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Hyaluronan based materials with catanionic sugar-derived surfactants as drug delivery systems
Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Hyaluronan based materials with catanionic sugar-derived surfactants as drug delivery systems F. Roig a , M. Blanzat b , C. Solans c,d , J. Esquena c,d , M.J. García-Celma a,d,∗ a
Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain Laboratoire des IMRCP, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France c Institut de Química Avanc¸ada de Catalunya (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain d CIBER-BBN (Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina), Jordi Girona 18-26, 08034 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 23 December 2016 Received in revised form 17 January 2018 Accepted 20 January 2018 Keywords: Hyaluronan Catanionic surfactant Controlled release Crosslinked hydrogel Solid foam Highly concentrated emulsion Ketoprofen
a b s t r a c t In the present work novel drug delivery systems consisting in highly porous Hyaluronan foams for the administration of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, have been obtained. A sugar-derived surfactant associated with ketoprofen was prepared and incorporated into the porous hyaluronan materials. The association between a lactose derived surfactant, Lhyd12 , and ketoprofen was obtained by acid-base reaction and its physicochemical properties were studied. Tensiometric and dynamic light scattering (DLS) determinations showed the formation of catanionic surfactant aggregates, Lhyd12 /ketoprofen, in aqueous solution. Furthermore, the catanionic surfactants allowed greater solubilisation of ketoprofen. Hyaluronan porous materials were developed using butanediol diglycidyl ether as crosslinking agent. The profile release of Lhyd12 /ketoprofen from hyaluronan based materials shows differences as a function of the aggregation state of catanionic surfactant. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Soft matter drug delivery systems have recently received substantial attention, in particular from the nanomedicine field. In fact, these carriers increase drug bioavailability and activity, while decreasing its toxicity, because drug efficiency is often altered by its non-controlled biodistribution. The design of the appropriate drug delivery system is then important to optimize drug efficiency [1]. In order to meet the needs, a wide range of drug delivery systems have already been developed. These systems include matrix and vesicular carriers, with a large range of size and structures [2]. The use of a biocompatible polymer matrix is an interesting approach because it allows controlling the release of low molecular weight drugs for various routes of administration such as: oral, parenteral, ocular, etc. [3]. Furthermore, these materials have the following advantages: reduction of side effects and improvement of drug bioavailability, solubilization of lipophilic drugs, and
∗ Corresponding author at: Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028, Barcelona, Spain. E-mail address: [email protected] (M.J. García-Celma). https://doi.org/10.1016/j.colsurfb.2018.01.037 0927-7765/© 2018 Elsevier B.V. All rights reserved.
lower treatment costs [4]. Hyaluronan (HA), belongs to the family of glycosaminoglycans and consists on N-acetyl-d-glucosamine and D-glucuronic acid [5]. This polymer is an important component of the extracellular matrix of connective tissue and is found in various parts of the human body such as: skin, cartilage, vitreous humour and intra-articular joint fluid [6]. It also plays an important role in cartilage matrix stabilization, cell proliferation, control of morphogenesis, cancer metastases, inflammation processes and wound healing [7–12]. HA is degradable in vivo by enzymes such as hyaluronidase present in human tissues [13]. A suitable approach to avoid fast elimination from the human body could be the preparation of HA materials chemically crosslinked that show an increase in their resistance against hyaluronidase [14,15]. Polymeric materials can be obtained by crosslinking hydrophilic polymers in bulk [16] (hydrogels) or by the use of colloidal systems as templates for the preparation of materials with controlled porosity (solid foams). The incorporation of a polymer in the continuous phase of a highly concentrated emulsion, allows the preparation of porous materials with very high pore volume [17]. These materials have found a number of applications, including biomaterial engineered devices and drug delivery systems [18]. In addition, vesicular drug delivery systems made of catanionic surfactants have also proved their great contribution to drug solu-
F. Roig et al. / Colloids and Surfaces B: Biointerfaces 164 (2018) 218–223
bility in water and drug delivery by means of spontaneous vesicles formation [19–21]. These vesicles also showed an increase of the therapeutic effect of the drug together with a sustained diffusion through the skin [19,22]. The strategy adopted in this work was to evaluate the impact of the combination of both drug delivery systems by preparing a novel complex drug delivery system. These advanced materials consist in the association between a drug, ketoprofen (KP), and a sugar-derived surfactant to form an ion-pair, “catanionic” surfactant, which would later be incorporated into a crosslinked polymer matrix based on HA. To ensure the formation of a KP catanionic surfactant, the selected surfactant has to be capable of forming an ionic acid-base pair with the carboxylic acid of KP. This feature means that the surfactant requires the presence of a basic group that is able to react with the acid group of the drug. In addition, the surfactant has to be biocompatible and biodegradable without inducing toxicity to ensure biocompatibility of the ion pair within the body. Furthermore, the physicochemical characteristics of the surfactant should lead to drug solubilization. In this context, sugar-derived surfactants represent a suitable choice for this purpose. Apart from their biocompatibility and biodegradability, these surfactants can be obtained from sugars that are natural raw materials available in large quantities. The aim of this work was to develop a novel drug delivery system based on HA materials (hydrogels and solid foams) loaded with KP catanionic surfactants and determine the differences in the release behavior when the aggregates of catanionic surfactants are formed. 