Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2022 Nov 16;7(47):42845–42853. doi: 10.1021/acsomega.2c04834

Bijels as a Fluid Labyrinth for Drugs: The Effect of Nanoparticles on the Release Kinetics of Ethosuximide and Dimethyl Fumarate

Valeria Vanoli †, Giovanna Massobrio †, Fabio Pizzetti †, Andrea Mele †,‡, Filippo Rossi †,*, Franca Castiglione †,*
PMCID: PMC9713867  PMID: 36467913

Abstract

graphic file with name ao2c04834_0011.jpg

Bijels (bicontinuous interfacially jammed emulsion gels) raised an increasing interest as biomaterials for controlled drug delivery due to their biphasic nature organized in mesoscopic tortuous domains. Two bijel formulations were prepared and explored as delivery systems for both hydrophilic and lipophilic drugs, ethosuximide and dimethyl fumarate. The two bijel-like structures, based on polymerized ε-caprolactone/water, differ in the stabilizing nanoparticle hydroxyapatite (inorganic) and nanogel-based nanoparticles (organic). Diffusion nuclear magnetic resonance spectroscopy has been used to characterize the bijel structure and the transport behavior of the drug molecules confined within the water/organic interconnected domains. A reduced diffusion coefficient is observed for several concentrations of the drugs and both bijel formulations. Moreover, in vitro release profiles also reveal the effect of the microstructure and drug–nanoparticle interactions.

1. Introduction

Bicontinuous interfacially jammed emulsion gels (known as “bijels”) are a new class of soft materials composed of two non-miscible fluids, such as water and oil, confined in a bicontinuous morphology by a monolayer of colloidal nanoparticles with equal affinity for the two fluids.1−3 The simultaneous presence of interconnected polar and apolar domains makes these materials suitable for several technological applications including molecular encapsulation,4,5 tissue engineering,6−9 and cosmetic10,11 and drug delivery.12−15 The latter is one of the most relevant applications as due to the biphasic nature of these materials, they are able to carry and release both lipophilic and hydrophilic drugs in the same system. Similarly to organogels16,17 and hydrogles,18,19 also developed as platforms for the controlled release of active ingredients or vaccines through a variety of different formulations,20 it is possible to fine-tune the structural/release properties by adjusting the preparation conditions. The design and fabrication of a bijel-like structure relay on a careful control of several key physical parameters, such as the liquid–liquid surface tension, the size of the nanoparticles, their symmetric wettability by the two liquids, and the characteristic time for jamming the interface.21 The interfacial self-assembly of colloidal particles or nanoparticles (NP) at a liquid–liquid interface due to NP-liquid favorable interactions minimizes the free energy of the system and leads to the formation of jammed structures.22 In particular, the channel width of the biphasic domains can be systematically varied by modifying the radius, volume fraction, and concentration of the colloidal particles23 and/or nanoparticles.24,25 Several theoretical calculations have been performed to study the interplay of all these factors in obtaining stable biphasic bicontinuous structures. Jansen and Harting,26 using the multi-phase lattice Boltzmann method, demonstrated that the fluid ratio and particle wettability have a crucial effect on the formation of bijel-like structures versus Pickering emulsions. Subsequent simulations27 have shown how the extent of particle–particle interactions, particle wettability, size, and diffusion determines the microstructure of the bijels in bulk samples and thin fluid films.28

Recently, several experimental methodologies for preparing the bijel-like structure have been proposed. They are essentially based on two synthesis paths: (i) by quenching partially immiscible liquids such as alcohol/alkane or 2,6-lutidine/water to induce phase separation via spinodal decomposition1−3 and (ii) by inducing phase separation in oil/water mixtures and stabilizing the fluid–fluid interfaces using colloidal particles, nanoparticles,29 or a combination of nanoparticles and molecular surfactants.8 In this process, both the shape of the nanoparticles, spherical shape8,30 or rod-like nanoparticles,31 and the particle surface properties define the morphology of the bijel-like structures. Furthermore, particle–fluid and inter-particle interactions, due to colloidal forces, e.g., van der Waals attraction and electrostatic and steric repulsion, must also be considered in the design of the bijel formation pathways.

In our study, a bijel-like network based on polymerized ε-caprolactone/water32 and stabilized by polyethylene glycol (PEG)-Jeffamine nanogel (organic nanoparticles) is prepared and its structure, morphology, and stability are characterized for the first time. Moreover, we investigate the effect induced by two different colloidal nanoparticles: porous hydroxyapatite (HAp) and PEG-Jeffamine nanogel (NG) on the properties/morphology of these bijels as drug delivery systems. Regarding this application, we consider here two active pharmaceutical ingredients: (i) ethosuximide, a water-soluble drug used extensively in the treatment of epilepsy conditions33 and (ii) dimethyl fumarate, a drug with low solubility in water used to treat psoriasis,34 relapsing forms of multiple sclerosis (MS) and relapsing remitting MS (RRMS).35 The chemical structure of both drugs is displayed in Scheme 1.

Scheme 1. (a) Ethosuximide and (b) Dimethyl Fumarate Chemical Structure.

Scheme 1

Open in a new tab

The transport properties of both hydrophilic/lipophilic small drugs loaded in the water/organic bijel phases and the drug–nanoparticle interactions are investigated at the molecular level using high-resolution magic angle spinning (HR-MAS) diffusion nuclear magnetic resonance (NMR) techniques36−39 suitable for soft materials and gels also in the presence of nanoscale confinement.40 In particular, we consider the effect of the nanoparticle type and drug concentration on their transport properties in the bijel fluid network. Finally, the NMR results for drug diffusion are compared with the macroscopic release kinetics by the two biphasic porous materials. Our interest in the development of bijel-like materials is motivated by the importance of multi-drug delivery systems.

2. Experimental Section

2.1. Materials

Calcium hydroxide (Ca(OH)2, ≥98%) and orthophosphoric acid (H3PO4, ≥85%) were purchased by Carlo Erba Reagents (Carlo Erba Reagents, Milano, Italy). ε-Caprolactone (CL, ≥99%) and 1,5,7triazabicyclo[4.4.0]dec-5-ene (TBD, ≥ 95%) were purchased by Sigma-Aldrich, Germany. Arabic gum (AG) was kindly gifted from the research group of Prof. Ciofani from Scuola Superiore di Studi Universitari e Perfezionamento Sant’Anna, CRIM Lab-Center for Applied Research in Micro and Nano Engineering. Jeffamine (Elastamine RE-2000) was sold by Huntsman Corporation (Woodlands, Texas, USA). Polyethylene glycol 8000 (PEG8000, MW = 8 kDa), carbodiimidazole (CDI, ≥ 98%), deuterium oxide (99.9 atom % D), PBS (Dulbecco’s phosphate-buffered saline solution pH = 7.4, 0.1 M), ethosuximide (Etho, MW = 141.168 g/mol), and dimethyl fumarate (DMF, MW = 144.127 g/mol) were purchased from Sigma-Aldrich, Germany. Fluorescein sodium salt (SF, MW = 412.3 g/mol) and rhodamine B (RhB, MW = 479.02 g/mol) were purchased from Merck (Deisenhofen, Germany). All reagents and solvents were used without further purification. Solvents were of analytical laboratory grade. Synthetized products were stored at 4 °C in the dark until used.

2.2. Materials Synthesis

2.2.1. Hydroxyapatite Nanoparticle (HAp) Synthesis

Hydroxyapatite nanoparticles were synthesized according to the procedure reported by Kumar et al.41 A 0.2 M solution of Ca(OH)2 dissolved in distilled water (24.9 mL) was stirred at 400 rpm and heated at 100 °C for 1 h. Then, a 0.12 M solution of H3PO4 (0.2 mL) was added to the water solution at a controlled rate of 4 mL/min using a dripping funnel. At the end of the addition, the pH was checked to be near neutrality with a litmus test and the mixture was left reacting for 2 h. After resting overnight, the solution underwent five centrifugation cycles at 5000 rpm of 3 min each, with supernatant removal and particle dispersion between each of them. The product was dried at 200 °C in an oven for 6 h and manually grinded with a pestle. The hydroxyapatite appears as a white fine powder, and the yield was 301 mg (81%). To stabilize nanoparticle dispersion preventing aggregation, a solution of Arabic gum (AG) in distilled water was heated at 37 °C for 1 h under moderate stirring and was successively added to the nanoparticles to achieve a 1:1 weight ratio.

The dispersion was sonicated for 12 h in an ultrasonic bath (Bandelin Sonorex) to make the AG graft onto the NP surface. Several colloidal dispersions of HAp were stabilized at a concentration of 20 mg/mL. For each of them, the DLS analysis was employed to evaluate the NP diameter.

2.2.2. PEG-Jeffamine Nanoparticle (NG) Synthesis

A solution of Jeffamine (2.5 g, 1.25 mmol) in H2O (5 mL) was added dropwise to a stirred solution of PEG-CDI (1 g, 0.125 mmol) in DCM (3 mL). The mixture was sonicated with a bath sonicator for 30 min at room temperature to obtain nanogels (NGs) and left stirring overnight in an open flask to let the DCM evaporate. The result was dialyzed against distilled water in an RC membrane with a 3.5 kDa cutoff for 72 h. Finally, the dialyzed product was lyophilized for 72 h, until all the remaining water was removed. The overall process is represented in Figure 1, and the yield was 0.706 g (70%). DLS analysis was performed to evaluate the hydrodynamic radius.

Figure 1.

Figure 1

Open in a new tab

Schematic representation of PEG-Jeffamine nanoparticle synthesis.

2.2.3. Bijel Preparation

A catalytic amount of TBD (12.5 mg) was added to a mixture of ε-caprolactone/ethanol (0.5 mL/55 μL), as reported by Marino et al.42 The solution was mixed at 1000 rpm for 8–15 min. Subsequently, 375 μL of the colloidal dispersions (the HA- or the NG-based one) was added, and the shaking speed was increased to 1700 rpm for 40 s to reassure a good distribution before the speed was reduced back to 1000 rpm until the complete polymerization was reached. As a container for the sample, a syringe (volume of 6 mL) with one end severed was chosen to extrude the sample without breaking its structure. The bijel preparation pathway is shown in Figure 2. The final morphological and structural features will be assessed by scanning electron microscopy, differential scanning calorimetry, and 1H HR-MAS NMR spectroscopy.

Figure 2.

Figure 2

Open in a new tab

Schematic representation of the bijel preparation pathway.

2.2.4. Bijel Drug Loading

Ethosuximide (Etho) and dimethyl fumarate (DMF) were chosen as loading drugs for the aqueous phase and for the organic phase, respectively.

2.2.4.1. Samples for HR-MAS NMR Spectroscopy

The hydrophilic drug was dissolved directly in the aqueous solution of NPs, whereas the hydrophobic one was added to the caprolactone prior to polymerization. Ethosuximed-loaded bijels having drug concentrations of 25, 50, and 100 mg/mL were prepared. Dimethyl fumarate-loaded bijels were separately prepared with two different drug concentrations of 12.5 and 25 mg/mL. The two drugs Etho and DMF were loaded into the bijels at different concentrations due to their diverse solubility in the water/organic phase. Given the high solubility of Etho in water (190 g/L at 25 °C), it was possible to load the bijels with three different concentrations of the drug and because the solubility of DMF in ε-caprolactone is lower, it was possible to prepare the bijels loaded with only two lower drug concentrations.

2.2.4.2. Samples for Release Tests

Hydrophilic drug-mimetic compounds (fluorescein sodium salt and rhodamine B) and ethosuximide were dissolved in the aqueous phase with a concentration of 1 mg/mL, and dimethyl fumarate was also dissolved in caprolactone (1 mg/mL). Afterward, the bijel synthesis was carried out following the procedure just described.

2.3. Sample Preparation

2.3.1. Solution State NMR Spectroscopy

Several liquid solutions composed by ethosuximide dissolved in D2O at various concentrations (C1 = 25, C2 = 50, and C3 = 100 mg/mL) and dimethyl fumarate dissolved in caprolactone at various concentrations (C4 = 12.5 and C5 = 25 mg/mL) were prepared for NMR analysis. The solutions were transferred in a 5 mm NMR tube.

2.4. Measurements

2.4.1. Dynamic Light Scattering (DLS)

DLS measurements were performed with a Malvern Zetasizer Nano ZS at a scattering angle of 173° (backscatter) to identify the hydrodynamic diameters and the polydispersity index (PDI) of the NPs produced. The temperature was kept at 25 °C, and an equilibration time of 20 s was provided before each measurement. Each sample was analyzed three times, and the results were averaged.

2.4.2. Scanning Electron Microscopy (SEM)

Environmental scanning electron microscopy analysis was performed on gold-sputtered samples at 10 kV with Evo 50 EP instrumentation (Zeiss, Jena, Germany). To preserve the actual morphology of the product, freeze-drying (for 150 h) was applied to remove all the liquid phases by sublimation. With this technique, the polymer chains are expected to retain the same conformation they had in wet conditions.

2.4.3. Differential Scanning Calorimetry (DSC)

Thermal property analysis was performed on a Mettler Toledo DSC polymer machine calibrated with indium and zinc standards. The heating rate was 10 °C/min under nitrogen flow. The melting point and glass transition temperature were derived from the first heating curve.

2.4.4. NMR Spectroscopy

The 1H HR-MAS NMR spectra of each gel sample were recorded using a Bruker Avance DRX spectrometer operating at 500 MHz proton frequency, equipped with a dual 1H/13C high-resolution magic angle spinning (HR-MAS) probe head for semisolid samples. This approach relies on the fast rotation of the sample at the magic angle (θ = 54.78° with respect to the z direction of the stray field of the NMR magnet), which averages the dipole–dipole interactions and susceptibility distortions, allowing to record spectra with high resolution. The bijel sample was carefully transferred in a 4 mm ZrO2 rotor (12 μL volume), ensuring that the gel texture was not destroyed. All the 1H HR-MAS spectra were acquired with a spinning rate of 4 kHz. The 1H NMR spectra of the liquid samples were also recorded by using a conventional high-resolution NMR probe and standard 5 mm glass NMR tubes. The temperature was set and controlled at 305 K with an air flow of 535 L·h–1 to avoid any temperature fluctuations due to sample heating during the magnetic-field pulse gradients.

Diffusion coefficients of the liquid and gel samples were measured using diffusion-ordered spectroscopy (DOSY) methods. The experiments were performed using the bipolar pulse longitudinal eddy current delay (BPPLED) pulse sequence.43 A pulsed gradient unit to produce magnetic-field pulse gradients in the z direction up to 53 G·cm–1 was used. The durations of the magnetic-field pulse gradients (δ) and the diffusion times (Δ) were optimized for each sample to obtain complete dephasing of the signals with the maximum gradient strength. The acquisition parameters for variable observation time measurements were δ = 0.5–1.9 ms and Δ = 10–60 ms in a step of 10 ms; for all the other samples, Δ was set to 50 ms while the δ value was 1.7 ms. In each experiment, a series of 28 spectra with 16 K points were collected with a D1 relaxation delay of 10 s. The pulse gradients were increased from 2 to 95% of the maximum gradient strength in a linear ramp. Each experiment of the gel samples was repeated in triplicate.

2.4.5. Release Studies

The bijel drug-loaded samples were submerged in 2 mL of PBS (0.1 M, pH 7.4) and incubated at 30 °C. At time points of 5 min, 30 min, 1 h, 1 h and 30 min, 2 h, 3 h, 4 h, 6 h, 7.25 h, 24 h, and 32 h, 1000 μL of the released media was withdrawn for UV measurements (UV–vis spectrophotometer V-630). At each measure, 1000 μL of fresh PBS was added to maintain the volume. The released amount of drug was measured spectrophotometrically at the wavelength corresponding to the peak of absorbance (ethosuximide = 244 nm, dimethyl fumarate = 236 nm) and determined referring to the standard calibration curve for each of them. The Lambert–Beer law was used to construct the release curves, ensuring that the absorbance measured was always in the linear range. Each measurement was repeated three times. The release study of the fluorophore drug-like molecules was performed following the procedure described in ref (32).

3. Results and Discussion

3.1. Characterization of the Bijel-like Structures

Several analytical techniques have been used to thoroughly investigate the morphology, properties, and behavior of the bijels produced with the two different nanoparticles HAp and NG.

3.1.1. SEM

Figure 3 shows SEM images useful for characterizing the internal morphology of the samples. The observed morphology is the result of the interpenetration of the organic and aqueous phase during the preparation of the samples. The resulting pores (darkest zones in the images) are originated by the removal of the aqueous phase during freeze-drying necessary to perform the SEM analysis. The internal structure of bijel/NG strongly resembles that of the matrix obtained with HAp nanoparticles. However, the structure of bijel/HAp is more jagged with respect to the other one, which presents smoother shapes. The particles are not directly observable in these pictures being of nanometric size.

Figure 3.

Figure 3

Open in a new tab

SEM images of the bijels/HAp (a, c) and bijels/NG (b, d) at different magnifications.

3.1.2. Thermal Properties

The melting (Tm) and crystallization points (Tc) of the products have been investigated by performing DSC analyses: the results showed a Tm of the two systems of 50 and 40 °C and Tc of 25 and 19 °C for the bijel/HAp and bijel/NG samples, respectively (Figures S1 and S2). These are above physiological temperatures, indicating that both could be used as drug delivery systems without dissolving once injected. This has been further confirmed through stability tests that simulated body conditions: both systems showed good resistance at 37 °C, lasting up to 2 weeks (see Figure 4).

Figure 4.

Figure 4

Open in a new tab

Nanogel-based samples at t0, 6 h, 8 h, 24 h, 50 h, 1 week, and 2 weeks (from left to right). The tests have been carried out in quadruplicate.

3.1.3. DLS

DLS analyses were performed to evaluate NG nanoparticle dimensions and, therefore, their ability to stabilize a colloidal dispersion in water. The NG nanoparticles had a mean size dimension of 434.6 nm with a polydispersity index (PdI) of 0.6, while HAp nanoparticles had a mean size of 372.9 nm, PdI = 0.27 (Figures S3 and S4).

3.1.4. HR-MAS NMR Structure and Diffusion

HR-MAS NMR spectroscopy, due to the magic angle spinning set-up, allows us to obtain the high-resolution spectra of soft materials such as gels, thus enabling structural investigation of drug–polymer interactions together with the dynamic properties of the drug molecules loaded in the gel phase. The 1H HR-MAS spectra of ethosuximide-loaded bijel/HAp and bijel/NG are reported in Figure 5b,c together with the 1H spectrum of the drug dissolved in D2O (trace a) and peak assignment. The 1H HR-MAS spectra of the gel phase both show high-resolution sharp lines for all the species with some well-defined peaks of the drug (protons 1, (3 + 3′), and 4; see Figure 5 for atom labels). A comparison between the HR-MAS versus high-resolution spectra shows that, overall, the NMR spectrum of ethosuxymide is retained upon encapsulation of the drug in the bijel, meaning that no changes in the spin systems are observed. This finding confirms that the entrapment process does not alter the chemical structure of the drug, thus allowing its biological activity. Conversely, some selective chemical shift variations are observed in the NMR spectrum of ethosuximide in the bijel compared to the reference in D2O. In particular, protons (3 + 3′) and 4 undergo an upfield chemical shift change (0.16 and 0.05 ppm, respectively), while no changes are observed for the methyl 1. These results suggest that the drug is likely to interact with the nanoparticle surface mainly via the heterocyclic ring through non-covalent interactions, which selectively change the magnetic environment of the mentioned protons without modification of the drug’s molecular structure. The 1H HR-MAS spectra of dimethyl fumarate loaded in bijel/HAp and bijel/NG (Figure S5) also show chemical shift variations compared to the high-resolution spectrum. Moreover, no effects on the spectral features, due to the different stabilizing nanoparticles, are observed in the bijels.

Figure 5.

Figure 5

Open in a new tab

(Left panel) (a) 1H spectrum of ethosuximide dissolved in D2O and peak assignment; (b, c) 1H HR-MAS spectra of ethosuximide loaded in bijel/NG and bijel/HAp, respectively. (Right panel) 1H HR-MAS DOSY map of NG-based bijel.

DOSY NMR has been used to characterize the bijel-like structure and observe the diffusion motion of the drug molecules in the water/organic phases. The technique, based on the application of magnetic-field gradients, is able to encode the molecular motion by carrying out a set of experiments with a linearly increasing gradient pulse strength. The final output of the experiment is a 2D map encoding the chemical shift of the observed species in the horizontal axis versus their diffusion coefficient, D, in the vertical dimension.44−46

The signal attenuation, I(q, Δ), for a given interpulse delay, Δ, is related to the gradient strength, g, and to the mean square displacement (MSD), ⟨z2⟩, of each diffusing species according to

3.1.4. 1

where q = (γgδ)/2π, γ is the gyromagnetic ratio of the observed nucleus, and δ is the length of the gradient pulse. For isotropic solutions, the mean square displacement shows a linear dependency with diffusion time Δ, as given by the equation

3.1.4. 2

where D is the diffusion coefficient. The above equation holds strictly for unrestricted Gaussian motion, while for restricted anomalous diffusion, a more general power law applies.47,48

The 1H DOSY map of NG-based bijel, as shown in Figure 5b, clearly indicates the presence of two distinct diffusing species ascribed to the organic phase/water solution. The diffusion coefficient of water in bijels was Dw = 2.7 × 10–9 m2/s, consistent with literature data for free water solutions at 305 K.49 The organic phase (PCL) exhibits a slower motion with a DPCL value of 8.9 × 10–11 m2/s. Moreover, DOSY experiments carried out at variable observation time Δ in the range of 10–60 ms show a linear regression of the MSD versus Δ for the diffusing water molecules (see Figure S6, Supporting Information). This is consistent with the diffusion process being Gaussian and unrestricted over the length scale lD accessible by DOSY NMR experiments. The observed diffusion length scale, within which lD = (2DΔ)1/2 covers the range of 7–18 μm, ensures the bicontinuous structure of the gels. Similar results were obtained for NG- and HAp-based bijels when loaded with the two drugs, ethosuximide and dimethyl fumarate in the water/organic phase, thus ensuring that the bicontinuous structure consisting of two immiscible domains is maintained even after drug loading.

Furthermore, the diffusion coefficient of ethosuximide and dimethyl fumarate loaded in both bijel/HAp and bijel/NG at different drug concentrations has been measured and the data are reported in Figure 6 together with the drug diffusion in pure water/organic solution for comparison. The results indicate that the diffusivity of the drugs in both bijel formulations is always slower than in free aqueous/organic solutions for all the concentrations, thus indicating the presence of confined diffusion within the bijel interconnected domains. Specifically, the decrease in diffusivity seems to become more distinct for the bijels stabilized with nanogel nanoparticles.

Figure 6.

Figure 6

Open in a new tab

Diffusion coefficients measured via 1H DOSY NMR at 305 K of (a) ethosuximide dissolved in D2O and loaded in HAp- and NG-based bijels; (b) dimethyl fumarate dissolved in caprolactone (CL) and loaded in HAp- and NG-based bijels. The experimental data are estimated to be accurate about ±2 to 10%.

The diffusion motion through bicontinuous structures and porous materials is known to depend on the microstructural topology50,51 usually described by the porosity, ϕ, and tortuosity, τ. Tortuosity is related to the distribution and interconnectivity of the pores or domains that may prolong the diffusional pathways and therefore limit the molecular motion over length scales even larger than the pore/domain sizes. Moreover, additional interactions of the drug molecules with the nanoparticle surface can modify their transport properties properly described by an effective diffusion coefficient, Deff, lower than the diffusion in the bulk solvent, D0. The parameter τeff defined as the ratio of Deff over D0 reflects both the geometric tortuosity of the bijel structure and the nanoparticle surface-drug interactions.52Table 1 collects the values of τeff, obtained from the diffusion data of Figure 6, for all our systems.

Table 1. Values of τeff for Ethosuximide and Dimethyl Fumarate in the Two Bijel Formulations.
C[mg/mL] τeff (bijel/HAp) τeff (bijel/NG)
Ethosuximide
C1 (25) 0.16 0.15
C2 (50) 0.37 0.17
C3 (100) 0.31 0.11
Dimethyl fumarate
C4 (12.5) 0.32 0.25
C5 (25) 0.27 0.19
Open in a new tab

Clearly, a reduction of the diffusion coefficient relative to its value D0, due to geometric tortuosity and physico-chemical interactions, is specific for each drug type/concentration and nanoparticle used in the bijel formulation. At the lower ethosuximide concentration, τeff is similar in both bijel formulations, while as the drug concentration increases, τeff is lower for bijel/NG than that for bijel/HAp, indicating both the presence of more convoluted domains and/or stronger drug–nanoparticle interactions in the 3D bijel/NG network. Similarly, dimethyl fumarate in bijel/NG formulation also shows lower τeff values regardless of drug concentration. These results suggest that the tortuosity and interactions induced by the two bijel formulations are different and affect the drug motion in the millisecond time scale.

3.2. Release Kinetics

Two aqueous hydrophilic drug mimetics, namely, rhodamine B (RhB) and sodium fluorescein (SF), have been selected due to their similarity to many analgesic and anti-inflammatory drugs.32 The release profiles of these model compounds can be observed in Figure 7. As far as RhB is concerned, no substantial differences in the cumulative release can be noticed, while for SF, some discrepancies are present when shifting from the HAp to the NG bijel formulations. However, these differences will not be discussed in detail since these results are only reported as reference. It is worth mentioning that rhodamine B is a cationic species, while the fluorescein molecule has negative charges, so we can evaluate the effect of the molecular charges on the release kinetics. A sustained release was observed for both compounds. The different rates and extents of SF release can be due to the presence of SF-NG nanoparticle non-covalent interactions.

Figure 7.

Figure 7

Open in a new tab

In vitro release profiles of SF and RhB from bijel/HAp and bijel/NG in the time range of 0–72 h. The chemical structure of SF and RhB molecules is also shown. Data SF-HAp are taken from ref (32).

Once the procedure has been optimized on SF and RhB, the release kinetics of ethosuximide and dimethyl fumarate has been performed. Figure 8 shows their cumulative release as a function of time.

Figure 8.

Figure 8

Open in a new tab

In vitro release profiles of (a) ethosuximide and (b) dimethyl fumarate from bijel/HAp and bijel/NG in the time range of 0–32 h.

Initial burst releases of 25 and 17% were observed after 30 min for ethosuximide released from bijel/HAp and bijel/NP, respectively; after that, a sustained drug release was observed. The initial burst release is due to the drug molecules located on the particle surface or to a possible aggregation, while the drug included in the bijel mesoscopic domains was released in a prolonged manner. Similarly to what was observed for fluorescein, the presence of NG nanoparticles significantly decreases the percentage of the drug released, and after 32 h, the cumulative amount of ethosuximide released by bijel/NG is 69% compared to 95% by bijel/HAp. The slow release kinetics can be attributed to drug-NG nanoparticle interactions that prevent the drug from moving away from the bijel matrix. These results are consistent with a slower drug diffusion in the bijel/NG measured in the NMR time scale. Conversely, the release profile of dimethyl fumarate (Figure 8b) is very slow over time and only 13% (bijel/HAp) and 20% (bijel/NG) of the drug were released within 32 h due to the lower affinity of DMF for the aqueous solution. In this case, the presence of the nanogel nanoparticles in the porous structure induces a slightly faster release kinetics of DMF. Overall, these results highlight that the bijel structure/nanoparticles have an impact on the macroscopic release and could be used to prolong the effect of the medicine.

To further investigate the drug release mechanism from bijels, the experimental release profile ft (up to 60% cumulative release) for both drugs and bijel formulations was analyzed using the Korsmeyer–Peppas model:53−55

3.2. 3

where k is a constant, Mt and M∞ are the fractions of the drug released at time t and at infinite time, respectively, and n is the diffusion exponent indicative of the drug transport mechanism. The fitting results are summarized in Table 2; all values of n are lower than 0.45, thus suggesting that Fickian diffusion drives the drugs released from both bijel formulations. For all data sets, the correlation coefficient (R2) was rather high (min. 0.96 up to 0.98; data are shown in the Supporting Information, Figures S7 and S8). A similar release profile from bijels has also been reported for drug-like molecules31 and by Singh and Kumar56 for moxifloxacin, in which the mechanism of drug release also followed the Fickian diffusion.

Table 2. Parameters (k and n) Obtained by Fitting the Drug Release Data.

sample k n
etho-bijel/NG 38.49 0.439
etho-bijel/HAp 22.17 0.323
DMF-bijel/NG 10.05 0.209
DMF-bijel/HAp 4.78 0.295
Open in a new tab

4. Conclusions

Biphasic water/oil porous structures have been successfully prepared and characterized. In these systems, fluid–fluid interfaces were stabilized by either hydroxyapatite (inorganic) or nanogel (organic) nanoparticles. The microstructures of such materials and nanoparticle surface interactions play a key role in the mass transport of small molecules such as active ingredients through the tortuous biphasic domains. The diffusion coefficients of the drugs in the bijel network, measured with HR-MAS NMR techniques, are reduced compared to diffusion in bulk solution, and this reduction depends on both the type of nanoparticles and drug concentration.

Furthermore, the in vitro release study showed sustained release of both ethosuximide and dimethyl fumarate from bijel structures, which followed the Fickian mechanism of diffusion despite the effect of the drug–nanoparticle non-covalent interactions that modify the amount of drug released over time. The microstructure/nanoparticle type allows us to design the desired properties of these materials as multi-drug delivery systems.

Acknowledgments

Authors would like to thank Prof. Maurizio Masi for fruitful discussion.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c04834.

  • DSC plot, DLS analysis, NMR spectra of DMF, dynamics of H2O in bijels, and release data analysis (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c04834_si_001.pdf (437.9KB, pdf)

References

  1. Stratford K.; Adhikari R.; Pagonabarraga I.; Desplat J.-C.; Cates M. E. Colloidal jamming at interfaces: A route to fluid-bicontinuous gels. Science 2005, 309, 2198–2201. 10.1126/science.1116589. [DOI] [PubMed] [Google Scholar]
  2. Cates M. E.; Clegg P. S. Bijels: a new class of soft materials. Soft Matter 2008, 4, 2132–2138. 10.1039/b807312k. [DOI] [Google Scholar]
  3. Herzig E. M.; White K. A.; Schofield A. B.; Poon W. C. K.; Clegg P. S. Bicontinuous emulsions stabilized solely by colloidal particles. Nat. Mater. 2007, 6, 966–971. 10.1038/nmat2055. [DOI] [PubMed] [Google Scholar]
  4. Tavacoli J. W.; Thijssen J. H. J.; Schofield A. B.; Clegg P. S. Novel, robust, and versatile bijels of nitromethane, ethanediol, and colloidal silica: capsules, subten-micrometer domains, and mechanical properties. Adv. Funct. Mater. 2011, 21, 2020–2027. 10.1002/adfm.201002562. [DOI] [Google Scholar]
  5. Lee M. N.; Mohraz A. Bicontinuous macroporous materials from bijel templates. Adv. Mater. 2010, 22, 4836–4841. 10.1002/adma.201001696. [DOI] [PubMed] [Google Scholar]
  6. Levesque S. G.; Lim R. M.; Shoichet M. S. Macroporous interconnected dextran scaffolds of controlled porosity for tissue-engineering applications. Biomaterials 2005, 26, 7436–7446. 10.1016/j.biomaterials.2005.05.054. [DOI] [PubMed] [Google Scholar]
  7. Martina M.; Subramanyam G.; Weaver J. C.; Hutmacher D. W.; Morse D. E.; Valiyaveettil S. Developing macroporous bicontinuous materials as scaffolds for tissue engineering. Biomaterials 2005, 26, 5609–5616. 10.1016/j.biomaterials.2005.02.011. [DOI] [PubMed] [Google Scholar]
  8. Cai D.; Clegg P. S.; Li T.; Rumble K. A.; Tavacoli J. W. Bijels formed by direct mixing. Soft Matter 2017, 13, 4824–4829. 10.1039/C7SM00897J. [DOI] [PubMed] [Google Scholar]
  9. Witt J. A.; Mumm D. R.; Mohraz A. Microstructural tunability of cocontinuous bijel-derived electrodes to provide high energy and power densities. J. Mater. Chem. A 2016, 4, 1000–1007. 10.1039/C5TA06260H. [DOI] [Google Scholar]
  10. Lupi F. R.; Shakeel A.; Greco V.; Rossi C. O.; Baldino N.; Gabriele D. A rheological and microstructural characterisation of bigels for cosmetic and pharmaceutical uses. Mater. Sci. Eng. C. 2016, 69, 358–365. 10.1016/j.msec.2016.06.098. [DOI] [PubMed] [Google Scholar]
  11. Lupi F. R.; Gentile L.; Gabriele D.; Mazzulla S.; Baldino N.; de Cindio B. Olive oil and hyperthermal water bigels for cosmetic uses. J. Colloid Interface Sci. 2015, 459, 70–78. 10.1016/j.jcis.2015.08.013. [DOI] [PubMed] [Google Scholar]
  12. Andonova V.; Peneva P.; Georgiev G. S.; Toncheva V. T.; Apostolova E.; Peychev Z.; Dimitrova S.; Katsarova M.; Petrova N.; Kassarova M. Ketoprofen-loaded polymer carriers in bigel formulation: An approach to enhancing drug photostability in topical application forms. Int. J. Nanomed. 2017, 12, 6221–6238. 10.2147/IJN.S140934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ivaskiene M.; Mazurkeviciute A.; Ramanauskiene K.; Ivaskiene M.; Grigonis A.; Briedis V. Topical antifungal bigels: Formulation, characterization and evaluation. Acta Pharm. 2018, 68, 223–233. 10.2478/acph-2018-0014. [DOI] [PubMed] [Google Scholar]
  14. Hamed R.; Mahmoud N. N.; Alnadi S. H.; Alkilani A. Z.; Hussein G. Diclofenac diethylamine nanosystems-loaded bigels for topical delivery: development, rheological characterization, and release studies. Drug Dev. Ind. Pharm. 2020, 46, 1705–1715. 10.1080/03639045.2020.1820038. [DOI] [PubMed] [Google Scholar]
  15. Martín-Illana A.; Notario-Pérez F.; Cazorla-Luna R.; Ruiz-Caro R.; Bonferoni M. C.; Tamayo A.; Veiga M. D. Bigels as drug delivery systems: From their components to their applications. Drug Discovery Today 2022, 27, 1008–1026. 10.1016/j.drudis.2021.12.011. [DOI] [PubMed] [Google Scholar]
  16. Murdan S. Organogels in drug delivery. Expert Opin. Drug Delivery 2005, 2, 489–505. 10.1517/17425247.2.3.489. [DOI] [PubMed] [Google Scholar]
  17. Esposito C. L.; Kirilov P.; Roullin V. G. Organogels, promising drug delivery systems: an update of state-of-the-art and recent applications. J. Controlled Release 2018, 271, 1–20. 10.1016/j.jconrel.2017.12.019. [DOI] [PubMed] [Google Scholar]
  18. Zhang Y.; Khademhosseini A. Advances in engineering hydrogels. Science 2017, 356, eaaf3627. 10.1126/science.aaf3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peppas N. A.; Bures P.; Leobandung W.; Ichikawa H. Hydrogels in pharmaceutical formulations. Eur. J. Pharm. Biopharm. 2000, 50, 27–46. 10.1016/S0939-6411(00)00090-4. [DOI] [PubMed] [Google Scholar]
  20. Dreiss C. A. Hydrogel design strategies for drug delivery. Curr. Opin. Colloid Interface Sci. 2020, 48, 1–17. 10.1016/j.cocis.2020.02.001. [DOI] [Google Scholar]
  21. Welch P. M.; Lee M. N.; Parra-Vasquez A. N. G.; Welch C. F. Jammed limit of bijel structure formation. Langmuir 2017, 33, 13133–13138. 10.1021/acs.langmuir.7b02805. [DOI] [PubMed] [Google Scholar]
  22. Cui M.; Miesch C.; Kosif I.; Nie H.; Kim P. Y.; Kim H.; Emrick T.; Russel T. P. Transition in dynamics as nanoparticles jam at the liquid/liquid interface. Nano Lett. 2017, 17, 6855–6862. 10.1021/acs.nanolett.7b03159. [DOI] [PubMed] [Google Scholar]
  23. Boeker A.; He J.; Emrick T.; Russell T. P. Self-assembly of nanoparticles at interfaces. Soft Matter 2007, 3, 1231–1248. 10.1039/b706609k. [DOI] [PubMed] [Google Scholar]
  24. Haase M. F.; Stebe K. J.; Lee D. Continuous fabrication of hierarchical and asymmetric bijel microparticles, fibers, and membranes by Solvent Transfer-Induced Phase Separation (STRIPS). Adv. Mater. 2015, 27, 7065–7071. 10.1002/adma.201503509. [DOI] [PubMed] [Google Scholar]
  25. Russell J. T.; Lin Y.; Boeker A.; Su L.; Carl P.; Zett H.; He J.; Sill K.; Tangirala R.; Emrick T.; