B) (n = 3). Zeta potential of PLGA nanoparticles containing GA (A) and PS80-coated PLGA nanoparticles containing GA (B) (n = 3).

presence on nanoparticles surface caused hemolysis. PS80 can act as a surfactant in formulations and consequently can induce hemolysis, justifying these results. In graph, NP-PLGA/PS80 (1) present more hemolysis than other two blank nanoparticles due to the higher volume used for incubation (the volume of blank nanoparticles used

was the same of drug-loaded nanoparticles), thus, presenting more PS80. Our results indicate PLGA-GA nanoparticles were compatible with erythrocytes, but NP-PLGA/PS80 can be capable to cause membrane damage with a concentration-dependent profile due to PS80 presence.

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the lowest antioxidant activity, being significantly inferior (p b 0.05) to NP-PLGA-GA and free GA. This result corroborates with its very slow release profile found in release in vitro assay. It suggests that GA loaded in coated nanoparticles confront additional barrier to be release, presenting more prolonged release than only PLGA nanoparticles, reflecting in its in vitro antioxidant activity reduced. Although this result, in vivo we can expect a better activity of drugloaded nanoparticles, since nanoparticles can improve biopharmaceutical and pharmacokinetic properties of drug-loading, resulting in pharmacological responses more evident than free drug. However, in vivo studies are necessary to confirm it and its possible antioxidant and neuroprotective effects. 4. Conclusions

Fig. 7. Hemolysis percentage of GA, PS80 coated PLGA nanoparticles (blank, the same weight/volume of nanoparticles containing GA) and PS80-coated PLGA nanoparticles containing GA. [1] refers to GA concentration of 80 μg/mL, [2] of 40 μg/mL and [3] of 20 μg/mL. a, b mean ± SD (n = 3) analyzed each 24 h, where equal words means statistic equality (ANOVA with Tukey posttest and α 0,05).

3.5.2. Determination of antioxidant activity in vitro Antioxidant activity of GA and nanoparticles containing GA was evaluated by colorimetric measure of radical cation ABTS•+ and results of radical inhibition percentage are showed in Table 1. GA presented stronger scavenger activity in all concentrations tested and a decrease of its activity was observed only after 48 h in the lower concentration. After 2 h, in higher GA concentration, NP-PLGA-GA showed similar percentage of radical inhibition to be obtained by free GA, in higher GA concentration, and it was maintained until 24 h (p N 0.05). The burst effect of PLGA nanoparticles can explain the initial antioxidant activity, but the slow drug release is not able to produce the same intensity of activity of free drug in vitro, and thus explain the decrease in antioxidant activity after 48 h. NP-PLGA/PS80-GA presented

Nanoparticles were successfully developed by a single-emulsion solvent evaporation technique with mean particle size in the range of 220 nm, low polydispersity index and drug encapsulation efficiency of around 25%. The encapsulated drug was converted to an amorphous state in the polymer matrix and no interactions between drug and polymer occurred after nanoencapsulation. In vitro release study revealed a prolonged gallic acid release with a byphasic profile controlled by diffusion. PS80 coated nanoparticles presented more prolonged gallic acid release and thus inferior antioxidant activity than PLGA nanoparticles. PLGA nanoparticles did not show cytotoxicity over erythrocytes whereas PS80-coated ones were hemolytic depending on PS80 amount. Declaration of interest The authors state no conflict of interest in this study. Acknowledgements This study was supported by CAPES in the form of a scholarship to A.C.S. Alves. References

Table 1 ABTS•+ radical inhibition percentage of GA, PLGA nanoparticles containing GA and PS80 coated PLGA nanoparticles containing GA. GA final concentration 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL 15.62 μg/mL 7.81 μg/mL 3.90 μg/mL a, b, c

NP-PLGA-GA 0h 85.44 ± 3.80b 66.62 ± 11.19b 37.47 ± 5.14b 2h 96.00 ± 4.78a 73.82 ± 10.76b 49.58 ± 10.79b 4h 97.67 ± 4.03a 80.96 ± 9.36b 45.60 ± 6.82b 8h 94.07 ± 8.10a 64.46 ± 11.96b 39.53 ± 3.93b 24 h 87.27 ± 7.57a 51.74 ± 8.65b 30.77 ± 6.31b 48 h 83.37 ± 6.34b 39.89 ± 8.45b 24.67 ± 3.71b

NP-PLGA/PS80-GA

GA

46.42 ± 7.18c 25.26 ± 6.56c 15.60 ± 5.62c

99.83 ± 0.29a 99.87 ± 0.00a 99.37 ± 0.25a

49.35 ± 10.14b 30.67 ± 1.45c 18.22 ± 4.79c

98.93 ± 0.46a 99.16 ± 0.07a 99.08 ± 0.52a

54.09 ± 6.42b 30.48 ± 2.28c 17.11 ± 0.92c

99.48 ± 0.14a 99.64 ± 0.00a 99.48 ± 0.49a

42.45 ± 6.74b 24.29 ± 2.44c 15.48 ± 1.08c

99.48 ± 0.14a 99.64 ± 0.00a 99.48 ± 0.49a

38.13 ± 3.52b 22.64 ± 4.12c 15.44 ± 1.18c

99.12 ± 0.30a 94.47 ± 0.62a 92.07 ± 0.79a

30.94 ± 4.41c 16.04 ± 1.36c 13.03 ± 1.68c

99.50 ± 0.12a 93.56 ± 0.45a 69.01 ± 2.01a

mean ± SD (n = 3) analyzed each 24 h, where equal words signify statistic equality (ANOVA with Tukey posttest and α 0,05).

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