2. Experimental 2.1. Materials Hyaluronic acid sodium salt from Streptococcus equi. of molecular weight around 2 million Daltons with 97% purity was obtained from Sigma-Aldrich. The chemical crosslinker butanediol diglycidyl ether (BDDE) with a molecular weight 202.25 g mol−1 with 95% purity was obtained from Sigma-Aldrich. Nonionic surfactant Cremophor RH455 (CRH 455) with an HLB between 14 and 16 was obtained from BASF. Miglyol 812, medium chain triglycerides, was obtained from Fagron. Ketoprofen (C16 H14 O3 ), (KP), non-steroidal anti-inflammatory drug (NSAIDs) used as anionic precursor surfactant, was from Fagron with 99.8% purity. Phosphate buffer solution pH 7.4, (PBS) was prepared from: KH2 PO4 from Fagron Iberica ® S.A.V, Na2 HPO4 from Probus S.A, NaCl from Acofarma , and Milli® Q deionized water. Cellulose tubular membrane was purchased from Orange Scientific. Its properties are a 12.000-14.000 Da nominal MWCO, and 20 m wall thickness. Mobile phase for HPLC (pH 3.0) was prepared from 45% of aqueous phase comprising: Citric ® ® acid from Acofarma with 99.5% purity, NaCl from Acofarma , NaOH ® ® from Acofarma , and Milli-Q deionized water; and 55% of organic phase acetonitrile obtained from Carlo Erba Reagents, with 99.9% purity. 2.2. Methods 2.2.1. Synthesis of KP catanionic surfactant The catanionic sugar-derived surfactant was obtained by an acid–base reaction between equimolar amounts (0.66 mmol) of cationic and anionic precursor surfactants in 30 mL of water. Ndodecylamino-1-deoxylactitol, designated as Lhyd12 , was used as cationic precursor surfactant. Lhyd12 was obtained as previously described from a reductive amination of dodecylamine with lactose [23,24]. An NSAID, KP was used as the anionic precursor surfactant. The two components were added in ultrapure water and stirred for 24 h. The resulting homogeneous solution was lyophilized. After lyophilization a white powder corresponding to the catanionic
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Fig. 1. Molecular structure of KP catanionic surfactant as a result of ionic association between Lhyd12 and ketoprofen.
sugar-derived surfactant was obtained with a quantitative yield (Fig. 1). 2.2.2. Physicochemical characterization of KP catanionic surfactant 2.2.2.1. Fourier transform − infrared spectroscopy (FT-IR). Infrared spectra were obtained with a Perkin-Elmer IR FT 1760X. KBr discs with a concentration of 0.5 w/w% were prepared of KP catanionic surfactant. 2.2.2.2. Surface tension measurements. The values of surface tension as a function of catanionic surfactant concentration were measured by the Wilhelmy plate method using a Kruss Tensiometer Easy Dyne at 25.0 ◦ C ± 0.1 ◦ C. The catanionic solutions were prepared by dissolving weighted amounts of dry catanionic associations in ultrapure water. Solutions were stirred at room temperature during a few minutes. 2.2.2.3. Dynamic light scattering (DLS). Dynamic light scattering was performed using a Malvern Zetasizer Nano-ZS, ZEN3600, with a measuring range of 0.5 nm to 10 m. The light source used was a He-Ne laser with a wavelength of 633 nm. The temperature was regulated at 25.0 ◦ C with a Peltier with an accuracy of ± 0.1 ◦ C. The measuring angle was 173◦ . Samples of aqueous solutions of KP catanionic surfactant (3.5 × 10−3 M) were introduced into cells (pathway, 10 mm). The deconvolution of the measured intensity autocorrelation function of the samples was realized with the multiple narrow modes program that uses a non-negatively constrained least squares (NNLS) fitting algorithm to obtain the distribution of diffusion coefficients (D) of the solutes. The apparent equivalent hydrodynamic diameter (dh ) was determined using the Stokes-Einstein equation. Hydrodynamic diameter values were obtained from three different runs. 2.2.3. Preparation of chemically crosslinked hydrogels For the preparation of HA hydrogels, 50 mg of sodium hyaluronate were introduced into test tubes of 12 × 75 mm, to which 500 L of crosslinking solution, consisting of BDDE (5% v/v) in alkaline media (0.2 M NaOH), were added. Then, the HA and the crosslinking solution were stirred with a vortex till a homogeneous mixing. The resulting mixture was incubated at 25 ◦ C for 24 h and the HA crosslinked hydrogel was obtained. The epoxy groups of BDDE react with the hydroxyls present in the HA polymer [25]. 2.2.4. In vitro cell viability analysis Cell viability in the presence free BDDE crosslinker, BDDE crosslinked hyaluronan hydrogels and KP catanionic surfactant was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay [26]. For each assay, HeLa cells were seeded in Dulbecco’s modified Eagle’s medium supplemented with FBS and antibiotics. Then, the culture medium was replaced with samples at the required concentrations. 100 L of BDDE solution at the same concentration that in the hydrogel and the corresponding dilutions 1:5 and 1:10 v/v, 100 L
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Fig. 2. IR spectrum in KBr tablet method for: a) an equimolar mixture of Lhyd12 and KP, and b) KP catanionic surfactant.
of KP catanionic surfactant (0.14, 0.7 and 1.18 mM) or 25 mg of hydrogel and the corresponding dilutions 1:5 and 1:10 p/v were incubated in this medium. After incubation, the medium was withdrawn and cells were seeded with fresh medium and incubated again. Then, MTT reagent solution was added and DMSO was used to dissolve the formazan crystals. Absorbance was measured at a = 590 nm. The results were expressed as viability percentages. 2.2.5. Preparation of chemically crosslinked solid foam For the preparation of HA solid foam, a O/W highly concentrated emulsion (HIPRE) was used as template. The components of the HIPRE were: aqueous component, a castor oil derivative as surfactant (CRH 455), and an oil derived from caprylic and capric fatty acids (Miglyol 812) as oil component. The HIPRE with HA (5% in water) in the continuous phase was formed with a high-performance dispersing equipment (Ultaturrax). After HIPRE formation, BDDE as crosslinker agent was introduced and kept in 25 ◦ C for 24 h to crosslinking HA. After crosslinking reaction of HA in the continuous phase of HIPRE, the surfactant and oil were removed by solvent extraction with ethanol and water for 12 h. Finally the HA solid foam was freeze-dryied. 2.2.6. Determination of the specific surface area of the solid foam The specific surface area of the solid foam was determined by Nitrogen sorption experiments. Nitrogen adsorption and desorption isotherms at 77 K of the solid foam were obtained by using an AUTOSORBTM IQ instrument. According to the BET theory [27], the specific surface area of the solid foam was determined fitting the BET equation to the adsorption curve. 2.2.7. Incorporation of the Lhyd12 /ketoprofen into the HA materials The method used to incorporate the KP catanionic surfactant into the HA materials was the permeation method: the HA materials (hydrogels and solid foams) were prepared and then immersed
in a volume of 2 mL solution of KP catanionic surfactant in water, which was completely absorbed by HA materials.
2.2.8. Release studies of ketoprofen from HA materials In vitro release studies were carried out in a dissolution tester. The equipment used was an Elite 8TM dissolution tester from Hanson Research Corporation (USA). It consists of 8 dissolution vessels immersed in a thermostatic bath. Each dissolution vessel had a capacity of 150 mL and a setup for semisolid formulations called Ointment cellwas incorporated in each vessel. A cellulose membrane was placed in each ointment cell to separate the hydrogel from the receptor solution. The receptor solution consisted of 100 mL of PBS (pH 7.4). The temperature of the receptor solution was 37 ◦ C. The stirring speed of the paddles in each dissolution vessel was 25 rpm. The HA materials were placed in the ointment cell, as described previously [28]. Then, 150 mL of receptor solution (PBS) were placed into the glass vessel. In order to determine the amount of drug released as a function of time, 0.5 mL of receptor solution has been removed for analysis and the same amount of PBS solution was replaced. The release study lasted 24 h. Three replicates of each formulation were assayed.
2.2.9. Determination of ketoprofen concentration by HPLC KP released was analyzed by HPLC. The chromatographic system ® consisted on Shimadzu equipment with a column Kromasil 1005C18 purchased from Akzo Nobel with dimensions of 250 × 4.6 mm and pore size of 5 m, and a UV detector set at 233 nm for ketoprofen determination. Separation was carried out at room temperature using 55% acetonitrile and 45% aqueous phase (pH 3.0), as a mobile phase, with flow rate of 1 mL/min, and injection volume of 50 L. The KP retention time was about 7 min.
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tered at 130 nm with a polydispersity index of 0.2, as shown as Supporting information. All these results are in good agreement with the physicochemical features of vesicles already reported for these catanionic associations [19].
3.2. Preparation of drug delivery systems based on HA materials
Fig. 3. Aqueous solution surface tension as a function of the logarithm of the concentration of KP catanionic surfactant.
