Tamoxifen-loaded poly(L-lactide) nanoparticles: Development, characterization and in vitro evaluation of cytotoxicity

Materials Science and Engineering C 60 (2016) 135–142

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Tamoxifen-loaded poly(L-lactide) nanoparticles: Development, characterization and in vitro evaluation of cytotoxicity Clescila Altmeyer, Thaysa Ksiaskiewcz Karam, Najeh Maissar Khalil, Rubiana Mara Mainardes ⁎ Universidade Estadual do Centro-Oeste/UNICENTRO, Laboratory of Pharmaceutical Nanotechnology, 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 26 June 2015 Received in revised form 1 November 2015 Accepted 6 November 2015 Available online 10 November 2015 Keywords: Nanoparticles Poly(L-lactide) Hemolysis Cytotoxicity Tamoxifen

a b s t r a c t In this study, poly(L-lactide) (PLA) nanoparticles containing Tamoxifen (Tmx) were developed using an emulsion/solvent evaporation method, observing the influence of surfactants and their concentrations on mean particle size and drug entrapment. Nanoparticles were characterized in terms of size, morphology, polydispersity, interaction drug-polymer and in vitro drug release profile. Cytotoxicity over erythrocytes and tumor cells was assessed. The optimized formulation employed as surfactant 1% polyvinyl alcohol. Mean particle size was 155 ± 4 nm (n = 3) and Tmx encapsulation efficiency was 85 ± 8% (n = 3). The in vitro release profile revealed a biphasic release pattern diffusion-controlled with approximately 24% of drug released in 24 h followed by a sustained release up to 120 h (30% of Tmx released). PLA nanoparticles containing Tmx presented a very low index of hemolysis (less than 10%), in contrast to free Tmx that was significantly hemolytic. Tmx-loaded PLA nanoparticles showed IC50 value 2-fold higher than free Tmx, but considering the prolonged Tmx release from nanoparticles, cytotoxicity on tumor cells was maintained after nanoencapsulation. Thus, PLA nanoparticles are promising carriers for controlled delivery of Tmx with potential application in cancer treatment. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tamoxifen [trans-1(4-β-dimethylaminoethoxy-phenyl) 1,2diphenylbut-1-ene] (Tmx) has been prescribed to treat patients with breast cancer. It's employed for the long-term (3–5 years) prophylactic therapy in high-risk and pre- and post-menopausal women as well [1,2]. Tmx is selective estrogen receptor modulator (SERM) and belongs to a class of non-steroidal triphenylethylene derivatives. This drug shows potential effects in patients who possess estrogen receptors positive in breast cancer cells by competing with estrogen [3]. One of the problems in cancer chemotherapy is the difficulty in administration of therapeutic drug concentrations and target to the tumor site without toxicity to healthy cells [4,5]. Tmx exhibits high bioavailability upon oral administration, with a half-life of 7 days, and its metabolites may have half-life up to 14 days. Commercially, Tmx is available in salt form of tamoxifen citrate, administered orally as tablet in a daily dose of 10–20 mg [6,7]. Therapy with Tmx has some major side effects, such as the high risk of causing endometrial cancer, oxidative stress mediated hepatotoxicity, multifocal hepatic fatty infiltration, submassive hepatic necrosis, hemolytic anemia, thrombocytopenia and leucopenia [8–12]. Thus, an alternative delivery system is necessary for oral chronic therapy of Tmx with

⁎ Corresponding author. E-mail address: [email protected] (R.M. Mainardes).

http://dx.doi.org/10.1016/j.msec.2015.11.019 0928-4931/© 2015 Elsevier B.V. All rights reserved.

reduced side effects especially hepatotoxicity and hemolytic anemia, to provide greater patient adherence to treatment. Drug delivery systems-based nanoparticles are established to modulate the physicochemical properties of drugs, resulting in improved pharmacokinetic profile, therapeutic efficacy and reduced toxicity [13, 14]. Nanoparticles present reduced size and high surface area that allow them to permeate through biological barriers, in addition provide stability in biological systems [15,16]. The use of nanoparticles promotes prolonged and controlled drug release preventing oscillations of the drug concentration in the bloodstream [17]. Thus, leads to the possibility of reducing the frequency of dosing of the drug, providing better convenience and patient cooperation. The biodistribution of the drug in nanoparticles is increased and the ability of nanoparticles to be uptaked by cells became then promising in improve the drug concentration in tumor cells, mainly due to the enhanced permeability and retention (EPR effect) to tumor tissue. Conventional nanoparticles (without surface modification) promote a passive targeting to tumor cells by the EPR effect, being more permeable to tumor cells (due to more fenestrations of the tissue) and also more retained due to lack of lymphatic around the tumor region, reducing the exposure of the drug to healthy cells [18–20]. Polymeric nanoparticles (NPs) made of biodegradable and biocompatible polymers, could be used as interesting controlled-release system for Tmx and applied for cancer therapy, exploring the EPR effect exerted by nanoparticles and reducing the drug toxicity [21–24]. Poly (L-lactide) (PLA) is a biodegradable polymer with good biocompatibility and widely employed for loading and encapsulation of variety of drugs [25–27].

