Sponges carrying self-microemulsifying drug delivery systems

International Journal of Pharmaceutics 458 (2013) 208–217

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Sponges carrying self-microemulsifying drug delivery systems Elinor Josef a,b , Havazelet Bianco-Peled b,c,∗ a

Inter-Departmental Program for Biotechnology, Technion – Israel Institute of Technology, Haifa 32000, Israel The Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 32000, Israel c Department of Chemical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel b

a r t i c l e

i n f o

Article history: Received 8 July 2013 Received in revised form 9 September 2013 Accepted 15 September 2013 Available online 3 October 2013 Keywords: Self-microemulsifying drug delivery systems Alginate Solubility Sponge Solid

a b s t r a c t Self-microemulsifying drug delivery systems (SMEDDS) increase the solubility of lipophilic drugs. One barrier to their wide application is their liquid nature. We report on a new method to solidify SMEDDS—their incorporation in sponges made from a hydrophilic natural polymer. Using different freeze-drying schemes, sponges were prepared from alginate gels containing microemulsions. The sponges’ structures were studied with scanning electron microscopy and small angle X-ray scattering. The oil droplets survived the drying process, and SMEDDS were present as 9 nm-sized objects in the dried sponges. The sponges were rehydrated in water, and evidence of the presence of SMEDDS in the rehydrated sponges was found. A model hydrophobic molecule, Nile red, was soluble in all dry and rehydrated sponges. SMEDDS containing Nile red were gradually released from the sponges, at a rate that depended on the drying method. The equilibrium water uptake of the sponges was also found to be influenced by the drying scheme. The combination of SMEDDS and sponges may be a way to overcome the disadvantages of each component separately, provide a solid dosage form for SMEDDS that can sustain the release of drugs and also enable utilization of hydrophilic sponges for the delivery of hydrophobic drugs. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Self-emulsifying drug delivery systems (SEDDS) are one method used to increase the solubility and bioavailability of hydrophobic drugs (Kohli et al., 2010; Neslihan Gursoy and Benita, 2004). SEDDS are typically a mixture of surfactant, oil and drug, which form oil-in-water (o/w) emulsions following mild agitation and dilution with water (Neslihan Gursoy and Benita, 2004; Pouton, 1997). SEDDS that are formed with droplet sizes smaller than 200 nm are called self-microemulsifying drug delivery systems (SMEDDS). SEDDS formulations are often liquid or semisolid and must be encapsulated in soft or hard gelatin capsules (Neslihan Gursoy and Benita, 2004). Some lipids and commonly used surfactants, such as polysorbates, may interact with the shell and compromise its mechanical properties (Alexander, 2012; Mallikarjun and Babu, 2011; Mistry and Sheth, 2011). It is, therefore, of interest to prevent the interaction or find an alternative to filled-capsule preparations (Talegaonkar et al., 2008), for example, by designing a solid dosage form of SEDDS. Conventional solidification of SEDDS converts liquid or semisolid ingredients into powders. This can be achieved by having the SEDDS absorbed by porous solid carriers such as crosslinked

∗ Corresponding author at: Department of Chemical Engineering, Technion – Israel Institute of Technology, Technion City, Haifa 32000, Israel. Tel.: +972 4 829 3588; fax: +972 4 829 5672. E-mail address: [email protected] (H. Bianco-Peled). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.09.024

silicon dioxide, magnesium aluminum silicate, and calcium silicate (Beg et al., 2012, 2013; Kang et al., 2011). Other methods include melt-mixing (Li et al., 2009), extrusion/spheronization (Setthacheewakul et al., 2011; Yanyu et al., 2013) and spray drying (Mallikarjun and Babu, 2011; Oh et al., 2011; Onoue et al., 2012). These powders can be further processed into other solid dosage forms or packed into capsules (Kohli et al., 2010). Other forms of solid SEDDS—aside from granules, tablets, pellets, and capsules—have not been studied extensively (Tang et al., 2008). In this work we present a new approach for solidification of SMEDDS—their incorporation in hydrophilic sponges—and demonstrate the utility of this method using alginate sponges. Macroporous sponges have been developed for tissue engineering, bone regeneration, wound dressings and local delivery of drugs. They provide a method for improved mass transport compared to nonporous hydrogels (Andersen et al., 2012). Common techniques for producing these sponges include a drying step using a hydrogel or a polymer solution such as freeze-drying or solvent evaporation (Jayakumar et al., 2011). Drugs that are not soluble in hydrogels or aqueous solutions cannot be integrated simply, and uniformly distributed in sponges. Previous studies on the delivery of the hydrophobic compound Curcumin to facilitate wound healing used ethanol in chitosan–alginate sponges (Dai et al., 2009) or cyclodextrins in slightly hydrated alginate foams (Hegge et al., 2010) to improve the distribution of Curcumin. We suggest that combining SMEDDS and sponges could provide a convenient solid dosage form for SMEDDS and enable the incorporation of hydrophobic drugs