3. Results and discussion 3.1. Synthesis and characterization of KP catanionic surfactant A catanionic sugar-derived surfactant based on the association between lactose-derived surfactant, Lhyd12 , and KP has been obtained by acid-base reaction as described previously [19]. This reaction consists on the proton transfer between KP and the basic aminosugar. Therefore, the carboxylic acid of KP was transformed to carboxylate. The formation of the catanionic surfactant was first assessed by infrared spectroscopy comparing the spectra of the initial reagents with that of the association. Fig. 2 shows the disappearance of group (C O) and (OC OH) corresponding to carboxylic acid, 1692 cm−1 and 1228 cm−1 respectively, and the appearance of the group (COO-st as) at 1576 cm−1 and (COO-st sy) to 1393 cm−1 for the carboxylate. To study the behavior of the KP catanionic surfactant in aqueous solution, the surface activity was determined by surface tension measurements. The plot of the aqueous solution surface tension as a function of the logarithm of the concentration of KP catanionic surfactant (Fig. 3) allowed to obtain the critical aggregation concentration (CAC) which was around 1.5 mM. The size distribution of the aggregates was verified by Dynamic Light Scattering (DLS) at a concentration of 3.5 mM. The results showed the formation of a large distribution of aggregate population with a diameter cen-
The HA materials (hydrogels and solid foams) were obtained as described in the experimental section, and either free KP or KP catanionic surfactant were added into the polymer matrix. Due to poor solubility of free KP in water, its incorporation was performed using alcoholic solution and phosphate buffered solution (pH 7.4) into solid foams and hydrogels respectively. However, the increase in the solubility for KP in the association of KP catanionic surfactant, allows its incorporation by swelling the polymer matrix with water (containing KP catanionic surfactant). SEM images of the two HA based materials show differences in their structure. Freeze-dried hydrogels exhibit a disordered layered structure (Fig. 4a), which is commonly observed after lyophilization, and it can be attributed to the growth of ice crystals during freezing [29]. However, high porous solid foams were obtained by crosslinking in the continuous phase of the HIPREs (Fig. 4b). The solid foams showed smaller, more homogeneous and well interconnected pores. Their pore size is between 1 and 5 m, being the largest macropores around 30 m, and the smallest around 0.5 m. Also, the specific surface area of the solid foam was determined by Nitrogen sorption experiments (Fig. 5), and the material show a high specific surface area, around 20 m2 /g. This porous texture is a replica of the structure of the highly concentrated emulsion used as template. Cytotoxicity studies were performed in order to determine the biocompatibility of the prepared materials. The MTT test [26] was applied to study the influence of various concentrations of the free crosslinker BDDE, BDDE crosslinked hyaluronan hydrogels and catanionic surfactant on HeLa cells viability, as an indication of toxicity. HeLa cells are a versatile model cell line that is widely used in nanomedicine tests. When the cells viability is higher than 80%, it can be considered that the sample assayed induces low cytotoxicity. The results showed the toxicity of the free crosslinker BDDE (cell viability lower than 11%), in contrast to the catanionic surfactant (cell viability higher than 90%) and the BDDE crosslinked Hyaluronan hydrogel (cell viability higher than 85%), an indication of absence of free BDDE in the medium. As the catanionic surfactant and the BDDE crosslinked HA hydrogels assayed induce low cytotoxicity in HeLa human cell line, they could be proposed as good
Fig. 4. SEM images corresponding to a) freeze-dried hydrogels, and b) solid foams based on HA.
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Fig. 5. Nitrogen adsorption-desorption isotherms at 77 K of a HA based solid foam. Fig. 7. KP release profiles from HA hydrogels: KP catanionic surfactant without aggregation (CAC), and free KP.
Fig. 6. KP release profiles as a function of HA materials: in solution, from HA hydrogels, and from HA solid foams.
candidates for implant developments. The details of cytotoxicity studies performed are showed as Supporting information. 3.3. Release studies of KP catanionic surfactant incorporated in HA materials The main purpose was to study the influence of the material structure based on HA (hydrogels and solid foams) and the aggregation state of catanionic surfactant (CAC) in KP release. Although drug release experiments are not an indication of in vivo behavior, they constitute a good approach to compare different formulations. For comparative purposes, a KP solution was also studied as a reference, to probe that the active is not retained in the cellulose membrane used. Firstly, we studied the release properties of KP from hydrogels and solid foams based on HA. The results are shown in Fig. 4. Whereas 80% of KP was released after 24 h from hydrogels, only about 20% was reached from solid foams (Fig. 6). The release profiles obtained, show a strong influence of the HA materials structure in KP diffusion. These differences may be due to the high capacity of HA based hydrogels to swell. The swelling of HA based hydrogels during the in vitro test helps the diffusion of KP to the receptor solu-
tion. However, for the HA based solid foams, the retention could be caused by the smaller pores present in these materials. The release of KP from HA solid foams is much lower than that obtained with other solids foams (e.g. polystyrene solid foams, that reach 70% after 24 h) [18]. Also we studied the release profile of KP catanionic surfactant as a function of their state of aggregation. The release of the KP catanionic surfactant incorporated to hydrogels and solid foams based on HA to PBS solution were performed with two concentrations: [KP catanionic surfactant] >CAC: 5.9 mM, and [KP catanionic surfactant] CAC), and KP catanionic surfactant without aggregation (< CAC) from hydrogels, took place with no lag-time but revealed slight differences (Fig. 7). Both showed a fast initial release which slows down progressively after several hours, but the maximum amount released was around 100% for KP catanionic surfactant without aggregation and 75% for KP catanionic surfactant aggregates, after 25 h. The release profiles of KP catanionic surfactant from hydrogels were compared to the release profile of hydrogel loaded with free KP (Fig. 7) which was closer to 70%. Similar trends were observed with KP catanionic surfactant from HA solid foams (Fig. 8). There is a slower release when KP catanionic surfactant is aggregated (around 20% after 24 h), while the release is faster when it is not aggregated (around 50% after 24 h). The difference observed in the release of KP catanionic surfactant depending on their state of aggregation may be caused by the influence of the polar head consisting of lactose present in KP catanionic surfactant, which helps the diffusion of the ion pair KP catanionic surfactant to the receptor solution. This effect cannot be observed when KP cationic surfactant forms vesicles. KP is retained in the HA matrix in a similar way that occurs with free KP. 4. Conclusions Catanionic surfactants have been obtained by the association between sugar-derived surfactants and NSAIDs, KP. The synthesis of these systems by acid-base reaction is quick and easy to obtain. The use of sugar-derived surfactants allows the preparation of biocompatible and biodegradable systems, and increases the solubility of lipid-soluble drugs. The characterization of KP catanionic surfactant assemblies by DLS, showed that they form vesicles in aqueous solution above
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.colsurfb.2018.01. 037. References
Fig. 8. KP release profiles from HA solid foams: KP catanionic surfactant without aggregation (CAC), and free KP.