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In this context, Tmx-loaded PLA nanoparticles were developed, characterized and evaluated for in vitro release characteristics, cytotoxicity over red blood cells and HeLa cell line.

Instruments, UK). The samples were appropriately diluted with KCl 0.1 mM and placed in the electrophoretic cell where a potential of ± 150 mV was applied. Three measurements were made for each sample and the zeta potential was calculated.

2. Materials and methods 2.1. Materials Tamoxifen citrate (Tmx), poly (L-lactide) (PLA, MW: 85–160 kDa), polyvinyl alcohol (PVA, MW 31 KDa, 88% hydrolyzed), poloxamer-188 and polysorbate-80 were purchased from Sigma-Aldrich (USA). Dimethyl sulfoxide (DMSO) and ethyl acetate were purchased from Biotec® (Brazil) and dichloromethane was obtained from FMaia® (Brazil). HPLC-grade methanol was purchased from JTBaker® (USA). Water was purified using a Milli-Q Plus system (Millipore®). MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide), Dulbecco's modified Iscove's medium (DMIM) and antibiotics (penicillin and streptomycin) were purchased from Sigma-Aldrich (USA). All other reagents used were of analytical grade and of the highest purity. 2.2. Preparation of Tmx-loaded PLA nanoparticles PLA nanoparticles containing Tmx (Tmx-NPs) were obtained by an emulsion/solvent evaporation method, as reported earlier [28] with some modifications. Briefly, 50 mg of PLA was dissolved in 1 mL of dichloromethane and added to an organic solution (100 μL of DMSO and 900 μL of ethyl acetate) containing Tmx (5 mg), which was emulsified into a 10 mL of aqueous solution containing the stabilizer (PVA, poloxamer 188 or polysorbate-80, at 1 or 2%), and sonicated over 6 min to produce an oil-in-water (O/W) emulsion. The emulsion was subjected to evaporation under vacuum, with continuous stirring at 37 °C for 20 min. The nanoparticles were isolated from the nonencapsulated drug by ultracentrifugation (19,975 × g, 30 min, 4 °C) and washed with ultrapure water. The precipitate was resuspended in 200 μL of sucrose solution (5%, w/v) and freeze-dried. The resultant supernatants were collected for further analyses. 2.3. Physicochemical characterization of PLA-nanoparticles containing Tmx 2.3.1. Determination of Tmx encapsulation efficiency The amount of Tmx incorporated into nanoparticles was determined indirectly. The supernatant containing free Tmx obtained from the ultracentrifugation of nanoparticles was appropriately diluted in methanol, and the samples were analyzed spectrophotometrically at 254 nm (Jasco V630 BIO). The encapsulation efficiency (EE) was determined in triplicate from the following equation (Eq. (1)). %EE ¼ Ainitial –Afree =Ainitial  100

ð1Þ

Where, Ainitial is the amount of Tmx initially added to the formulation and Afree is the concentration of the unencapsulated drug quantified in the supernatant after ultracentrifugation. 2.3.2. Determination of particle size, polydispersity index and size distribution Mean particle size, polydispersity index and size distribution were determined using dynamic light scattering (DLS) (BIC 90 plus, Brookhaven Instruments Corp.) after its dispersion in water. The analyses were performed at a scattering angle of 90° and a temperature of 25 °C. For each sample, the mean particle diameter, polydispersity and size distribution and the standard deviation of ten determinations were calculated. 2.3.3. Zeta potential The zeta potential of the nanoparticles was measured on the basis of electrophoretic mobility under an electric field (Nano ZS90, Malvern

