L-81 Binary Hydrogels as Drug-Delivery Systems

Oct 24, 2014 - We investigated the structure of the binary mixture of Pluronic F-127 (PL F-127) and Pluronic L-81 (PL L-81), as hydrogels for sumatrip...
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Pluronics F‑127/L-81 Binary Hydrogels as Drug-Delivery Systems: Influence of Physicochemical Aspects on Release Kinetics and Cytotoxicity Alisson Oshiro,† Deyse C. da Silva,† Joyce C. de Mello,† Vivian W. R. de Moraes,† Leide P. Cavalcanti,‡ Margareth K. K. D. Franco,§ Melissa I. Alkschbirs,∥ Leonardo F. Fraceto,⊥ Fabiano Yokaichiya,§ Tiago Rodrigues,† and Daniele R. de Araujo*,† †

Human and Natural Sciences Center, ABC Federal University, Santo André, SP 09210-580, Brazil Brazilian Synchrotron Light Laboratory, Campinas, SP 13083-970, Brazil § Nuclear and Energy Research Institute, São Paulo, SP 05508-000, Brazil ∥ Chemistry Institute, State University of Campinas, Campinas, SP 13083-970, Brazil ⊥ Department of Environmental Engineering, “Júlio de Mesquita Filho” State University, Sorocaba, SP 18087-180, Brazil ‡

S Supporting Information *

ABSTRACT: We investigated the structure of the binary mixture of Pluronic F-127 (PL F-127) and Pluronic L-81 (PL L-81), as hydrogels for sumatriptan delivery and investigated the mixture possible use via subcutaneous route for future applications as a long-acting antimigraine formulation. We studied the drug−micelle interaction by dynamic light scattering and differential scanning calorimetry, sol−gel process by rheology, and small-angle X-ray scattering (SAXS). We also employed pharmaceutical formulation aspects by dissolution rate, release profile, and cytotoxicity studies for apoptosis and/or necrosis in fibroblasts (3T3) and neural cells (Neuro 2a). Micellar hydrodynamic diameter studies revealed the formation of binary PL-micelles by association of PL F-127/PL L-81. The mixed micelle and binary hydrogels formation was also verified by only one phase transition temperature for all formulations, even in the presence of sumatriptan. The characterization of the hydrogel supramolecular organization by SAXS, rheology studies, and in vitro dissolution/release results showed a probable relationship between the transition of the lamellar to the hexagonal phase and the lower release constant values observed, indicating that PL L-81 participates in micelle-hydrogel formation and aggregation processes. Furthermore, the reduced cytotoxicity (annexin V-fluorescein isothiocyanate positive staining), with minor PL L-81 concentration, points to its potential use for the development of binary PL-systems containing sumatriptan capable of modulating the gelation process. This use may employ the minimum PL concentration and be interesting for pharmaceutical applications, particularly for migraine treatment.

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INTRODUCTION Poloxamers or Pluronics (PL) are triblock copolymers, composed of poly(ethylene oxide) (PEO) and polipropylene oxide (PPO) units, characterized by their amphiphilic properties and different values of hydrophilic−lipophilic balance (HLB), which depends on the number of PEO and PPO units1 (Figure 1). PL self-assembly in response to concentration and temperature is the most important aspect to be considered for the development of drug-delivery systems.2−5 Temperatureinduced gelation occurs after the micellization process, when sufficient amounts of PL produce high-viscosity hydrogels, due to the transition from the liquid to the soft solid phase, allowing for the incorporation of hydrophilic and hydrophobic drugs.6−8 Those copolymers have been extensively studied for the development of modified release pharmaceutical formulations and are described as low toxicity excipients, that can be used as © 2014 American Chemical Society

component of intravenous, inhalatory, oral, suspension, ophthalmic, and topical formulations.1,9 The main advantage of PL-based delivery systems is their ability to self-assemble in semisolid hydrogels (as unique or binary systems) at close to body temperatures, promoting the gradual release of the incorporated drugs.10,11 In addition, PL with PPO block intermediate lengths (between 30 and 60 units) and HLB less than 20, such as PL P-85 (HLB = 16), PL L-61 (HLB = 3), and PL L-81 (HLB = 2), showed to be effective inhibitors of Pgp in microvessel endothelial cells from bovine brain.12 For this study, PL L-81 was selected as one of the binary system components (in Received: August 1, 2014 Revised: October 12, 2014 Published: October 24, 2014 13689