a critical aggregation concentration, the size of these aggregates being suitable for the drug release from a polymeric matrix system. Concerning the KP release from the hyaluronan materials, it appears that the presence of the catanionic assembly improve the drug release capacity of the material to PBS receptor solution. However, the release of the KP catanionic surfactant from HA materials depends on the structure of HA materials and on the aggregation state of the catanionic entity. The vesicles seem to be constrained within the polymer matrix limiting then the KP delivery. These results show that the KP release profile can be modulated through a change on the porosity of the HA material or by the aggregation state of the active principle. Acknowledgements The authors wish to acknowledge the sponsorship of the Spanish Ministry of Economy and Competitivity (CTQ2016-80645-R and CTQ2014-52687-C-1-P) and Generalitat de Catalunya (Grant 2014SGR1655). The authors also acknowledge Dr. M. Monge and M. Bover for technical support.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Hyaluronan based materials with catanionic sugar-derived surfactants as drug delivery systems F. Roig a , M. Blanzat b , C. Solans c,d , J. Esquena c,d , M.J. García-Celma a,d,∗ a
Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028 Barcelona, Spain Laboratoire des IMRCP, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France c Institut de Química Avanc¸ada de Catalunya (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain d CIBER-BBN (Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina), Jordi Girona 18-26, 08034 Barcelona, Spain b
a r t i c l e
i n f o
Article history: Received 23 December 2016 Received in revised form 17 January 2018 Accepted 20 January 2018 Keywords: Hyaluronan Catanionic surfactant Controlled release Crosslinked hydrogel Solid foam Highly concentrated emulsion Ketoprofen
a b s t r a c t In the present work novel drug delivery systems consisting in highly porous Hyaluronan foams for the administration of a non-steroidal anti-inflammatory drug (NSAID), ketoprofen, have been obtained. A sugar-derived surfactant associated with ketoprofen was prepared and incorporated into the porous hyaluronan materials. The association between a lactose derived surfactant, Lhyd12 , and ketoprofen was obtained by acid-base reaction and its physicochemical properties were studied. Tensiometric and dynamic light scattering (DLS) determinations showed the formation of catanionic surfactant aggregates, Lhyd12 /ketoprofen, in aqueous solution. Furthermore, the catanionic surfactants allowed greater solubilisation of ketoprofen. Hyaluronan porous materials were developed using butanediol diglycidyl ether as crosslinking agent. The profile release of Lhyd12 /ketoprofen from hyaluronan based materials shows differences as a function of the aggregation state of catanionic surfactant. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Soft matter drug delivery systems have recently received substantial attention, in particular from the nanomedicine field. In fact, these carriers increase drug bioavailability and activity, while decreasing its toxicity, because drug efficiency is often altered by its non-controlled biodistribution. The design of the appropriate drug delivery system is then important to optimize drug efficiency [1]. In order to meet the needs, a wide range of drug delivery systems have already been developed. These systems include matrix and vesicular carriers, with a large range of size and structures [2]. The use of a biocompatible polymer matrix is an interesting approach because it allows controlling the release of low molecular weight drugs for various routes of administration such as: oral, parenteral, ocular, etc. [3]. Furthermore, these materials have the following advantages: reduction of side effects and improvement of drug bioavailability, solubilization of lipophilic drugs, and
∗ Corresponding author at: Departament de Farmàcia i Tecnologia Farmacèutica i Fisicoquímica, IN2UB, Universitat de Barcelona, Joan XXIII s/n, 08028, Barcelona, Spain. E-mail address: [email protected] (M.J. García-Celma). https://doi.org/10.1016/j.colsurfb.2018.01.037 0927-7765/© 2018 Elsevier B.V. All rights reserved.
lower treatment costs [4]. Hyaluronan (HA), belongs to the family of glycosaminoglycans and consists on N-acetyl-d-glucosamine and D-glucuronic acid [5]. This polymer is an important component of the extracellular matrix of connective tissue and is found in various parts of the human body such as: skin, cartilage, vitreous humour and intra-articular joint fluid [6]. It also plays an important role in cartilage matrix stabilization, cell proliferation, control of morphogenesis, cancer metastases, inflammation processes and wound healing [7–12]. HA is degradable in vivo by enzymes such as hyaluronidase present in human tissues [13]. A suitable approach to avoid fast elimination from the human body could be the preparation of HA materials chemically crosslinked that show an increase in their resistance against hyaluronidase [14,15]. Polymeric materials can be obtained by crosslinking hydrophilic polymers in bulk [16] (hydrogels) or by the use of colloidal systems as templates for the preparation of materials with controlled porosity (solid foams). The incorporation of a polymer in the continuous phase of a highly concentrated emulsion, allows the preparation of porous materials with very high pore volume [17]. These materials have found a number of applications, including biomaterial engineered devices and drug delivery systems [18]. In addition, vesicular drug delivery systems made of catanionic surfactants have also proved their great contribution to drug solu-
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bility in water and drug delivery by means of spontaneous vesicles formation [19–21]. These vesicles also showed an increase of the therapeutic effect of the drug together with a sustained diffusion through the skin [19,22]. The strategy adopted in this work was to evaluate the impact of the combination of both drug delivery systems by preparing a novel complex drug delivery system. These advanced materials consist in the association between a drug, ketoprofen (KP), and a sugar-derived surfactant to form an ion-pair, “catanionic” surfactant, which would later be incorporated into a crosslinked polymer matrix based on HA. To ensure the formation of a KP catanionic surfactant, the selected surfactant has to be capable of forming an ionic acid-base pair with the carboxylic acid of KP. This feature means that the surfactant requires the presence of a basic group that is able to react with the acid group of the drug. In addition, the surfactant has to be biocompatible and biodegradable without inducing toxicity to ensure biocompatibility of the ion pair within the body. Furthermore, the physicochemical characteristics of the surfactant should lead to drug solubilization. In this context, sugar-derived surfactants represent a suitable choice for this purpose. Apart from their biocompatibility and biodegradability, these surfactants can be obtained from sugars that are natural raw materials available in large quantities. The aim of this work was to develop a novel drug delivery system based on HA materials (hydrogels and solid foams) loaded with KP catanionic surfactants and determine the differences in the release behavior when the aggregates of catanionic surfactants are formed. 2. Experimental 2.1. Materials Hyaluronic acid sodium salt from Streptococcus equi. of molecular weight around 2 million Daltons with 97% purity was obtained from Sigma-Aldrich. The chemical crosslinker butanediol diglycidyl ether (BDDE) with a molecular weight 202.25 g mol−1 with 95% purity was obtained from Sigma-Aldrich. Nonionic surfactant Cremophor RH455 (CRH 455) with an HLB between 14 and 16 was obtained from BASF. Miglyol 812, medium chain triglycerides, was obtained from Fagron. Ketoprofen (C16 H14 O3 ), (KP), non-steroidal anti-inflammatory drug (NSAIDs) used as anionic precursor surfactant, was from Fagron with 99.8% purity. Phosphate buffer solution pH 7.4, (PBS) was prepared from: KH2 PO4 from Fagron Iberica ® S.A.V, Na2 HPO4 from Probus S.A, NaCl from Acofarma , and Milli® Q deionized water. Cellulose tubular membrane was purchased from Orange Scientific. Its properties are a 12.000-14.000 Da nominal MWCO, and 20 m wall thickness. Mobile phase for HPLC (pH 3.0) was prepared from 45% of aqueous phase comprising: Citric ® ® acid from Acofarma with 99.5% purity, NaCl from Acofarma , NaOH ® ® from Acofarma , and Milli-Q deionized water; and 55% of organic phase acetonitrile obtained from Carlo Erba Reagents, with 99.9% purity. 2.2. Methods 2.2.1. Synthesis of KP catanionic surfactant The catanionic sugar-derived surfactant was obtained by an acid–base reaction between equimolar amounts (0.66 mmol) of cationic and anionic precursor surfactants in 30 mL of water. Ndodecylamino-1-deoxylactitol, designated as Lhyd12 , was used as cationic precursor surfactant. Lhyd12 was obtained as previously described from a reductive amination of dodecylamine with lactose [23,24]. An NSAID, KP was used as the anionic precursor surfactant. The two components were added in ultrapure water and stirred for 24 h. The resulting homogeneous solution was lyophilized. After lyophilization a white powder corresponding to the catanionic
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Fig. 1. Molecular structure of KP catanionic surfactant as a result of ionic association between Lhyd12 and ketoprofen.
sugar-derived surfactant was obtained with a quantitative yield (Fig. 1). 2.2.2. Physicochemical characterization of KP catanionic surfactant 2.2.2.1. Fourier transform − infrared spectroscopy (FT-IR). Infrared spectra were obtained with a Perkin-Elmer IR FT 1760X. KBr discs with a concentration of 0.5 w/w% were prepared of KP catanionic surfactant. 2.2.2.2. Surface tension measurements. The values of surface tension as a function of catanionic surfactant concentration were measured by the Wilhelmy plate method using a Kruss Tensiometer Easy Dyne at 25.0 ◦ C ± 0.1 ◦ C. The catanionic solutions were prepared by dissolving weighted amounts of dry catanionic associations in ultrapure water. Solutions were stirred at room temperature during a few minutes. 2.2.2.3. Dynamic light scattering (DLS). Dynamic light scattering was performed using a Malvern Zetasizer Nano-ZS, ZEN3600, with a measuring range of 0.5 nm to 10 m. The light source used was a He-Ne laser with a wavelength of 633 nm. The temperature was regulated at 25.0 ◦ C with a Peltier with an accuracy of ± 0.1 ◦ C. The measuring angle was 173◦ . Samples of aqueous solutions of KP catanionic surfactant (3.5 × 10−3 M) were introduced into cells (pathway, 10 mm). The deconvolution of the measured intensity autocorrelation function of the samples was realized with the multiple narrow modes program that uses a non-negatively constrained least squares (NNLS) fitting algorithm to obtain the distribution of diffusion coefficients (D) of the solutes. The apparent equivalent hydrodynamic diameter (dh ) was determined using the Stokes-Einstein equation. Hydrodynamic diameter values were obtained from three different runs. 2.2.3. Preparation of chemically crosslinked hydrogels For the preparation of HA hydrogels, 50 mg of sodium hyaluronate were introduced into test tubes of 12 × 75 mm, to which 500 L of crosslinking solution, consisting of BDDE (5% v/v) in alkaline media (0.2 M NaOH), were added. Then, the HA and the crosslinking solution were stirred with a vortex till a homogeneous mixing. The resulting mixture was incubated at 25 ◦ C for 24 h and the HA crosslinked hydrogel was obtained. The epoxy groups of BDDE react with the hydroxyls present in the HA polymer [25]. 2.2.4. In vitro cell viability analysis Cell viability in the presence free BDDE crosslinker, BDDE crosslinked hyaluronan hydrogels and KP catanionic surfactant was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay [26]. For each assay, HeLa cells were seeded in Dulbecco’s modified Eagle’s medium supplemented with FBS and antibiotics. Then, the culture medium was replaced with samples at the required concentrations. 100 L of BDDE solution at the same concentration that in the hydrogel and the corresponding dilutions 1:5 and 1:10 v/v, 100 L
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Fig. 2. IR spectrum in KBr tablet method for: a) an equimolar mixture of Lhyd12 and KP, and b) KP catanionic surfactant.
of KP catanionic surfactant (0.14, 0.7 and 1.18 mM) or 25 mg of hydrogel and the corresponding dilutions 1:5 and 1:10 p/v were incubated in this medium. After incubation, the medium was withdrawn and cells were seeded with fresh medium and incubated again. Then, MTT reagent solution was added and DMSO was used to dissolve the formazan crystals. Absorbance was measured at a = 590 nm. The results were expressed as viability percentages. 2.2.5. Preparation of chemically crosslinked solid foam For the preparation of HA solid foam, a O/W highly concentrated emulsion (HIPRE) was used as template. The components of the HIPRE were: aqueous component, a castor oil derivative as surfactant (CRH 455), and an oil derived from caprylic and capric fatty acids (Miglyol 812) as oil component. The HIPRE with HA (5% in water) in the continuous phase was formed with a high-performance dispersing equipment (Ultaturrax). After HIPRE formation, BDDE as crosslinker agent was introduced and kept in 25 ◦ C for 24 h to crosslinking HA. After crosslinking reaction of HA in the continuous phase of HIPRE, the surfactant and oil were removed by solvent extraction with ethanol and water for 12 h. Finally the HA solid foam was freeze-dryied. 2.2.6. Determination of the specific surface area of the solid foam The specific surface area of the solid foam was determined by Nitrogen sorption experiments. Nitrogen adsorption and desorption isotherms at 77 K of the solid foam were obtained by using an AUTOSORBTM IQ instrument. According to the BET theory [27], the specific surface area of the solid foam was determined fitting the BET equation to the adsorption curve. 2.2.7. Incorporation of the Lhyd12 /ketoprofen into the HA materials The method used to incorporate the KP catanionic surfactant into the HA materials was the permeation method: the HA materials (hydrogels and solid foams) were prepared and then immersed
in a volume of 2 mL solution of KP catanionic surfactant in water, which was completely absorbed by HA materials.
2.2.8. Release studies of ketoprofen from HA materials In vitro release studies were carried out in a dissolution tester. The equipment used was an Elite 8TM dissolution tester from Hanson Research Corporation (USA). It consists of 8 dissolution vessels immersed in a thermostatic bath. Each dissolution vessel had a capacity of 150 mL and a setup for semisolid formulations called Ointment cellwas incorporated in each vessel. A cellulose membrane was placed in each ointment cell to separate the hydrogel from the receptor solution. The receptor solution consisted of 100 mL of PBS (pH 7.4). The temperature of the receptor solution was 37 ◦ C. The stirring speed of the paddles in each dissolution vessel was 25 rpm. The HA materials were placed in the ointment cell, as described previously [28]. Then, 150 mL of receptor solution (PBS) were placed into the glass vessel. In order to determine the amount of drug released as a function of time, 0.5 mL of receptor solution has been removed for analysis and the same amount of PBS solution was replaced. The release study lasted 24 h. Three replicates of each formulation were assayed.
2.2.9. Determination of ketoprofen concentration by HPLC KP released was analyzed by HPLC. The chromatographic system ® consisted on Shimadzu equipment with a column Kromasil 1005C18 purchased from Akzo Nobel with dimensions of 250 × 4.6 mm and pore size of 5 m, and a UV detector set at 233 nm for ketoprofen determination. Separation was carried out at room temperature using 55% acetonitrile and 45% aqueous phase (pH 3.0), as a mobile phase, with flow rate of 1 mL/min, and injection volume of 50 L. The KP retention time was about 7 min.
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tered at 130 nm with a polydispersity index of 0.2, as shown as Supporting information. All these results are in good agreement with the physicochemical features of vesicles already reported for these catanionic associations [19].
3.2. Preparation of drug delivery systems based on HA materials
Fig. 3. Aqueous solution surface tension as a function of the logarithm of the concentration of KP catanionic surfactant.