2.3.4. Particle morphology Morphology of PLA nanoparticles and Tmx-NPs was verified by transmission electron microscopy (TEM – JEOL, JEM 1400). A fresh solution of nanoparticles was diluted in water, then dropped in a carbon coated copper grid, contrasted negatively by a 2% solution of uranyl acetate followed by air drying, and an acceleration voltage of 80 kV was used. 2.3.5. Fourier-transform infrared (FT-IR) spectroscopy Fourier-transform infrared spectroscopy (FT-IR) of Tmx, PLA and Tmx-NPs was determined by Nicolet IR200 FT-IR Spectrometer (Thermo Scientific, US) using the KBr disk method. The scanning range used was from 400 to 4000 cm-1. 2.3.6. X-ray diffraction (XRD) The XRD patterns of the pure Tmx, PLA and Tmx-NPs were obtained using the X-ray diffractometer (D2 Phaser, Bruker, Germany) using CuKα radiation. Samples were scanning angle over the 2θ ranged from 10 to 70°, scanning rate of 0.05°/min, in a wavelength 1.54 Å, at 30 kV tube voltages and 10 mA tube current. 2.3.7. Differential scanning calorimetry Differential scanning calorimetry (DSC) thermogram of the pure Tmx, PLA and Tmx-NPs was obtained by SDT Q600 Simultaneous DSC-TGA (TA Instruments, US), calibrated with metallic zinc, calcium oxalate and sapphire standards. Samples were weighed, placed into sealed hermetically aluminum pans under atmosphere of nitrogen. The scanning rate was 10 °C/min covering temperature range of 30 to 450 °C and nitrogen gas was introduced at the flow rate of 100 mL/min. 2.4. In vitro release assay In vitro release of Tmx from PLA nanoparticles was performed in phosphate buffer saline (PBS) 50 mM, pH 7.4, containing PVA (1%, w/v). PVA was used to assure sink conditions. The study was performed using lyophilized PLA nanoparticles containing 1 mg of Tmx. The Tmx-NPs were suspended in 12 mL of PBS solution and incubated in an orbital shaking incubator at 37 °C and 150 rpm. At predetermined time intervals, the tubes were subjected to ultracentrifugation (19,975 × g) for 15 min. The resultant supernatants were analyzed by a spectrophotometer UV at 254 nm. Precipitates were redispersed in the buffer and incubated until further use. The cumulative concentration of Tmx released was calculated as the mean ± standard deviation and was plotted against time (h) (n = 3). The kinetic analysis of the release data was using mathematics models, such as zero order, first order, second order, Higuchi and Korsmeyer–Peppas [29] through MicroMath Scientist® software. 2.5. Hemolysis assay Hemolysis assay was assessed as method described elsewhere [30] with some modifications. The experimental protocol was approved by the Institutional Human Ethics Committee of the Universidade Estadual do Centro-Oeste, Brazil (Registration no. 023067/2014). Human venous blood obtained from healthy volunteers was collected in tubes containing heparin (10 μL). Whole blood (10 mL) was centrifuged for 5 min at 1200 × g, and the supernatant and buffy coat were removed and discarded. The red blood cells (RBC) were washed three times with solution PBS (pH 7.4, 10 mM and 0.86% NaCl) containing glucose (1.08 mg/mL) and the antibiotics penicillin G (0.3 mg/mL) and streptomycin (0.5 mg/mL). After, RBC were resuspended in PBS at 2%

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hematocrit. Subsequently, the RBC suspension was incubated with either free Tmx citrate (4.4 μM or 1.1 μM) or Tmx-NPs (4.4 μM or 1.1 μM of Tmx) (n = 3), distilled water (positive control), PBS, ethanol and blank nanoparticles. Assay was incubated for 37 °C in a shaking incubator for 4, 12, 24, 48, 72 and 96 h. At pre-determined time intervals, one aliquot was withdrawn and intact RBC was removed by centrifugation for 5 min at 1200 × g/4 °C. The supernatants were collected for analysis of the extent of hemolysis by measuring the absorbance of the hemoglobin at 540 nm. The percentage of hemolysis was calculated using the following Eq. (2): %Hemolysis ¼ Absa =Abscp  100

ð2Þ

Where Absa is absorbance of the sample and Abscp is absorbance of positive control, representing 100% of hemolysis. 2.6. In vitro cytotoxicity assay Human cervix carcinoma HeLa cells (ATCC CCL 2) were used as model of as a tumor cell line. The cell lines were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 10,000 IU mL−1 penicillin and 10 mg mL−1 streptomycin in a humidified environment with 5% CO2 at 37 °C. For the assays, 96-well plates were used. In each well, 1 × 104 cells were plated containing culture medium and incubated for 24 h at 37 °C in 5% CO2. After this period, the medium was removed and the cells were incubated 24, 48, 72 and 96 h with free Tmx citrate or Tmx-NPs (60, 30, 15, 7.5, 3.75, 1.875 μM), ethanol and blank nanoparticles. After the incubation period, the cells were washed with PBS twice and cell viability, was assessed by MTT assay, based on the cellular conversion of a tetrazolium salt into a formazan in the presence of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) [31]. The MTT (1 mg/mL) was added to each well, followed by 3 h incubation at 37 °C, 5% CO2. MTT solution was removed then 50 μL of ethanol and 150 μL of a solution containing PBS and isopropanol (1:2) were added to each well in order to solubilize the formed crystals. The absorbance of each well was read on a microplate reader (SpectraMax, Molecular Device) at 570 nm and 630 nm being proportional to the number of live cells. Experiments were performed in triplicate for each concentration of drugs used and the results were presented as mean ± S.D. Viability cellular was calculated using the following Eq. (3) and the half maximal inhibitory concentration (IC50) was determined as cytotoxicity parameter. %Viability ¼ Absa  100=Absc ;

ð3Þ

Where Absa is absorbance of the sample treated with drugs and Absc is absorbance of control group cell.