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(PL-sumatriptan) aqueous solutions (20 mM Hepes buffer with 154 mM NaCl, at pH 7.4) were prepared according to the cold method.25 Sumatriptan (12 mg.mL−1) was dispersed in different solutions of Pluronic F-127 (Sigma-Aldrich, St. Louis, MO, PL F-127) at 18 and 20% (w/w) alone or in binary mixtures containing Pluronic L-81 (Sigma-Aldrich, PL L-81) in concentrations ranging from 0.6 to 3.2% (w/w), kept at 4 °C under magnetic stirring (100 rpm). After dissolution, each solution was equilibrated overnight at 4 °C. The concentration of PL F-127 alone or in association was selected to obtain a thermoreversible gel at the minimum possible polymer concentration. The sumatriptan concentration was chosen according to commercially available formulations for subcutaneous use. The PL F-127 concentration range was selected according to previous results from sol−gel transition temperature results, since there is no gel phase formation below 15% PL F-127. The design of the hydrogels formulations was carried out by adjusting the copolymer concentrations. Different formulations, F1 (PL F-127, 18%), F2 (PL F-127/PL L-81 18/2.6%), F3 (PL F-127/PL L-81 18/3.2%), F4 (PL F-127 20%), F5 (PL F-127/PL L-81 20/0.6%), and F6 (PL F-127/PL L-81 20/1.2%), were assembled following the final PL concentrations of 18 or 20% w/ w and 20.6 or 21.2% w/w for PL F-127 isolated or in binary systems PL F127/PL L-81. The method for preparation of the micelles is described in the Supporting Information. Differential Scanning Calorimetry (DSC) Analysis. DSC experiments were performed with a TA Instruments (New Castle, DE) Q-200 DSC apparatus. PL hydrogels were weighed (50 mg) and placed in sealed aluminum pans, and the samples underwent three successive thermal cycles of heating and cooling from 0 to 50 °C at a rate of 5 °C/min with an empty pan as a reference. All of the analyses were performed in triplicate, and thermograms represented by heat flux (kJ·mol−1) versus temperature (°C). We calculated the thermodynamic parameters for the micellization process. Micellization thermodynamic parameters such as the Gibbs free energy (ΔG°), enthalpy variation (ΔH°, determined by the area under the phase-transition peak on the heating cycle), and entropy (ΔS°) were determined for the systems, according to previous studies.20,21,26

Figure 1. Chemical structures of sumatriptan succinate (A) and Pluronics (B). Note that (x) refers to the number of ethylene oxide (EO) and (y) to the propylene oxide units (PO).

association with PL F127), since it presents intermediate lipophilicity (classified as group II, such as PL L-64, P-85, and P-105)12 due to its lower molecular weight (compared with other PL) and a higher PPO in relation to PEO chains (∼7:1 PPO/PEO ratio).12,13 On the other hand, PL F-127 is a hydrophilic copolymer with high molecular weight (12 600 g· mol−1) and a lower PPO in relation to the PEO chains (∼1:3 PPO/PEO ratio) which confers a higher HLB value (22) when compared to PL L-81 (2).12,13 The binary PL-system proposed here (PL F-127 and PL L-81) may have benefits regarding the control of drug release associated with an interesting biological property of the adjuvant copolymer (L-81). In this work, sumatriptan (Figure 1) was selected as drug model since is a hydroplilic indol derivative, which presents selective activity as serotonin 5-HT agonist at the 5-HT1B and 5-HT1D receptors on the central nervous system. Sumatriptan exhibits multiple mechanisms of action, since targets were described within brain blood vessels and in both central and peripheral trigeminal sensory fibers.14 For this reason, sumatriptan was the first triptan molecule with high impact on the treatment of migraine.15,16 In fact, previous reports in the literature have been presented the use of PL binary systems based on hydrophilic− hydrophobic copolymers association. However, those studies have been devoted to solubilization, micellization, or selfassembling fields3,7,17,18 and also their applications for targeting or controlled release.19,20 In particular, these investigations have focused on poorly soluble drugs.21,22 For sumatriptan, PL-based thermoreversible hydrogels have been reported only for nasal23 and transdermal delivery.24 However, these studies used a single PL type and did not employ PL L-81. In addition, based on our knowledge, there are no studies investigating the structure of the binary mixture PL F-127/PL L-81 and the influence of its physicochemical properties on sumatriptan delivery, including its possible use by subcutaneous route, as a long-acting antimigraine formulation. We also studied the drug−micelle interaction by dynamic light scattering and differential scanning calorimetry, sol−gel process by rheology, and small-angle X-ray scattering (SAXS), which made it possible to investigate the influence of the composition or PL proportion and the drug incorporation on hydrogel structure. Additionally, we also extended our investigation to pharmaceutical formulation aspects such as dissolution rate, release profile, and cytotoxicity studies.

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ΔG° = RTCMT ln(x)

(1) (2)

ΔG° = ΔH ° − T ΔS° −1

−1

where R is the gas law constant (8.31 J·mol ·K ), T is the critical micelle temperature or temperature for micellization (Tm) in K, and x is the concentration of the polymer in mole fraction units. Sol−Gel Phase Transition Temperature: Tube Inversion Method. Vials containing 1 g of hydrogels of PL F-127 alone or in combination with PL L-81 were incubated in a circulating water bath (Quimis Ltda., Brazil) at 0 °C for 20 min, to allow the samples to equilibrate thermally with their environment.7 Next, the temperature was gradually increased (2 °C/10 min) ranging from 0 to 60 °C. At temperature set points, the samples were removed from the water bath (for a maximum of 30 s to prevent phase transition due to external temperature) and inverted to observe the liquid/gel behavior. The samples were classified into three categories: (i) liquid was observed when the sample was moved rapidly in the direction of gravity; (ii) a viscous-liquid or soft gel was seen when the sample was moved slowly down in the direction of gravity; and (iii) a hard gel was observed when the sample remained on the bottom of the vial. The temperature at which a sample was classified as a hard gel was called the sol−gel point. In addition, sample precipitation and color changes were evaluated. All experiments were performed in triplicate, and the results were expressed as gelation temperatures. Rheology. The rheological behavior of hydrogels samples were performed using a Haake Mars-III (Thermo Fischer Scientific, San Jose, CA) rheometer at the National Laboratory of Synchrotron Light (LNLS, Campinas, SP, Brazil). Measurements were performed at 37 °C using a plate−plate geometry (P35 methyl), a sample volume of 1 mL, a gap between the plates of 1 mm, and a voltage from 2 to 6 Pa (viscosity region was determined by linear dynamic deformation scan).

EXPERIMENTAL SECTION

Hydrogel and Micelles Preparation. To prepare the hydrogels, Pluronics−sumatriptan succinate (Libbs Pharm, São Paulo, Brazil) 13690

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Table 1. Temperatures (Tm), Enthalpies (ΔH°), Free Energies (ΔG°), and Entropies (ΔS°) of Micellization and Sol−Gel Transition Temperature (Tsol−gel) for PL F-127 Isolated or in Association with PL L-81 formulations

Tm (°C)

ΔH° (kJ·mol−1)

ΔG° (kJ·mol−1)

ΔS° (kJ·mol·K−1)

Tsol−gel (°C)

F1 F1-sumatriptan F2 F2-sumatriptan F3 F3-sumatriptan F4 F4-sumatriptan F5 F5-sumatriptan F6 F6-sumatriptan

15.4 15.6 13.8 13.7 13.7 13.9 14.6 15.0 13.8 15.1 14.6 15.0

30.1 35.6 24.2 67.4 89.8 92.7 24.6 45.4 25.4 62.0 52.3 51.4

−10.2 −10.2 −10.3 −10.3 −10.2 −10.2 −9.9 −9.9 −10.3 −10.3 −10.2 −10.3

0.14 0.16 0.12 0.27 0.28 0.36 0.12 0.19 0.12 0.25 0.22 0.21

32 34 26 28 24 26 28 30 26 28 24 26

Mt = KKPt n M∞

The oscillatory measurements allowed us to obtain parameters related to the elastic modulus (G′) and the viscous modulus (G″). Haake Rheo WinT version 4.3 software was used to analyze the data. Small-Angle X-ray Scattering (SAXS). The SAXS experiments were performed at the SAXS 1 beamline at the LNLS. We used an incident beam energy of 8.3 keV (λ = 1.488 Å) with a distance between the sample and the detector of 1007 mm (the MarCCD detector had a diameter of 165 mm). The measuring range (brand measuring range) was 0.13−3.34 nm−1, and the measurements were performed at two different temperatures (25 and 37 °C). In Vitro Dissolution and Release Assays. Hydrogels samples were weighed (1 g), placed in plastic microtubes with diameters of 1 cm, and incubated in a water bath at 37 °C for 12 h. Next, 1 mL of 20 mM Hepes with 154 mM NaCl (pH 7.4) was added at regular intervals from 30 min to 24 h, and the samples were centrifuged (13 000g, 10 min). The supernatant liquid was removed, and the remaining hydrogel was weighed. The weight of the remaining hydrogel at each time point was expressed as a percentage of the original mass. In vitro release assays were performed using a membrane diffusion model in vertical Franz-type cells with a permeation area of 1.76 cm2 (Automatized Microette Plus, Hanson Research, Chatsworth, CA), with artificial membranes (cellulose acetate sheets, MWCO 1000 Da., Spectrum Lab., Rancho Dominguez, CA) acting as a barrier. The donor compartment was filled with 1 g of the different hydrogel formulations containing sumatriptan at 12 mg· mL−1. The receptor compartment was filled with 20 mM Hepes with 154 mM NaCl buffer, pH 7.4, at 37 °C under constant magnetic stirring (350 rpm). Aliquots from the receptor compartment (at the same time intervals described for the dissolution assays) were withdrawn (1 mL) and analyzed by UV−vis spectrophotometry (282 nm, y = 0.05152 + 0.01162x, R2 = 0.99966 with quantification (0.0353 μg·mL−1) and detection limits (0.107 μ·.mL−1) obtained from a previous analytical curve. The data were expressed as a percentage of the sumatriptan released for each sample (n = 3 replicates/ experiment). The in vitro release profiles were analyzed according to zero-order, Higuchi, and Korsmeyer−Peppas models, as described by the following equations, respectively: Q t = Q 0 + K 0t