3. Results and discussion 3.1. Synthesis and characterization of KP catanionic surfactant A catanionic sugar-derived surfactant based on the association between lactose-derived surfactant, Lhyd12 , and KP has been obtained by acid-base reaction as described previously [19]. This reaction consists on the proton transfer between KP and the basic aminosugar. Therefore, the carboxylic acid of KP was transformed to carboxylate. The formation of the catanionic surfactant was first assessed by infrared spectroscopy comparing the spectra of the initial reagents with that of the association. Fig. 2 shows the disappearance of group (C O) and (OC OH) corresponding to carboxylic acid, 1692 cm−1 and 1228 cm−1 respectively, and the appearance of the group (COO-st as) at 1576 cm−1 and (COO-st sy) to 1393 cm−1 for the carboxylate. To study the behavior of the KP catanionic surfactant in aqueous solution, the surface activity was determined by surface tension measurements. The plot of the aqueous solution surface tension as a function of the logarithm of the concentration of KP catanionic surfactant (Fig. 3) allowed to obtain the critical aggregation concentration (CAC) which was around 1.5 mM. The size distribution of the aggregates was verified by Dynamic Light Scattering (DLS) at a concentration of 3.5 mM. The results showed the formation of a large distribution of aggregate population with a diameter cen-
The HA materials (hydrogels and solid foams) were obtained as described in the experimental section, and either free KP or KP catanionic surfactant were added into the polymer matrix. Due to poor solubility of free KP in water, its incorporation was performed using alcoholic solution and phosphate buffered solution (pH 7.4) into solid foams and hydrogels respectively. However, the increase in the solubility for KP in the association of KP catanionic surfactant, allows its incorporation by swelling the polymer matrix with water (containing KP catanionic surfactant). SEM images of the two HA based materials show differences in their structure. Freeze-dried hydrogels exhibit a disordered layered structure (Fig. 4a), which is commonly observed after lyophilization, and it can be attributed to the growth of ice crystals during freezing [29]. However, high porous solid foams were obtained by crosslinking in the continuous phase of the HIPREs (Fig. 4b). The solid foams showed smaller, more homogeneous and well interconnected pores. Their pore size is between 1 and 5 m, being the largest macropores around 30 m, and the smallest around 0.5 m. Also, the specific surface area of the solid foam was determined by Nitrogen sorption experiments (Fig. 5), and the material show a high specific surface area, around 20 m2 /g. This porous texture is a replica of the structure of the highly concentrated emulsion used as template. Cytotoxicity studies were performed in order to determine the biocompatibility of the prepared materials. The MTT test [26] was applied to study the influence of various concentrations of the free crosslinker BDDE, BDDE crosslinked hyaluronan hydrogels and catanionic surfactant on HeLa cells viability, as an indication of toxicity. HeLa cells are a versatile model cell line that is widely used in nanomedicine tests. When the cells viability is higher than 80%, it can be considered that the sample assayed induces low cytotoxicity. The results showed the toxicity of the free crosslinker BDDE (cell viability lower than 11%), in contrast to the catanionic surfactant (cell viability higher than 90%) and the BDDE crosslinked Hyaluronan hydrogel (cell viability higher than 85%), an indication of absence of free BDDE in the medium. As the catanionic surfactant and the BDDE crosslinked HA hydrogels assayed induce low cytotoxicity in HeLa human cell line, they could be proposed as good
Fig. 4. SEM images corresponding to a) freeze-dried hydrogels, and b) solid foams based on HA.
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Fig. 5. Nitrogen adsorption-desorption isotherms at 77 K of a HA based solid foam. Fig. 7. KP release profiles from HA hydrogels: KP catanionic surfactant without aggregation (
Fig. 6. KP release profiles as a function of HA materials: in solution, from HA hydrogels, and from HA solid foams.
candidates for implant developments. The details of cytotoxicity studies performed are showed as Supporting information. 3.3. Release studies of KP catanionic surfactant incorporated in HA materials The main purpose was to study the influence of the material structure based on HA (hydrogels and solid foams) and the aggregation state of catanionic surfactant (
tion. However, for the HA based solid foams, the retention could be caused by the smaller pores present in these materials. The release of KP from HA solid foams is much lower than that obtained with other solids foams (e.g. polystyrene solid foams, that reach 70% after 24 h) [18]. Also we studied the release profile of KP catanionic surfactant as a function of their state of aggregation. The release of the KP catanionic surfactant incorporated to hydrogels and solid foams based on HA to PBS solution were performed with two concentrations: [KP catanionic surfactant] >CAC: 5.9 mM, and [KP catanionic surfactant]
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.colsurfb.2018.01. 037. References
Fig. 8. KP release profiles from HA solid foams: KP catanionic surfactant without aggregation (
a critical aggregation concentration, the size of these aggregates being suitable for the drug release from a polymeric matrix system. Concerning the KP release from the hyaluronan materials, it appears that the presence of the catanionic assembly improve the drug release capacity of the material to PBS receptor solution. However, the release of the KP catanionic surfactant from HA materials depends on the structure of HA materials and on the aggregation state of the catanionic entity. The vesicles seem to be constrained within the polymer matrix limiting then the KP delivery. These results show that the KP release profile can be modulated through a change on the porosity of the HA material or by the aggregation state of the active principle. Acknowledgements The authors wish to acknowledge the sponsorship of the Spanish Ministry of Economy and Competitivity (CTQ2016-80645-R and CTQ2014-52687-C-1-P) and Generalitat de Catalunya (Grant 2014SGR1655). The authors also acknowledge Dr. M. Monge and M. Bover for technical support.
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