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3. Results 3.1. Preparation of Tmx-loaded PLA nanoparticles The effects of different surfactants and their concentrations on the entrapment drug efficiency, mean particle size, polydispersity index (PDI), size distribution and zeta potential have been summarized in Table 1. All surfactants used produced nanoparticles with size below 200 nm, but formulations used PVA (1 or 2%) produced the smaller mean particle size compared to nanoparticles composed of polysorbate 80 or poloxamer 188 (p b 0.05). PDI and size distribution were not influenced by surfactant and/or its concentration. The PDI remained below 0.25 and all formulations presented a monomodal profile of size distribution. Zeta potential varied from −4 to −21 mV, and nanoparticles composed of poloxamer 188 presented the lower zeta potential. Encapsulation efficiency varied from 45 to 85%, and it was the most representative parameter influenced by the surfactant. Nanoparticles which used 1% PVA or 2% PVA presented higher encapsulation efficiency (p b 0.05) compared to formulations composed of poloxamer 18 or polysorbate80. The formulations based on PVA (1 and 2%) exhibited the best parameters concerning mean size, zeta potential and encapsulation efficiency, however the composed by PVA 1% (F3) was chosen as the optimized formulation for the following assays since it uses low surfactant content.

3.2. Morphology TEM images of blank PLA nanoparticles (Fig. 1A) and optimized Tmx-NPs (F3) (Fig. 1B) showed spherical regular shape and smooth surface, and also confirmed the reduced size of particles corroborating DLS analysis. Also, drug encapsulation did not affect the surface morphology of PLA nanoparticles.

3.3. Fourier-transform infrared (FT-IR) spectroscopy Tmx-PLA interaction was evaluated by using FT-IR. Fig. 2 shows FT-IR spectra of free Tmx citrate, PLA and optimized Tmx-NPs (composed by 1% PVA). The FT-IR spectra of free Tmx (Fig. 2A) showed characteristics bands at 1236 cm− 1 of quaternary ammonium by NH stretching, aromatic C–C (stretch) at 1596 cm− 1, monosubstituted aromatic benzene at 711 cm− 1 , carbonyl band of citrate at 1737 cm− 1 and 10 hydroxyl at 3406 cm− 1. Spectra of PLA polymer have ester group characterized as the main band at 1762 cm− 1 and hydroxyl at 3461 cm− 1 (Fig. 2B). The spectra of Tmx-NPs (Fig. 2C) exhibited the characteristic peaks of PLA and Tmx, but the Tmx peaks were exhibited in lower intensity and some were not evidenced (1596 cm − 1), due to ratio of PLA:Tmx in nanoparticles and the dilution in KBr. The spectra 2C show at 2350 cm − 1, the asymmetrical stretch of O=C=O band, possibly resulted from measuring conditions.

Table 1 Nanoparticles parameters. Formulation

Mean particle size (nm)

Polydispersity index

Size distribution

Encapsulation efficiency (%)

Zeta potential (mV)

F1 F2 F3 F4 F5 F6

186 ± 4a 190 ± 2a 155 ± 4b 152 ± 10b 183 ± 1a 179 ± 9a

0.18 ± 0.02a 0.21 ± 0.01a 0.18 ± 0.02a 0.18 ± 0.01a 0.19 ± 0.01a 0.20 ± 0.01a

83–613 nm (100%) 31–527 nm (100%) 77–412 nm (100%) 67–379 nm (100%) 54–396 nm (100%) 75–496 nm (100%)

59 ± 7a 51 ± 9a 85 ± 8b 79 ± 4b 52 ± 6a 45 ± 3a

−4.1 ± 0.5 −12.6 ± 0.5 −21.7 ± 1.4 −17.8 ± 0.6 −21.1 ± 1.3 −14.9 ± 0.9

Notes: F1 — nanoparticle formulation with 1% poloxamer 188 and F2 — with 2% poloxamer 188, F3 — nanoparticle formulation with 1% PVA and F4 — with 2% PVA, F5 — nanoparticle formulation with 1% polysorbate-80 and F6 — with 2% polysorbate-80. a, b, same letters represent statistical equality with p N 0.05 and different letters mean statistically significant difference, p b 0.05. Values are expressed as mean ± standard deviation (n = 3).

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Fig. 1. TEM images of blank PLA nanoparticles (A) and PLA nanoparticles containing tamoxifen.

3.4. X-ray diffraction (XRD)

3.5. Differential scanning calorimetry (DSC)

The XRD patterns of Tmx, PLA and Tmx-NPs are shown in Fig. 3. The XRD pattern of Tmx (Fig. 3A) showed several intense diffraction peaks in the region 10° at 40°, characterizing its crystalline structure. Contrary, the XRD patterns of PLA (Fig. 3B) and Tmx-NPs (Fig. 3C) did not exhibit diffraction peaks, showing only broad bands, characterizing amorphous state of polymer and drug-loaded nanoparticles.