where Mt/M∞ is the fraction of drug released at time t, KKP is a rate constant, and n is the release exponent. An n value of 0.45 represents Fickian diffusion, 0.45 < n < 0.89 is anomalous (non-Fickian) diffusion, n = 0.89 is case-II transport, and n > 0.89 super case-II transport. Cell Cultures. Neuro-2a (neuroblast cells from the brain of Mus musculus)28 and 3T3 (fibroblast; spontaneous immortalization of Mus musculus)29 cells were grown in RPMI-1640 medium (Sigma Chem. Co., St. Louis, MO), pH 7.2, supplemented with 10% fetal bovine serum (Gibco FBS, Invitrogen, Grand Island, NY), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Life Technologies), in a 5% CO2 atmosphere at 37 °C (Panasonic MCO-19AIC, Japan). For the experiments, the cells were washed twice with CMF-BSS (calcium- and magnesium-free buffered saline solution) and detached from the flasks with trypsin/EDTA (Gibco, Life Technologies), followed by the addition of supplemented RPMI medium in order to inactivate the trypsin. After centrifugation at 160g for 10 min, the cells were suspended in the supplemented RPMI medium and then plated for the subsequent assays. Cytotoxicity Assays. The cytotoxicity of the formulations was screened by using the MTT reduction test and trypan blue dye exclusion assays. For the MTT assay, Neuro-2a or 3T3 cells (2.6 × 105 cells/cm2) were incubated in 96-well microplates for 24 h in the presence of the drug and copolymers in a final volume of 0.2 mL. After the addition of 0.25 mg·mL−1 MTT to each well, plates were incubated for 4 h. Next, 100 μL of 10% SDS (prepared in 0.01 M HCl) was added to dissolve the formazan crystals, and the plates were read at 570 nm and at 630 nm as a reference (BiochromAsys Expert Plus Microplate Reader, Biochrom Ltd., U.K.). The cell viability was calculated in relation to the control. Considering that hydrogels were prepared in aqueous solution, control (considered as 100%) was prepared using the same volume of water (2 μL) in each well. Also, MTT was added in blank for zeroing the multiplate reader to achieve 0% of viability, that is, absence of cellular reduction of MTT. For the trypan blue exclusion assay, cells (2.6 × 105 cells/cm2) were added to 24-well microplates in the presence of copolymers for 24 h. The cells were washed twice with CMF-BSS, detached from the flasks with trypsin/EDTA, and suspended in the RPMI media. After the addition of 0.016% trypan blue in the cell suspension, the cells were counted using a hemocytometer in a inverted optical microscope (Leica Microsystems, Germany). The percentage of viable cells was calculated in relation to the control, considered to be 100%. Annexin V-FITC/PI Double-Staining Flow Cytometry Analysis. After incubation in the same conditions as the MTT test, Neuro2a and 3T3 cells were washed twice with CMF-BSS, detached from the flasks with trypsin/EDTA, and combined with supplemented RPMI medium. After centrifugation at 160g for 10 min, the cells were suspended in the binding buffer (0.01 M Hepes, 0.14 M NaCl, 2.5 mM CaCl2, pH 7.4) and 3.0 μL Annexin V−fluorescein isothiocyanate

(3)

where Qt is the cumulative amount of drug released at time t, Q0 is the initial amount of drug, K0 is the zero-order release constant, and t is time.

Q t = KHt 1/2

(5)

(4)

where the rate of drug release is linear as a function of square root of time and the drug is the only component that diffuses through the medium.27 In this equation, the release mechanism is described as a diffusion process according to Fick’s law. KH is the coefficient of release, and Qt is the amount of drug released. 13691

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(Annexin V-FITC, BD Biosciences, CA, USA) plus 5.0 μg/mL PI (BD Biosciences) was added. The cells were incubated at room temperature for 20 min. After the addition of 0.3 mL of binding buffer, the analysis was performed in a FACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva software (10 000 events were collected per sample). The data are presented as media ± SEM of the replicates (n = 3). Statistical Analysis. Statistical analysis was performed by one-way ANOVA with a Tukey−Kramer post hoc test and significance defined as p < 0.05.

form micellar aggregates, with ΔG° values slightly more negative compared with the systems in which PL F-127 was isolated. These results suggest that the insertion of PL L-81 monomers form a mixed system with PL F-127. Finally, the variations in entropy (ΔS°) for binary systems were slightly higher than those observed for isolated systems with only PL F127. Once the formation of micelles is directed by the dehydration of PPO units, the presence of a more hydrophobic copolymer, such as PL L-81, promotes micellization due to the orientation of water molecules in direction to the micellar corona. Thus, when micellization occurs, high ΔS° values are observed in relation to the isolated PL F-127, reflecting the formation of a mixed system.30,31 In addition to DSC analysis, other information about micelles, such as their micellar hydrodynamic diameter, is presented in the Supporting Information. Sol−Gel Transition Temperature. Initially, the sol−gel temperature was determined for PL F-127 at 15, 20, 25, and 30% w/w. No sol−gel transition occurred for the isolated system consisting of 15% PL F-127. However, for 25 and 30% PL F-127, the temperatures obtained were 20 and 16 °C, respectively. In this study, the formulations were designed for gelling close to body temperature, because of our intent of using the formulations as subcutaneous injection. It is interesting to note that if the gelation temperature is higher than 37 °C, a liquid pharmaceutical form remains at the body temperature, resulting in fast clearance from the injection site. For this reason, the use of PL F-127 at 18% (F1) and 20% (F4), intermediate copolymer concentrations, was considered for the formulation design, since their sol−gel transition temperatures were 32 and 28 °C, respectively, in the absence of sumatriptan. After incorporation of sumatriptan, the sol−gel temperatures were 34 and 30 °C, in agreement with other studies.23 The sol−gel temperature for isolated PL F-127 (F1 and F4) decreased for high concentrations of the copolymer (20%). Similar results were also observed for binary systems, as a function of increasing PL L-81 concentrations (F2−F6). The formation of mixed micelles with PL L-81 leads to a reduction in the gelling temperature for all concentrations tested (Table 1). On the other hand, the incorporation of sumatriptan increased the sol−gel temperatures for all formulations by 2−3 °C. In fact, the influence of pharmaceutical incorporation into PL micelles has been reported in the literature by several authors. Sharma and Bhatia2 studied the effects of antiinflammatory drugs (naproxen and indomethacin) in PL F-127 micelles and reported that hydrophobic drugs shift the liquidto-gel boundary to lower temperatures. This fact is due to the PPO unit aggregation on the micellar core. Similar results were also reported by other authors,7 since the gelation behavior was found to be dependent on the aqueous solubility of the drug. However, sumatriptan is a hydrophilic drug with log P = 1.2 (determined between the octanol and phosphate buffer phases, at pH 6.8)32 and exhibits three pKa values: pKa1 (succinic acid) = 4.21−5.67, pKa2 (tertiary amine group) = 9.63, and pKa3 (sulfonamide group) > 12. The drug ionization and the fact that our formulations were prepared at pH 7.4 (with the ionized form of sumatriptan dominating) favor the interaction of the drug with the hydrated POE units on the micellar corona and may possibly reduce micellar interactions at lower temperatures. Rheological Analysis. In order to study the rheological behavior of isolated PL F-127 and binary systems with PL L-81,

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RESULTS AND DISCUSSION Thermal Reversibility and Thermodynamics. DSC analysis (Table 1) showed that the temperature for micellization (Tm) was similar for all formulations. Thermal analysis showed one endothermic peak, relative to the phase transition for all formulations, even those containing sumatriptan. However, the phase transition peaks were shifted during the cooling and heating cycles. This fact was also observed by other authors,30 considering that the kinetics of association are different since the micelle formation involves a PPO block dehydration process and micelle dissociation is dependent on PPO blocks hydration. The formulations containing only PL F-127 (F1 and F4) presented lower ΔH° values depended on the copolymer concentration, as observed for PL F-127 20% (24.6 kJ·mol−1) compared with PL F-127 18% (30.0 kJ·mol−1), indicating that for high concentrations of PL F-127 low ΔH° values are observed during the formation of micelles. These results are in accordance with other authors who found that the enthalpy of the micellization was proportional to the concentration of PL F-127.4 For binary systems, the enthalpy of micelles formation changed after the addition of PL L-81. At 2.6 w/w % PL L-81 associated with PL F-127 18 w/w % (F2), a ΔH° value of 13.8 kJ·mol−1 was reported. For the same formulation, after sumatriptan incorporation, the enthalpy variation increased to 37 kJ·mol−1, indicating a greater interference of the drug on self-assembly and micelles formation (Table 1). Comparisons among the PL F-127 isolated systems and its binary mixture with PL L-81 also showed similar results, indicating that the incorporation of sumatriptan contributes to increasing the enthalpy variation. This fact can be explained by the tendency of micelles to “package” after the addition of a salt, such as sumatriptan succinate, due to dehydration of units in the PPO micellar core. Furthermore, in the case of binary systems, the presence of PL L-81 also resulted in high ΔH° values, indicating the formation of a more hydrophobic mixed micellar system (when compared with PL F-127 alone) with selfassembly changes evoked by the possible inclusion of sumatriptan succinate (a hydrophilic drug). For the F2 and F5 samples, the incorporation of PL L-81 in two different proportions (PL F-127/PL L-81), but with same polymer total concentration, yielded similar ΔH° values. However, comparisons between F3 and F6 showed that despite the samples having the same polymer total concentration, the different ratios of copolymers and high concentration of PL L-81 induced high ΔH° values, possibly related to the high concentration of PL L-81 compared with other formulations. ΔG° values were calculated from the temperature micellization peak (Table 1). Although isolated PL L-81 does not show a tendency to form ordered structures, such as spherical micelles (due to the reduced number of PEO in relation to the number of PPO units),26 in binary systems PL L-81 tends to 13692

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A comparison between G′ and G″ values reveals viscoelastic behavior for the different formulations, since the values of G′ are 15−20 times higher than those observed for G″. These results are in agreement with the findings of other authors that showed that the elastic properties of an injectable hydrogel should predominate over its viscous properties, thereby facilitating injection and making it possible to describe differences in the dissolution of the gel and the release of the drug administration site.34−36 Similarly, published reports revealed an increase in viscosity, changes in viscoelastic properties and sol−gel transition temperature values after the addition of drugs as salts.33,34 On the other hand, the addition of sumatriptan in hydrogels of PL F-127 did not alter the viscoelastic properties, which can be explained by the high copolymer concentration into the hydrogel. Hydrogel Phase Behavior: SAXS. Figure 3 highlights some of the data about SAXS studies. The diffraction pattern of

we performed experiments to determine G′ and G″, depending on the frequency at 37 °C, where the formulations appeared as hydrogels. Figure 2 shows variations in G′ and G″ versus frequency for the binary systems consisting of PL F-127 and PL L-81.

Figure 2. Rheograms for hydrogels composed of PL F-127/PL L-81 (20/0.6% w/w) at 37 °C before (A) and after (B) sumatriptan incorporation.

For all systems, we observed that high G′ values in relation to G″ values would be favored by an increase in the interactions between the micellar aggregates, which are dependent on the types of interaction forces and the intermicellar distance. The hydrogels, described below, were compared according to the presence or absence of sumatriptan. The results show that a temperature of 37 °C is recommended for the development of delivery systems corresponding to the formation of the gel structure,33 since the values of the elastic modulus were higher than those observed for the viscous modulus at all formulations, ranging from 7500 to 14 700 and 320 to 920 for G′ and G″, respectively. In general, gels containing PL F-127 in combination with L81 are viscoelastic materials, based on values determined for the elastic modulus (a characteristic of solid material) and the viscous modulus characteristic of liquid materials.34 However, it is noteworthy that such behavior is temperature dependent and that it is possible to infer that for the chosen temperature (37 °C) and the values of G′ (for the frequency range studied), the G′ values were generally higher than those observed for G″.

Figure 3. SAXS profiles for the binary system PL F-127/PL L-81 at 25 °C (A) and 37 °C (B) before and after sumatriptan incorporation.

the hexagonal phase shows relative positions of 1:31/2:41/2:71/2:91/2. On the other hand, the phase lamellar diffraction pattern shows relative positions of 1:41/2:91/2. The results presented in this work indicate that the scattering peak at a position of 91/2 was not visible due to the initial settings adopted for our measurements, but it was possible to observe the structures in lamellar and hexagonal phases according to temperature (Table S2, Supporting Information). 13693

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According to a previous study,37 the formation of a hexagonal phase occurs with PL F-127 concentrations only between 63 and 80% (w/v) at 25 °C. The measurements performed for samples with concentrations between 25 and 63% would show phase centered cubic structure. The method of preparation used by authors of the previous study included a step of removing impurities using a hydrophobic solvent (hexane) that could be induced structural changes. We observed that the sol− gel transition temperature values for the systems containing PL L-81 copolymer (with longer PPO blocks compared with PL F127) were lower than those determined for isolated systems (PL F-127), which contributes to the formation of the hexagonal phase at 37 °C. In fact, a previous study reported the supramolecular organization in a hexagonal phase (in water) for the binary system PL F88/PL P-85 (two PL with different HLB values) at concentrations above ∼25% (at 36−38 °C),3 in agreement with our results for the PL F-127/PL L-81 system. After sumatriptan incorporation, the SAXS patterns for PL F-127 isolated or in binary systems with PL L-81 were similar to those observed in the absence of the drug, indicating that the drug does not interfere with the micellization and gelation phenomena, similar to what we infer from the DSC, sol−gel transition temperature, and micellar hydrodynamic diameter results. Figure 4 shows a representative model for the

the dissolution (or erosion) of the gels and the micellar disruption would be able to control the release of the drug.1 Dissolution assays were carried out for 24 h, expressed as a percentage of the residual gel. Next, the data were compared with the percentage of sumatriptan released. Figure 5 shows the

Figure 5. Release profile for sumatriptan in solution (A); percentages of residual gel and sumatriptan released for binary hydrogels (B) (n = 3/formulation).

results from the dissolution profiles for hydrogels after the addition of sumatriptan. In general, the dissolution of hydrogels composed only of PL F-127 occurred rapidly; almost 50% of the hydrogels were dissolved after 2 and 4 h for PL F-127 18% and 20%, respectively. On the other hand, for binary systems, half of the hydrogels dissolution was achieved only after 6 h (PL F-127/PL L-81 at 20/0.6 or 1.2%) and 8 h (PL F-127/PL L-81 at 18/2.6 or 3.2%), indicating that the presence of PL L81 at higher concentrations is able to control the hydrogel dissolution. After 24 h, differences were also in terms of the percentages of residual hydrogel for PL F-127 18% (4.3 ± 0.9%) and PL F-127 20% (9.4 ± 1.3%) when compared with PL F-127/PL L-81 at 20/0.6 or 1.2% (24 ± 1.6% and 16.6 ± 1.3%, respectively) and PL F-127/PL L-81 at 18/2.6 or 3.2% with 27.5 ± 3.2% (p < 0.001). In general, the fast dissolution of PL F-127 is related to the relatively high aqueous solubility (due to the PPO/PEO ∼1:3 ratio) compared with PL L-81 (PPO/PEO ∼7:1 ratio). For the in vitro release assays, the release of sumatriptan in solution was progressive and the total drug release (100%) was observed after 4 h. However, the sumatriptan release profile for PL F-127 isolated or in association with PL L-81 was regular, presenting low percentage release values compared with sumatriptan in an aqueous solution even after 24 h (p < 0.01). The sumatriptan release kinetics constant (Krel) from all formulations was calculated, and, according to correlation coefficient values, the sumatriptan mechanism release follows a Fickian diffusion model. Next, Krel values were compared to the sumatriptan solution (commercially available for subcutaneous

Figure 4. Schematic representation of the lamellar phase and the transition to hexagonal supramolecular structure for PL F-127 isolated or in association with PL L-81. The possible disposition of sumatriptan molecule into the micellar corona and intermicellar spaces is also represented.

PL F-127 isolated or the binary mixture of PL F-127/PL L-81 systems and the possible disposition of the sumatriptan, interacting with the micellar corona, into the intermicellar spaces. In Vitro Dissolution and Release Assays. In order to study the mechanisms involved on sumatriptan controlled release, we performed in vitro dissolution assays using a membraneless model (at pH 7.4). Our goal was to determine if 13694

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Table 2. − Release Constant Values and Correlation Coefficients Obtained for PL F-127 and PL F-127/PL L-81 Hydrogelsa zero-order formulations sumatriptan (solution) F1 F2 F3 F4 F5 F6 a

Higuchi

K0 (%·h−1)

R2

± ± ± ± ± ± ±

0.9094 0.8315 0.9691 0.9583 0.8870 0.9897 0.9454

16.7 11.2 11.7 12.6 11.0 11.1 11.8

0.7 2.9 1.2 1.5 2.3 0.6 1.0

KH (%·h−1/2) 48.6 37.5 37.5 40.3 36.5 35.5 37.8

±2.6 ± 7.1 ± 3.3 ± 2.9 ± 4.5 ± 2.7 ± 3.8

Korsmeyer−Peppas R2

KKP (%·h−n)

R2

0.9738 0.9252 0.9808 0.9866 0.9647 0.9832 0.9893

nd 1.95 ± 0.33 (n = 0.87) 1.26 ± 0.32 (n = 0.64) 0.98 ± 0.30 (n = 1.79) 2.12 ± 0.30 (n = 1.13) 0.82 ± 0.27 (n = 3.11) 1.0 ± 0.30 (n = 1.73)

nd 0.7383 0.9332 0.9693 0.7451 0.9758 0.9794

Data expressed as mean ± SD (n = 3/formulation); nd = not determined.

physiological and metabolic differences between these cells (Figure 6).

injection), as shown in Table 2. The results show that the hydrogel formulations were able to control the sumatriptan release, since lower Krel values were observed for the binary systems. Additionally, we observed a relationship between the dissolution and release profiles, since a low percentage of remaining hydrogel was related to a high sumatriptan percentage of release, controlled by concentration of copolymers and the insertion of PL L-81 into the formulations. Despite this study being an in vitro evaluation, we can suggest that the possible in vivo mechanisms involved on the sumatriptan gradual release are dependent on the dissolution and erosion of the hydrogels into the interstitial liquid and are associated with the drug diffusion across the hydrogel matrix. Cytotoxicity Evaluation. Considering the subcutaneous route for the hydrogels injection and also the effect of sumatriptan on the central nervous system, two cellular models were select to the experiments: 3T3 fibroblasts and Neuro-2a cells. Cytotoxicity screening for the different formulations was performed by the MTT reduction test by varying the composition and concentration of the copolymers. The absence of cytotoxicity in both cell lines was observed for the F4 formulation within the concentration range tested, corroborating previous studies showing that PL F-127 does not exhibit cytotoxic effects.38,39 This formulation contains only PL F-127, and the addition of L81 to these systems resulted in a concentration-dependent decrease of the cell viability. For F5 formulation containing 0.6% L81, we observed only a slight effect on the cell viability (∼20%) that became more pronounced with increasing L81 content (see the Supporting Information, Figure S2, panels A and D). Additionally, sumatriptan and formulations F4 and F5 loaded with sumatriptan did not exhibit significant cytotoxicity (see the Supporting Information, Figure S2, panels B/C for 3T3 and panels E/F for Neuro-2a). The cytotoxicity of L81 has been previously reported.40 As observed in Figure 5, at a copolymer concentration of 0.1%, the cytotoxicity increased with increasing L81 concentration (F4 < F5 < F6 < F2 < F3) in 3T3 fibroblasts (panel A) and also in Neuro-2a cells (panel B). We found that low HLB PL such as L81 are able to be inserted into lipid bilayers due to their hydrophobicity, promoting changes in their structure and function,41 which could explain at least in part the observed cytotoxicity. Other authors42−45 have proposed that PL with low HLB are able to inhibit Pglycoprotein at low concentrations, resulting in an increased susceptibility to cell death. This fact could be related to the effect of sumatriptan-F5 formulation. Interestingly, sumatriptan did not exhibit cytotoxicity alone or in the F4 formulation in both cell models. Also, a slight difference between the results obtained with MTT and the trypan blue test was observed in the 3T3 cells, but not in the Neuro-2a cells, likely due to the

Figure 6. Effects of Pluronics-based formulations (0.1%) on the viability of 3T3 (A) and Neuro-2a (B) cells after 24 h of incubation. The percentage of viable cells was calculated in relation to control (C, untreated) considered as 100%. The data are presented as mean ± SEM of three independent experiments. *Statistically different from control (p < 0.05).

In order to confirm the effects on the cell viability and also to advance in the understanding of the cell death pathways elicited by the copolymer formulations, we used flow cytometry analysis with annexin V-FITC and propidium iodide (PI) double staining was used. Annexin V is a cell-impermeable protein with high affinity for phosphatidylserine (PE), a phospholipid located in the cytosolic surface of the cell membrane.46 PI is also a membrane-impermeable fluorescent probe for staining nuclear material, which is useful to evaluate plasma membrane disruption.47 Considering that the externalization of PE is an early event in the apoptotic signaling cascade and its fluorescent detection using FITC labeled annexin V and the evaluation of the integrity of plasma membrane are typically observed in necrotic cell death, this experimental delineation provides additional information about the cytotoxicity exhibited 13695

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sumatriptan associated with the possibility of prolonging the drug release has important clinical applications in terms of reduced side effects, patient compliance, and therapeutic effectiveness. In this work, we have presented a study of the micellization and gelation of two copolymers (PL F-127 and PL L-81). For the development of an injectable formulation, it is desirable that systems containing PL are liquids at room temperature (25 °C) in order to avoid patient discomfort and ensure fluidity during application. The mixed micelle formation was verified by micellar diameter and DSC studies, since only one phase transition was detected for all of the formulations even in the presence of sumatriptan. The DSC results also showed that the thermoreversible properties were preserved for all systems. The characterization of the hydrogel supramolecular organization by SAXS and the in vitro dissolution/release results showed a probable relationship between the transition of the lamellar to hexagonal phase and the lower Krel values observed. In addition, we observed that the presence of PL L-81 favors a shift to the hexagonal phase, indicating that this copolymer participates in the formation of micelles and the aggregation processes. Those results are also in agreement with the rheological analysis, since the addition of PL L-81 to the systems yielded a greater variation in G′ relative to G″. This supramolecular organization can facilitate incorporation of the drug into intermicellar spaces. Due to its hydrophilic characteristics, sumatriptan possibly interacts with the hydrated micellar corona region. For this reason, the retention of the drug in micelles cannot be attributed only to the presence of PL L-81, since formulations with high concentrations of this copolymer were found to yield a significant increase in their release percentages. This fact can be explained by the hydrophobicity of the micellar core evoking the displacement of sumatriptan molecules in the direction toward the micellar corona region. Thus, it is worthwhile to highlight that the contribution of the increased concentrations of PL F-127 in the system, a hydrophilic copolymer (with a large number of PEO units than PL L-81), which favors the hydration of the micellar corona and the likely permanence of the drug in this region. The reduced cytotoxicity for formulations with low L81 concentrations highlights potential use for the development of binary PL-systems capable of modulating the gelation process. These systems which would have minimal PL concentration would be interesting for pharmaceutical application particularly for migraine treatment.

by the formulations. Thus, as depicted in Figure 6, the absence of a fluorescent stain (An−/PI−), which represents viable cells, followed the same cytotoxicity pattern observed with MTT and trypan blue. Moreover, it is noteworthy that the increase in L81 content in the formulations, which in turns increased the cytotoxicity of the formulations, induced predominantly annexin V-FITC positive staining (An+/PI‑) suggestive of apoptosis. This finding will need to be investigated further, in terms of cell death mechanisms. Additionally, a comparative analysis of the two cell models used in this study revealed that the viability of 3T3 fibroblasts was slightly more affected that of Neuro-2a by the formulations, which was mainly revealed by the F2 formulation in the trypan blue assay (Figure 5, gray bars) and the flow cytometry analysis (Figure 7).

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ASSOCIATED CONTENT

S Supporting Information *

Figure 7. Annexin V-FITC/PI double-staining followed by flow cytometry analysis of 3T3 (A) and Neuro-2a (B) cells after incubation with formulations (0.1%) for 24 h. At least 10 000 cells were analyzed per sample, and the figure is representative of three independent experiments. An (annexin V-FITC) and PI (propidium iodide).

Micellar hydrodynamic diameter studies, SAXS reflections for PL F-127 and PL F-127/PL L-81 hydrogels. Effects of variation in the concentration of all formulations without sumatriptan on the viability of 3T3 and Neuro-2a cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSIONS AND PERSPECTIVES Migraine is characterized by attacks lasting from 4 to 72 h that have a typical moderate to severe pain intensity. In fact, the pain reduction provided by available formulations is clinically relevant, but the responses of available formulations are rather ineffective.15 Due to the relatively short duration of the analgesia, there is a need for improving of acute drug treatment for migraine. In this sense, the development of a hydrogel for

AUTHOR INFORMATION

Corresponding Author

*Mailing address: Human and Natural Sciences Center, ABC Federal University - UFABC. Av dos Estados, 5001, Bairro Bangú, Bloco A, Torre 3, Sala 623-3, Santo André, SP, Brasil, CEP 090210-580. E-mail: [email protected] or [email protected]. 13696

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant 2010/ 11475-1) and Conselho Nacional de Desenvolvimento ́ Cientifico e Tecnológico (CNPq, Grants 487619/2012-9, 300952/2010-4, 309612/2013-6). The authors are also grateful to the Brazilian Synchrotron Light Laboratory for SAXS facilities (SAXS 1 beamline). The authors declare no competing financial interest.

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