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in hydrophilic sponges. Solidification of SEDDS by absorption into sponges or foams has not been investigated to date. Unlike most SMEDDS which dissolve shortly after administration, SMEDDSsponges retain the drug over time hence can be used for local or sustained drug delivery rather than systemic delivery. Furthermore, SMEDDS-sponges are ready to use after drying and do not require an additional processing step such as capsule-filling. SMEDDS entities in alginate sponges were produced by drying composite gels, consisting of microemulsions embedded in crosslinked alginate. Composite gels were recently shown to be capable of loading and releasing various hydrophobic drugs (Josef et al., 2010, 2012, 2013). It is known that drying hydrogels by different techniques generates sponges with varied pore sizes, affecting the diffusion rate of solutes in the sponge. Thus, we investigated several drying techniques as a means to provide control over the release rate. Four process methods were investigated. The first three included lypophilization, after immersing the hydrogels in isopropanol or freezing either at −18 ◦ C or in liquid nitrogen. In addition, films were produced by vacuumdrying. Our aim was to produce SMEDDS by solvent evaporation of microemulsions. We hypothesized that upon introduction into a wet environment, the SMEDDS would revert to a microemulsion inside the sponge, and the sponge would reconstitute as a hydrogel, enabling a hydrophobic entity to be released together with the newly formed microemulsion droplets. We examined the release and swelling rates, and explored the micro- and nanostructure of the SMEDDS-sponges and their corresponding rehydrated sponges. The nanostructure of rehydrated sponges as a function of the drying technique is thoroughly discussed. Incorporation of SMEDDS in sponges could open the way for the design of an easyto-store, stable delivery dosage form capable of gradually releasing a hydrophobic drug.

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freezing in liquid nitrogen (N2 ), freezing at −18 ◦ C (FRZ), or placed in isopropanol for 15 min followed by freezing at −18 ◦ C (IPRO). Gels were then freeze-dried at −25 ◦ C. For comparison, a film of alginate with SMEDDS was prepared by drying in a vacuum oven (VAC). 2.3. Release studies 6 mL of double distilled water were added to each sample. Samples were placed in a 37 ◦ C water bath and shaken at a rate of 100 rpm. At each time interval, 0.2 mL of the surrounding medium was sampled and replaced with 0.2 mL of fresh water. The sample was measured by a spectrophotometer at a wavelength of 233 nm. A control sponge with no SMEDDS was used as blank, and SMEDDS concentrations were calculated from calibration curves. UV spectroscopy was carried out on a 96-well plate with a Synergy HT microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Results represent an average of four samples. 2.4. Water uptake Water uptake experiments were executed using the same conditions as the release studies. At given times, the sponges were removed from water, blotted with filter paper to eliminate excess water, and weighed. The water uptake St was calculated at each time point using the weight of the wet sponges (Mt ) and the weight of the initial dry sponges (M0 ): St = Mt − M0 /M0 . Total water uptake was defined as the water uptake at equilibrium. The fraction of water uptake at each time point was calculated by normalizing the water uptake in respect to the equilibrium uptake. Results represent an average of five samples. 2.5. Small angle X-ray scattering (SAXS)

2. Materials and methods 2.1. Materials D(+)-gluconic acid ␦-lactone (GDL), ethylene glycol-bis(2aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA), Sorbitan oleate (Span 80), and Isopropyl Myristate (IPM) were purchased from Fluka, Israel. CaCl2 was purchased from J. T. Baker, and Polysorbate 80 (Tween 80) from Merck, USA. Phenoxazon-9 (Nile red) and isopropanol were obtained from Sigma, Israel. Alginate (LF 200 FTS) was supplied by FMC Biopolymers, Drammen, Norway. All materials were used as received. 2.2. Sponge preparation The microemulsion was prepared by mixing Tween-80 (27.2 wt%/wt), Span-80 (0.8%) and IPM (5.4%), followed by dropwise addition of double distilled water (66.4%). Nile red was added at a concentration of 0.5 mg/mL. All ingredients are considered to be biocompatible. Alginate was dissolved in double distilled water; thereafter, the microemulsion (10 wt%/vol of final gel) was added and stirred with a magnetic stirrer. A calcium source in the form of a pre-prepared Ca-EGTA solution was introduced next, followed by a GDL solution. GDL induces slow release of calcium ions from the Ca-EGTA complex, allowing gelling of the alginate solution (Stokke et al., 2000). The Ca2+ :GDL molar ratio was 1:2. For the preparation of the Ca-EGTA solution, equal molar amounts of CaCl2 and EGTA were dissolved in water and the pH level was adjusted to 7 by adding 1 M NaOH. Final compositions were 10 mg/mL alginate and 20 mM calcium. Gels were cast by pouring 600 ␮L of the solution into a mold ring with a diameter of 1.4 cm and allowed to equilibrate for 24 h. Next, the gels were dried by different methods:

X-ray scattering was performed using a small-angle diffractometer (Molecular Metrology SAXS system with Cu K␣ radiation from a sealed microfocus tube, (MicroMax-002+S), two Göbel mirrors, and three-pinhole slits; the generator was powered at 45 kV and 0.9 mA). The scattering patterns were recorded by a 20 cm × 20 cm two-dimensional position-sensitive wire detector (gas-filled proportional type of Gabriel design with 200 ␮m resolution), which was positioned 150 cm behind the sample. The scattered intensity I(q) was recorded in the interval 0.08 < q < 2.5 nm−1 , where q is the scattering vector defined as q = (4/)sin(), 2 is the scattering angle, and  is the radiation wavelength (0.1542 nm). The sample or gel under study was sealed in a Linkam apparatus with poly-imide. Measurements were performed under vacuum at ambient temperature. The scattering curves were corrected for counting time, sample absorption, and scattering from the solvent. 2.6. High resolution scanning electron microscopy (SEM) Imaging was performed using a Zeiss Ultra-plus high resolution SEM equipped with a Schottky field-emission electron source. Micrographs were acquired using both secondary electrons (in-lens and Everhart-Thornley detectors) and backscattered electrons, at a relatively low accelerating voltage of 1 kV and at working distances of 2.3–5 mm. 3. Results and discussion 3.1. Macrostructure/microstructure Sponges containing SMEDDS were produced by freeze-drying gels containing microemulsions (which we name composite gels)

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in three ways: (1) Freezing at −18 ◦ C (FRZ), (2) freezing in liquid nitrogen (N2 ), and (3) placing in excess isopropanol for 15 min followed by freezing at −18 ◦ C (IPRO). For comparison, a film containing SMEDDS was produced by vacuum drying the composite gel (VAC). Nile red was entrapped within the microemulsion as a model hydrophobic drug. Control sponges were fabricated in the same manner from microemulsion-free alginate gels. The different freezing regimes were selected due to their ability to produce sponges with variations in pore size, which could enable different rates of drug release. Freezing at −18 ◦ C (FRZ) creates ice crystals that tear the gel, making large pores in the sponges (Kang et al., 1999). Freezing in liquid nitrogen (N2 ) is frequently applied in order to preserve the network structure. The fast cooling rate prevents the formation of large ice crystals, generating smaller macroscopic pores (Gutiérrez et al., 2008). Isopropanol (IPRO) is an anti-solvent for alginates. Upon immersion, the gel contracts, possibly producing another pore size. Vacuum drying (VAC) is expected to lead to a non-porous sample due to collapse of the polymeric network under capillary forces (Hirashima, 2011). Pictures of the dried control sponges and SMEDDS-sponges are presented in Fig. 1A and B, respectively. Fig. 1C and D exhibits the rehydrated sponges. The most prominent feature of the SMEDDSsponges is their ability to increase the solubility of Nile red both in dried and rehydrated sponges. The uniform red color of the SMEDDS-sponges indicates that the Nile red is homogenously dispersed, which suggests that the SMEDDS exist after rehydration. Nile red is commonly used in the literature as a model for hydrophobic drugs (Ben Yehuda Greenwald et al., 2013; Kurniasih et al., 2010). In a previous study on composite hydrogels embedding microemulsions we have shown that several hydrophobic molecules including Nile red and the drugs Progesterone, vitamin D3 , and Ketoprofen could be loaded and released from the hydrogels (Josef et al., 2013). All compounds were released from composite gels at the same rate, suggesting that the release is governed by the structure of the gel rather than the attributes of the drug itself. Thus we believe that the SMEDDS-sponges have the potential for delivering these drugs and possibly others as well. The sponges’ appearance depends on the drying method. Rehydrated sponges that were produced using the FRZ and IPRO schemes contain bubbles, which are not present in the control gel. The bubbles could originate from macroscopic damage caused by the large ice crystals formed while freezing at −18 ◦ C. In rehydrated IPRO sponges the bubbles are not distributed uniformly, possibly because the diffusion of isopropanol into the gel before drying causes a gradient of this solvent as a function of the depth and the radius in the gel. The coloring of IPRO rehydrated sponges appears to be fainter compared to the other sponges. The gel contracted in the presence of isopropanol, and some microemulsion droplets were released, decreasing the loading of the drug. A spectroscopic assessment of the concentration of oil droplets in the isopropanol after 15 min immersion reveals that about a quarter of the droplets in the gels were lost to the medium. However, the IPRO drying technique produces visually appealing sponges with a shape that most resembles the shape of the control gel. The sponges were imaged with HR-SEM. Comparison of the control sponges (Fig. 2A–C) and the SMEDDS-sponges (Fig. 2E–G) reveals that the SMEDDS fill most of the sponges’ voids. Therefore, the porosity of different drying schemes could only be inspected by inspecting the micrographs of the control sponges. SEM micrographs of SMEDDS spray-dried with colloidal silica or dextran as solid carriers revealed that the absorption of SMEDDS depends on the type of carrier (Oh et al., 2011). The rough surface of SMEDDSsponges suggests that similar to colloidal silica, the SMEDDS were absorbed or coated inside the pores of the sponges. Many studies have shown that pore size depends on the freezing scheme. For example, Thornton et al. (2004) reported that the

pore size of covalently crosslinked alginate gels was smaller when frozen in liquid nitrogen compared to freezing at −18 ◦ C. Zmora et al. (2002) found that pore size and shape are a function of the position in the gel. A literature search did not reveal any analyses of the pore size of alginate sponges fabricated from gels in which the water was replaced with isopropanol. From our SEM micrographs it can be deduced that the VAC-film contains no pores in the microrange, and that the pore size of the N2 -sponges is smaller than those in the IPRO- and FRZ-sponges. In SMEDDS-films produced by vacuum drying, the SMEDDS accumulate on one side of the film (Fig. 2H). SMEDDS were also observed on the vessel that held the films during vacuum drying, indicating migration of SMEDDS from the VAC-sponges. The pale red color of the rehydrated gel (Fig. 1D, fourth panel) supports this postulation. 3.2. Release of SMEDDS The impact of the drying method on the release of SMEDDS from sponges was investigated. The release from an as-prepared gel was also measured for comparison purposes. The experiment was carried out in double distilled water in order to eliminate the effect of salts on the integrity of the gel. The release reached equilibrium in 24 h, and the curves were normalized in respect to the release at the end of the experiment. The as-prepared gel had the slowest release rate, whereas all drying methods accelerated it (Fig. 3). Freezing at −18 ◦ C induced the fastest release, as can be seen in the corresponding SEM micrographs. The sponges here exhibited larger pores than those produced by other drying methods (except the IPRO-sponge). These sponges also exhibited the largest burst effect. The slowest release from the sponges is seen in the VAC-film and N2 -sponges. These samples follow a similar profile, although the VAC-film showed no microscopic pores in SEM while the N2 -sponges were porous. These VAC-film images may be a result of the film’s small thickness compared to other drying methods, leading to shorter diffusion length. Power law fitting is a common and simple method utilized to analyze the release mechanism. Power law can indicate whether the release behavior is controlled by swelling of the sponge, diffusion of the drug, or both (Ritger and Peppas, 1987). A single power law could not be fitted to any of the release profiles from t = 0 and up to 60% release, suggesting that more than one mechanism is involved, e.g., both swelling and diffusion play a role in determining the release rate. Instead of attempting to fit both mechanisms using multiple variables, a simpler analysis is offered. Once the swelling process has reached equilibrium, it is probable that the main mechanism of drug release is diffusion. The swelling kinetics of the various sponges will be discussed in Section 3.3; at this point we only note that after 2 h, the water uptake reached over 80% of its final value. Therefore, it could be expected that at times longer than ca. 2 h, the transport of the solute will be well-described by Fick’s Law, and a plot of the fraction of SMEDDS released as a function of the square root of time will emerge as linear. Fig. 3B demonstrates that indeed after 2 h, and in some sponges even before, the release profiles follow the t0.5 rule up to 60% release. Since the slope is proportional to the diffusion coefficient (Brazel and Peppas, 2000; Ritger and Peppas, 1987), it can be deduced that the drying technique influences the diffusion coefficient of the SMEDDS in the sponges. In the study by Zmora et al. (2002), where pore size varied when the alginate was frozen at different temperatures prior to lyophilization, the release of bovine serum albumin (BSA) was also measured. The release rate of BSA from alginate scaffolds was not affected by their pore microstructure but rather was similar for all freezing regimes. The loading method used by Zmora et al., however, was different than the one used in our study: in the former study, the sponges were first soaked in a solution containing

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Fig. 1. Pictures of the sponges: each panel represents a different drying technique. (A) Dry sponge without SMEDDS. (B) Dry SMEDDS-sponge. (C) Rehydrated sponge without SMEDDS. (D) Rehydrated SMEDDS-sponge.

BSA, and then the release from the rehydrated gels was examined. Sriamornsak (1999) studied the release of BSA from freeze-dried and air-dried pectinate gel beads. BSA was released more quickly from freeze-dried beads due to the higher porosity—equivalent to our results. Release of drugs from solid-SEDDS produced by different techniques have mostly shown a burst effect, whereas more than 80% of the drug was released within 10 min (Balakrishnan et al., 2009;

Beg et al., 2013; Li et al., 2009; Nekkanti et al., 2010; Oh et al., 2011; Yanyu et al., 2013) or up to an hour (Beg et al., 2012; Li et al., 2009; Yanyu et al., 2013). However, some systems exhibited a gradual release of a drug, similar to our results. Tetrahydrocurcumin was release from a solid-SEDDS floating device (Setthacheewakul et al., 2011). Tuning the composition of the solid carrier could prolong the release to hours. A control over the release rate was also achieved in coated SEDDS-pellets, where the thickness of

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Fig. 2. HR-SEM micrographs of dry sponges: (A–D) without SMEDDS, (E–H) with SMEDDS. (A and D) FRZ-sponge, (B and F) N2 -sponge, (C and G) IPRO-sponge, (D and H) VAC-film. Bars = 20 ␮m.

the coating determined the rate of drug release (Serratoni et al., 2007). 3.3. Water uptake In order to learn about the contribution of swelling to the release process, we studied the swelling kinetics of the various sponges. First, we compared the rate of water uptake of SMEDDSsponges and their corresponding control sponges (Fig. 4A–D). The

rehydration kinetics of the IPRO-SMEDDS-sponges and the IPROsponges were similar (Fig. 4C). Similar behavior was observed in as-prepared gel with entrapped microemulsions, where the microemulsions did not alter the swelling kinetics of the gel (Josef et al., 2013). However, the N2 -sponges, FRZ-sponges and VAC-films rehydrated faster than their SMEDDS-sponge counterparts (Fig. 4A, B, and D). It is possible that drying induces an interaction between the SMEDDS components and the polymer chains. Comparing the water uptake of SMEDDS-sponges dried by different techniques,

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SMEDDS-sponges and their rehydrated form. The scattering curves of SMEDDS-sponges and SMEDDS-film are shown in Fig. 5. For comparison, the scattering curve of a microemulsion before drying is also plotted. All SMEDDS-sponges exhibit a peak that is absent from their corresponding control sponges, and hence could be attributed to the presence of SMEDDS droplets in the sponge. In small angle scattering the size of a scattered object can be approximated by the q position of its peak. The position of the peak of SMEDDS-sponges corresponds to a size of 2␲/q ≈ 9 nm, close to the size of an asprepared microemulsion, 11 nm. Thus, it is evident that the drying process does not disintegrate the SMEDDS and that during freezedrying, the droplets rearrange into smaller droplets. This explains the solubility of Nile red in SMEDDS-sponges. The peak is located at the same position for all freeze-drying schemes, but when vacuumdrying is applied the peak shifts toward higher q’s, indicating the formation of even smaller droplets. In addition, the intensity of the peak in the SMEDDS-film decreases, suggesting that there are fewer droplets. This supports the SEM micrograph, where SMEDDS are observed only in part of the film, and strengthens our postulation that some of the SMEDDS migrate from the film during drying.

Fig. 3. Release profiles of SMEDDS from different sponges. FRZ-sponge, VACfilm, IPRO-sponge, N2 -sponge, as-prepared gel. (A) %SMEDDS released as a function of time. (B) %SMEDDS released as a function of the square root of time. Lines represent a linear fit.

one can observe that the rate is similar across all freeze-drying methods (Fig. 4E). The water absorption rate of the VAC-SMEDDSfilms is smaller, possibly due to its smaller porosity. The reaction of control sponges is similar, with the exception of the first hour when the water uptake of the FRZ-sponges is higher than that of the IPRO-sponges. The similar rehydration kinetics of SMEDDS-sponges dried by different techniques is not compatible with the variations in release rates or in the pore size of the different sponges. A possible explanation is that the water molecule is too small to be affected by the macrostructure of the sponge, i.e., the pores do not pose a barrier to the diffusion of water into the sponge. The SMEDDS droplets, on the other hand, are larger, and could be influenced by the macrostructure. The drying method modifies the equilibrium water uptake. The highest water uptake is found for the IPRO-sponges and the lowest for VAC-film (Fig. 4F). There is no significant difference between the values of the FRZ-sponges and the N2 -sponges. Since the drying method changes the equilibrium state of the final gel, this indicates that the structure of rehydrated sponges also depends on the drying method. 3.4. Nanostructure 3.4.1. Dry SMEDDS-sponges The structure of SMEDDS in the dry sponges and the question of their rearrangement into microemulsion droplets upon sponge hydration are not trivial and have not been addressed before. We used small angle X-ray scattering to probe the nanostructure of the

3.4.2. Rehydrated SMEDDS-sponges The similar structures of the dry SMEDDS-sponges do not explain the different release profiles or swelling degree. Therefore, the rehydrated sponges were investigated for possible variances in their structure. The scattering from these sponges reveal that the drying scheme indeed influences the structure of the gel (Fig. 5B). We have previously shown that in these polymer and crosslinker concentrations, the scattering from composite gels is the addition of the scattering from the gel and the scattering from the microemulsion (Josef et al., 2013). This could not be extended to rehydrated sponges. With an exception of the N2 -sponge, the presence of SMEDDS changes the structure of the rehydrated gel to some extent. Thus, the origin of the different rates of water uptake between SMEDDS-sponges and the corresponding SMEDDS-free sponges traces back to changes in the nanostructure. Since Nile red is solubilized in the rehydrated sponges (Fig. 1), the presence of microemulsion droplets in the gel is anticipated. SAXS plots of rehydrated N2 -SMEDDS-sponges and FRZ-SMEDDSsponges exhibit a small peak that is absent from the N2 -sponges and the FRZ-sponges (Fig. 5B), analogous to plots of as-prepared gels with and without microemulsion (bottom curve in Fig. 5B). Similarly to the dry sponges, the peak of rehydrated SMEDDS-sponges is positioned at higher q’s than the as-prepared composite gel, signifying shrinkage of the droplets following drying and rehydrating. We have previously shown that in SAXS, the scattering from the gel exceeds the scattering from the microemulsion (Josef et al., 2013). The low scattering from the microemulsion could explain why it is difficult to observe a peak in the IPRO-SMEDDS-sponge and VAC-SMEDDS-film drying techniques, even though droplets are observed in the dry sponge and Nile red remains soluble in the rehydrated sponges. Future studies aimed at verifying the size and shape of the reformed microemulsion could be executed in SANS, in which scattering from the microemulsion dominates. 3.4.3. Modeling rehydrated sponges The equilibrium water uptake of the rehydrated sponges varied as a function of the drying method, hinting at different structures in the rehydrated sponges. A more refined study of the impact of the drying method on the nanostructure of rehydrated sponges was carried out by fitting models to SAXS curves. Models can provide physical parameters of the gel. A broken rod model is commonly used to describe scattering from alginate gels (Stokke et al., 2000). The crosslinks in alginate gels are formed by chain–chain associations induced by the presence of calcium ions, the crosslinker. Chains can be stacked to create junction zones. The broken rod

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Fig. 4. (A–D) Kinetics of water uptake, a comparison between SMEDDS-sponges (filled symbols) and control sponges (empty symbols). (A) VAC-film, (B) N2 -sponge, (C) FRZ-sponge, VAC-film, IPRO-sponge, N2 -sponge. (F) Equilibrium water uptake of IPRO-sponge, (D) FRZ sponge, (E) kinetics of water uptake of SMEDDS-sponges, SMEDDS sponges (dark gray) and control sponges (light gray).

model defines two typical radiuses of junction zones that take the form of a rod, plus a term depicting the chains. Although this model fit the as-prepared gel, its fit to rehydrated sponges is not as good. Since the gel is physically crosslinked, it is reasonable to hypothesize that the crosslinks rearrange during drying and rehydration processes, causing some of them to lose their rod characteristics. Thus, we attempted a different approach, one that decomposes the total scattering from a gel to the scattering from the solution and an excess scattering function depicting the contribution of the crosslinks (Shibayama, 2008). The crosslinks were modeled by one form factor of a rod, similarly to the broken rod model, expressing the total intensity as: I(q) =

J 2 (q, r) kchain · Pchain + krod 1 2

1 · , q (q · r)

(1)

where kchain and krod are pre-factors related to the number of scatterers and their scattering contrast constants, r is the crosssectional radius of the rod, and Pchain is the form factor of the chains. Three models were tested to account for Pchain : (1) A Gaussian chain, frequently used to describe polymer chains (Pedersen, 1997), (2) an Orenstein–Zernike equation, typically used for semidilute polymer solutions (Shibayama, 2008), and (3) a semi-flexible chain with excluded volume effects (Chen et al., 2006; Pedersen and Schurtenberger, 1996), which was shown to fit alginate solutions (Josef and Bianco-Peled, 2012). A model consisting of a rod and a semi-flexible chain was used previously to describe scattering from pectin–calcium gels (Ventura et al., 2013). The first two models fit the data of the control gel and rehydrated sponges well and produced similar parameters, while the third model had a slightly inferior fit (results not shown). Hence, the following equation based on the Orenstein–Zernike model was chosen to represent rehydrated sponges: I(q) = kchain

1 2 1 + q2 chain

+ krod

J12 (q, r) (q · r)

2

·

1 , q

(2)

where  chain is the correlation length. Fig. 6 shows the fit of Eq. (2) to the control gel, rehydrated VAC-film, FRZ-sponge and N2 sponge. The rehydrated IPRO-sponge was an exception; none of the three models fit it. We postulate that the immersion in isopropanol caused all junction zones to lose their rod characteristics and take on a disordered aggregate form. We, therefore, replaced the rod form factor with a general aggregate function (Wu et al., 1990). The addition of an Orenstein–Zernike and Debye–Bueche function is regularly used to describe scattering from gels (Shibayama, 2008): I(q) = kchain

1 2 1 + chain q2

+ kagg

1 2 q2 ) (1 + agg

2

,

(3)

where  agg is a length scale characterizing the inhomogeneities in the gel. Eq. (3) produced a good fit to the rehydrated IPRO-sponge (Fig. 6), as well as to the control gel, and rehydrated VAC-film and N2 -sponge. The parameters obtained from fitting Eqs. (2) and (3) to the relevant sponges are displayed in Fig. 7. The pre-factors kchain , 3 3 kagg , and krod were normalized by chain , agg and r2 , respectively. Assuming that the scattering contrast of each component is the same for all samples, the ratio kchain /kagg represents the relative amount of chains and aggregates. The parameters calculated from fitting as-prepared gel, VACfilm and N2 -sponge to Eq. (2) or (3) are generally statistically indifferent and they will be discussed as one. The length scale of the aggregates  agg or r is larger in rehydrated VAC-, FRZ-, and N2 -sponges than in the as-prepared gel (Fig. 7A). A balance is maintained between the size of the aggregates in rehydrated samples and their relative number kchain /kagg (Fig. 7B): when the size of the aggregates increases, their relative number decreases. There are only small differences in the correlation length of the chains for the samples (Fig. 7C). We can conclude that the drying process causes further aggregation of the crosslinks, and the extent of aggregation depends on the drying method.

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Fig. 5. SAXS curves of SMEDDS-sponges and control sponges. (A) Dry sponges FRZ-SMEDDS-sponge, VAC-SMEDDS-film, IPRO-SMEDDS-sponge, N2 SMEDDS-sponge, FRZ-sponge, IPRO-sponge, as-prepared microemulsion (−). (B) Rehydrated sponges, from top to bottom: IPRO-sponge, FRZ-sponge, N2 -sponge, VAC-film, as-prepared alginate gel ( ) and composite gel ( ). The bottom curve of each couple of curves is the control sponge (empty symbols) and the top one is SMEDDS-sponge (filled symbols). Curves were shifted to coincide at the tail.

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Fig. 7. Parameters derived from fitting the scattering curves of sponges dried using different methods. Light gray columns – model of rod and network (Eq. (2)). Dark gray columns – model of aggregates and network (Eq. (3)). (A)  chain in the aggregates and network model or r radius of the rod in the rod and network model. (B) kchain /kagg (C)  chain .

IPRO-sponges do not follow the same trend. Their aggregates remain the same size as in the as-prepared gel; however, their number greatly increases. The major difference between IPRO-sponges and all other samples manifests in the correlation length  chain , which increases almost threefold, indicating a more open network. This is in line with the water uptake values, where IPRO-sponges absorb more than double the amount of water than other formulations. The distinct behavior of the IPRO-sponges could possibly be due to rearrangement of the crosslinks and chains even before drying, during the immersion in isopropanol, an alginate anti-solvent. The porous nature of this network in the nano-range is not manifested in the release profiles, where the FRZ-sponges release the droplets faster than IPRO-sponges. 4. Conclusions Fig. 6. SAXS curves of rehydrates sponges. Lines are fits to the aggregate and network model (Eq. (3)) for PRO-sponge and to the rod and network model (Eq. (2)) for the rest of the samples. From top to bottom: IPRO-sponge, FRZ-sponge, N2 -sponge, VAC-film, as-prepared alginate gel. Curves are shifted for better visualization.

In this work we designed a solid SMEDDS dosage form capable of sustained delivery of a model hydrophobic molecule. SMEDDS were incorporated in alginate sponges by freeze-drying gels

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containing microemulsions. Droplets of SMEDDS were present in the dried sponges and evidence of their existence in rehydrated sponges was found. Different drying schemes were investigated as a way to produce various pore sizes, and consequently, alter release rates. Immersion of the sponges in isopropanol, prior to freeze-drying, ensured retention of their original gel shape. SEM micrographs revealed that vacuum-dried gels had no microscopic porosity, and that sponges frozen with liquid nitrogen had the smallest pores of the freeze-dried sponges. The rate of water uptake was found to be slower when SMEDDS were present in the sponge, but was not affected by the type of freezedrying method. The total water uptake and release profiles of SMEDDS were dependent on the drying scheme. Small angle scattering from rehydrated SMEDDS-free sponges were analyzed and a model of a network and a rod was found to fit rehydrated sponges. For sponges produced by immersion in isopropanol prior to lyophilization, the aggregates lost their rod shape, and a model of a network and aggregates was fitted instead. These sponges possessed the highest degree of swelling, and a SAXS curve fitting model suggested that the reason was the large correlation length, indicative of a large mesh size. It is concluded that both the microscopic and nanoscopic porosity are affected by the drying scheme. SMEDDS-sponges could be used as-is for local and sustained delivery of hydrophobic drugs, without an additional processing step such as tablet compression or capsule filling. The concept of combining SMEDDS with sponges could be attempted for other hydrophilic polymers and enable their application of hydrophobic drugs delivery. Acknowledgement We thank Judith Schmidt for her help in obtaining HR-SEM images. References Alexander, A., 2012. A review on novel therapeutic strategies for the enhancement of solubility for hydrophobic drugs through lipid and surfactant based self micro emulsifying drug delivery system: a novel approach. Am. J. Drug Discov. Dev. 2, 143–183. Andersen, T., Melvik, J.E., Gåserød, O., Alsberg, E., Christensen, B.E., 2012. Ionically gelled alginate foams: physical properties controlled by operational and macromolecular parameters. Biomacromolecules 13, 3703–3710. Balakrishnan, P., Lee, B.-J., Oh, D.H., Kim, J.O., Hong, M.J., Jee, J.-P., Kim, J., Yoo, B.K., Woo, J.S., Yong, C.S., 2009. Enhanced oral bioavailability of dexibuprofen by a novel solid self-emulsifying drug delivery system (SEDDS). Eur. J. Pharm. Biopharm. 72, 539–545. Beg, S., Jena, S.S., Patra, C.N., Rizwan, M., Swain, S., Sruti, J., Rao, M.E.B., Singh, B., 2013. Development of solid self-nanoemulsifying granules (SSNEGs) of ondansetron hydrochloride with enhanced bioavailability potential. Colloids Surf. B: Biointerfaces 101, 414–423. Beg, S., Swain, S., Singh, H., Patra, C., Rao, M., 2012. Development, optimization, and characterization of solid self-nanoemulsifying drug delivery systems of valsartan using porous carriers. AAPS PharmSciTech 13, 1416–1427. Ben Yehuda Greenwald, M., Ben Sasson, S., Bianco-Peled, H., 2013. A new method for encapsulating hydrophobic compounds within cationic polymeric nanoparticles. J. Microencapsul., 1–9. Brazel, C.S., Peppas, N.A., 2000. Modeling of drug release from swellable polymers. Eur. J. Pharm. Biopharm. 49, 47–58. Chen, W.-R., Butler, P.D., Magid, L.J., 2006. Incorporating intermicellar interactions in the fitting of SANS data from cationic wormlike micelles. Langmuir 22, 6539–6548. Dai, M., Zheng, X., Xu, X., Kong, X., Li, X., Guo, G., Luo, F., Zhao, X., Wei, Y.Q., Qian, Z., 2009. Chitosan–alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat. J. Biomed. Biotechnol., 2009. Gutiérrez, M.C., Ferrer, M.L., del Monte, F., 2008. Ice-templated materials: sophisticated structures exhibiting enhanced functionalities obtained after unidirectional freezing and ice-segregation-induced self-assembly†. Chem. Mater. 20, 634–648. Hegge, A.B., Andersen, T., Melvik, J.E., Kristensen, S., Tønnesen, H.H., 2010. Evaluation of novel alginate foams as drug delivery systems in antimicrobial photodynamic therapy (aPDT) of infected wounds—an in vitro study: studies on curcumin and curcuminoides XL. J. Pharm. Sci. 99, 3499–3513.

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