Fig. 4 shows DSC curves of free Tmx, PLA and Tmx-NPs. The DSC curve of Tmx (Fig. 4A) and PLA (Fig. 4B) exhibited a melting peak at 143.26 °C and 172.3 °C, respectively. The DSC curve obtained for Tmx-NPs (Fig. 4C) indicated drug has an amorphous characteristic, since no melting peak of drug was observed, which confirms the absence of a drug crystalline structure, corroborating the XRD data.

Fig. 2. FT-IR spectra of free tamoxifen (A), pure PLA (B) and tamoxifen-loaded PLA nanoparticles (C).

Fig. 3. XRD spectra of free tamoxifen (A), pure PLA (B) and tamoxifen-loaded PLA nanoparticles (C).

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Fig. 5. In vitro release profile of tamoxifen from PLA nanoparticles (n = 3).

percentage of hemolysis are expressed in Fig. 6. Free Tmx, at 4.4 μM, caused hemolysis (15%) after 4 h of incubation and after 24 h the hemolysis was superior to 70%. In lower concentration (1.1 μM), Tmx exhibited high percentage of hemolysis after 48 h. In 96 h, both Tmx concentrations showed hemolysis superior to 80% (p N 0.05). In contrast, Tmx-NPs (4.4 or 1.1 μM), exhibited negligible hemolytic activity, independent of Tmx concentration or time of incubation (p b 0.05). Blank nanoparticles showed no erythrocyte lysis (data not shown) suggesting the polymer exhibit biocompatibility. Also, incubations with ethanol solvent and solution of PBS (50 mM, pH 7.4) showed no hemolysis, whereas incubation with distilled water showed 100% hemolysis at 4 h (data not shown).

3.8. In vitro cytotoxicity assay

Fig. 4. DSC curve of free tamoxifen (A), pure PLA (B) and tamoxifen-loaded PLA nanoparticles (C).

3.6. In vitro release assay The in vitro cumulative release profile of Tmx from PLA nanoparticles is shown in Fig. 5. Nanoparticles formulation exhibited a sustained release of Tmx. The obtained profile was characterized as a biphasic drug release with approximately 24% of drug released in the first 24 h followed by sustained release up to 120 h (30% of Tmx released). To predict the release kinetics, five drug release models (zero order, first order, second order, Higuchi and Korsmeyer–Peppas) were used. When we use the first four models (results expressed in Table 2), the release profile of Tmx from nanoparticles was best fitted with the second-order model. Application of the Korsmeyer–Peppas semi-empirical model resulted in release exponent (n = 0.25), indicating the diffusion as mechanism of drug release (Fickian transport) [29].

3.7. Hemolysis assay The effects of free Tmx citrate and Tmx-NPs in isolated human RBC were assessed as a function of incubation time (4, 12, 24, 48, 72 and 96 h) and Tmx concentration of 4.4 μM or 1.1 μM. The results of

The cytotoxicity of Tmx-NPs was assessed in HeLa (ATCC CCL2) cell line using the MTT assay. The results demonstrate free Tmx was effective to reduce the number of viable cells at four higher concentrations (60, 30, 15 and 7.5 μM), while Tmx-NPs showed effectiveness at three higher concentrations (60, 30 and 15 μM) (Fig. 7). In 24 h, cell viability was statistically similar between free Tmx and Tmx-NPs, at all concentrations of Tmx, with exception of 7.5 μM. While in 48 h, only the two highest concentrations of Tmx (60 and 30 μM) free and nanoencapsulated Tmx exhibited similar cytotoxicity (p b 0.05), and at other lower concentrations free Tmx was more effective. In 72 and 96 h, free and nanoencapsulated Tmx exhibited similar cytotoxicity (p b 0.05) at three highest drug concentrations (60, 30 and 15 μM). In the respective times, cell viability was lower than 10%. Blank nanoparticles showed no cytotoxicity in HeLa cells (data not shown). The IC50 values of free Tmx and Tmx-NPs were calculated and the results after 24, 48, 72 and 96 h are expressed in Table 3. The IC50 of free Tmx was 2-fold lower than the exhibited by Tmx-NPs. Table 2 Analysis of the release models of tamoxifen from PLA nanoparticles in phosphate buffered saline (50 mM, pH 7.4) with 1% PVA. Kinetic model

R

MSC

k (h−1)

Zero order First order Second order Higuchi

0.9080 0.9260 0.9995 0.9108

−0.91 −0.75 6.05 −0.93

0.3566 0.0046 α = 0.0013/β = 0.1685 0.2163

Notes: R = correlation coefficient, MSC = criterion for model selection k = constant release.

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C. Altmeyer et al. / Materials Science and Engineering C 60 (2016) 135–142 Table 3 IC50 values of the free tamoxifen and tamoxifen-loaded PLA nanoparticles at 24, 48, 72 and 96 h. Time (h)

[Free Tmx]

[Tmx-NPs]

24 48 72 96

4.76 ± 2.04 μM a 4.67 ± 0.32 μM a 4.30 ± 0.28 μM a 4.60 ± 0.27 μM a

9.27 ± 0.59 μM b 10.54 ± 1.76 μM b 9.21 ± 1.37 μM b 10.97 ± 1.53 μM b

a, b

, same letters represent statistical equality with p N 0.05 and different letters mean statistically significant difference, p b 0.05. Values are expressed as mean ± standard deviation (n = 3).

Fig. 6. Percentage of hemolysis of the free tamoxifen and tamoxifen-loaded PLA nanoparticles versus time. a, b, c, d, e, f, same letters represent statistical equality with p N 0.05 and different letters mean statistically significant difference, p b 0.05 (n = 3).

4. Discussion PLA nanoparticles containing Tmx formulated with different surfactants were successfully prepared by emulsion-solvent evaporation technique. In this method, the formation of a stabilized emulsion is a very critical step because the droplet size of the emulsion indicates the final nanoparticles size. The droplet size largely depends on the type and amount of the stabilizing agents because they disperse aqueous and

oil phases and provide the stabilization of droplets during the emulsification process [32]. The application of different surfactants (PVA, poloxamer-188 or polysorbate-80) influenced in Tmx encapsulation efficiency, zeta potential and mean particle size. Formulations comprised of PVA exhibited higher Tmx encapsulation, lower size and high negative zeta potential, but the amount of PVA tested did not result in alteration on the analyzed parameters. Poloxamer-188 and polysorbate-80 resulted in similar responses in nanoparticles characteristics, and also, the surfactant concentration tested did not influence the parameters. In this case, 1% of stabilizer was sufficient to stabilize the emulsion globules, and when the concentration was increased to 2%, probably only a small quantity of stabilizer was adsorbed at the interface and the excess remained in the continuous phase, and does not play any significant role, neither in the emulsification nor in the protection of the droplets [33]. The three surfactants used are classified as non-ionic surfactant, so, presents the same mechanism to stabilize the emulsion globules. Thus, the influence of surfactant in nanoparticles parameters could be explained based on the hydrophilic-lipophilic balance (HLB) of the

Fig. 7. Cell viability percentage of free tamoxifen and tamoxifen-loaded PLA nanoparticles on the HeLa cell line, in the times of (A) 24 (B) 48 (C) and 72 (D) 96 h. a, b, c, d, e, same letters represent statistical equality with p N 0.05 and different letters mean statistically significant difference, p b 0.05 (n = 3). Statistical analysis performed by time.

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surfactant. HLB is represented on a numerical scale the proportion of hydrophilic and lipophilic properties of an amphiphilic compound according to which values will increase as the substance becomes more hydrophilic [34,35]. The HLB values of poloxamer-188, polysorbate-80 and PVA are respectively 29 [36], 15 [37] and 10.9 [38]. Thus, compared with poloxamer-188 or polysorbate 80, PVA presents higher hydrophobic interactions, resulting in greater chemical affinity for the oil phase of the emulsion, being very adequate to stabilize O/A emulsions. Also, PVA presents antiaggregant properties producing a steric barrier on the surface of the emulsion globules during the process of obtaining of nanoparticles [39,40]. It has been showed the polysorbate-80 has the tendency to form unstable emulsions with low steric stabilization, and it is able to migrate to dichlorometane oil phase of emulsion and thus, decrease its surfactant properties [41,42]. The mean size (150 nm) obtained by formulations composed by PVA (F3 and F4) can be considered adequate considering a controlled drug delivery systems for cancer therapy, because for the transport and drug release in the bloodstream as well as extravasation and accumulation in the interstitial space of the tumor cell, is required nanoparticles with reduced mean particle size [43,44]. Regardless of the transport mechanism, the pore cutoff size of several tumor models has been reported ranging between 380 and 780 nm [45,46]. Experiments using liposomal vesicles of different mean size suggested the particle size of maximum 400 nm for extravasation into tumors [47], although other studies have shown that particles below 200 nm are more effective [46,48]. The size distribution analysis showed particles with size below 500 nm and the high encapsulation efficiency (around 80%) contribute to the viability of nanoparticles production. Zeta potential analysis demonstrated the negative surface of nanoparticles, being result of the presence of PLA terminal carboxyl groups [49]. This measurement allows predictions about the storage stability of a colloidal dispersion. It is currently admitted that zeta potentials above of |30|mv are required for stabilization avoiding aggregation of nanoparticles due to electric repulsion [50]. Nanoparticles composed of PVA or polysorbate 80 have the tendency to present higher physical stability than the composed of poloxamer 188, since they exhibited the lower zeta potential. FT-IR studies were performed to study the possible interaction between the drug and the polymer [51] in nanoparticles, and no peaks indicative of chemical interaction between Tmx and PLA were observed. The physical state of Tmx into nanoparticles was assessed by XRD and DSC analysis. In XRD, Tmx-loaded nanoparticles did not exhibit diffraction peaks, characterizing the amorphous state of drug in polymeric matrix. This result was corroborated with DSC analysis, which was observed the disappearance of endothermic peak of the Tmx in nanoparticles, evidencing entire drug molecularly dispersion in polymeric matrix in their amorphous state. The drug amorphization probably occurred due to relatively higher solid-state drug – polymer solubility, since both are hydrophobic, influencing the ability of the polymeric matrix to entrap drug in amorphous dispersed state, and also by the low drug loading and low particle size, favoring entrapment of drug amorphous [52]. The characteristic of solid state is important in controlling drug release. The crystalline states require high energy to separate the molecules that causes low aqueous solubility and consequently low physiological bioavailability. While the amorphous state shows molecules randomly arranged, that require low energy to separate them and drug solubility and bioavailability is superior [53,54]. In vitro drug release assay reveled Tmx was prolonged released from nanoparticles and exhibited second order release pattern with an initial burst effect followed by sustained drug release. This model present two release kinetic constants, the α and β constant, which represents, respectively, the dissolution rate of the fast step (burst effect) and the dissolution rate of slow step [55,56]. The burst effect is advantageous in certain applications where a rapid onset of drug action is desirable, and the prolonged release is important to maintain the drug effects. The initial burst effect of release is due to drug desorption from the

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particle surface and the sustained release can be characterized by drug diffusion through the polymeric matrix and or/polymer erosion [57]. Application of the Korsmeyer–Peppas model was performed to gain further insight into the mechanisms of drug release. In this case, the particles were considered as spheres [58]. For this geometric shape, n values of 0.43 or less indicate that the drug release is controlled by Fickian diffusion. Values of n between 0.43 and 0.85 indicate a mechanism known as anomalous transport (combination of drug diffusion and polymer chain relaxation as the solvent diffuses into the polymeric matrix). Finally, n = 0.85 indicates polymer relaxation [29]. The n value obtained (n = 0.25), suggests the mechanism of Tmx release followed Fickian diffusion. PVA was included in release medium to help maintain sink conditions, since Tmx is hydrophobic, but it presence can contribute to increase drug release, due to its surfactant property. After nanoparticles characterization, cytotoxicity over erythrocytes was evaluated. Tmx has a high toxic potential front human erythrocytes causing hemolytic anemia. Tmx induces defects in erythrocyte membrane structure, due to its interaction with membrane proteins and/or changes on the framework of cytoskeleton resulting in structural rupture [13,59]. In this study, the cytotoxic profile displayed by free Tmx citrate was mainly time dependent, since in low or high drug concentration, hemolysis was increasing along time, and after 72 h the percentage of hemolysis was similar for the two drug concentrations (1.1 and 4.4 μM) (p N 0.05). Tmx-loaded nanoparticles presented negligible hemolysis, independent of drug concentration or time of incubation. The slow and prolonged Tmx release from nanoparticles contributes to explain its low hemolytic potential. When incubated with free Tmx, cells were exposed with total drug since time 0 h, and thus, the damage occurred due to time of exposure, but when incubated with Tmx-NPs, the cells were slowly and progressively exposed to Tmx released, and thus, the damage was inhibited. The work of Silva et al. [59] shows that hemolysis of Tmx was in function of incubation time in all tested concentrations. If a correlation with the in vitro drug release assay was done, we would verify some discrepancies in the hemolysis results. In 24 h of hemolysis assay, nanoparticles containing 4.4 μM Tmx should release approximately 27% of Tmx (according to the in vitro release results), corresponding to a Tmx release nearly 1.1 μM, similar to the lowest free Tmx concentration assayed. At this time, both (free Tmx and Tmx-NPs) did not cause hemolysis but in following times free Tmx showed increased cytotoxicity while Tmx from nanoparticles did not cause hemolysis. These data show that possibly the Tmx amount released into hemolysis assay did not follow the same quantitative profile than the obtained in the vitro release assay, and thus, the amount of Tmx released in the hemolysis assay could be lower. A possible explanation for this result may be based on the surfactant properties of PVA, which present in the release medium in the in vitro release assay (to maintain sink conditions), enabled greater amount of Tmx released to the detriment of the released on the hemolysis assay, that did not use PVA due to its inherent hemolytic property. Thus, TmxNPs displayed negligible hemolysis due to prolonged drug release from nanoparticles and thus resulting in lower time of contact between erythrocytes and released drug. The results also suggest no interactions between nanoparticles and erythrocytes, since blank nanoparticles did not cause cellular damage. Probably due to negative surface charge of PLA nanoparticles, repulsive forces with negative charges membrane of erythrocytes could occur. Nanostructured systems can be used as a way to reduce the toxic potential of hemolytic drugs. Studies show use of nanoparticles reduce cytotoxicity of the drugs with high hemolytic potential, corroborating the results obtained in this study [28,60–66]. Free and nanoencapsulated Tmx were further evaluated for their in vitro cellular viability assay on HeLa cell line, by MTT assay. Free Tmx exhibited preferentially a concentration-dependent cytotoxicity, while the Tmx-NPs exhibited a time and concentration-dependent cytotoxicity for the four higher drug concentrations. This is a characteristic of prolonged drug release systems; the effect is increased

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with time. After 72 h, in the three higher drug concentrations (60, 30 and 15 μM), cell viability was similar between the cell treated with free and nanoencapsulated Tmx. Also, an important parameter for evaluating quantitatively the in vitro therapeutic effects of an anticancer drug is the IC50, which represents the drug concentration needed to kill 50% of the tumor cells at a designated time. In this study, the IC50 in HeLa cells of Tmx-NPs was 2-fold superior than the obtained by free Tmx. It can be explained due to slow drug release from nanoparticles. While free Tmx was fully available to interact to cells and promote its cytotoxic effect, that from nanoparticles was progressively released, reflecting in the IC50 value obtained. Considering the prolonged drug delivery, the results obtaining with PLA nanoparticles are expressive, since the effects of Tmx-NPs were similar to free Tmx in three higher concentrations. Our results show PLA nanoparticles maintains the drug cytotoxicity in tumor cells and reduces the drug cytotoxicity in normal cells (erythrocytes). 5. Conclusion In the obtaining of Tmx-loaded PLA nanoparticles, surfactant was an important factor influencing particle size, zeta potential and encapsulation efficiency. The concentration of surfactant tested showed to have no effect in nanoparticles parameters. Nanoparticles exhibited negligible hemolysis and maintained the cytotoxicity over HeLa cells, even presenting prolonged drug release. Thus, it is possible to suggest that the nanoparticles developed offer an alternative system for the controlled release of Tmx for the cancer treatment. However, in vivo studies are needed to evaluate this possibility. References [1] M. Clemons, S. Danson, A. Howell, Cancer Treat. Rev. 28 (2002) 165. [2] B. Fisher, J. Dignam, N. Wolmark, D.L. Wickerham, E.R. Fisher, E. Mamounas, R. Smith, M. Begovic, N.V. Dimitrov, R.G. Margolese, C.G. Kardinal, M.T. Kavanah, L. Fehrenbacher, R.H. Oishi, Lancet 353 (1999) 1993. [3] M.P. Goetz, A. Kamal, M.M. Ames, Clin. Pharmacol. Ther. 83 (2008) 160. [4] S.S. Feng, S. Chien, Chem. Eng. Sci. 58 (2003) 4087. [5] R. Siegel, D. Naishadham, A. Jemal, CA Cancer J. Clin. 63 (2013) 11. [6] M.C. Gamberini, C. Baraldi, A. Tinti, F. Palazzoli, V. Ferioli, J. Mol. Struct. 840 (2007) 29. [7] M.D. Johnson, H. Zuo, K.H. Lee, J.P. Trebley, J.M. Rae, R.V. Weatherman, Z. Desta, D.A. Flockhart, T.C. Skaar, Breast Cancer Res. Treat. 85 (2004) 151. [8] E.V. Mocanu, R.F. Harrison, Rev. Gynaecol. Pract. 4 (2004) 37. [9] M.H. Lee, J.W. Kim, J.H. Kim, K.S. Kang, G. Kong, M.O. Lee, Toxicol. Lett. 199 (2010) 416. [10] L.A. Stanley, P. Carthew, R. Davies, F. Higginson, E. Martin, J.A. Styles, Cancer Lett. 171 (2001) 27. [11] Cohen, Gynecol. Oncol. 94 (2004) 256. [12] M.M.C. Silva, V.M.C. Madeira, L.M. Almeida, J.B. Custódio, Biochim. Biophys. Acta 1464 (2000) 49. [13] P.P. Desai, A.A. Date, V.B. Patravale, Drug Discov. Today 9 (2012) 87. [14] N.M. Khalil, T.C. Do Nascimento, D.M. Casa, L.F. Dalmolin, A.C. De Mattos, I. Hoss, M.A. Romano, R.M. Mainardes, Colloids Surf. B: Biointerfaces 101 (2013) 353. [15] R.M. Mainardes, M.P. Gremião, J. Nanosci. Nanotechnol. 12 (2012) 8513. [16] Plapied, N. Duhem, A. Rieux, V. Préat, Curr. Opin. Colloid Interface Sci. 16 (2011) 228. [17] K. Thanki, R.P. Gangwal, A.T. Sangamwar, S. Jain, J. Control. Release 170 (2013) 15. [18] F. Danhier, O. Feron, V. préat, J. Control. Release 148 (2010) 135. [19] T. Asai, Biol. Pharm. Bull. 35 (2012) 1855. [20] L. Sun, Q. Wu, F. Peng, L. Liu, C. Gong, Colloids Surf. B: Biointerfaces 135 (2015) 56.

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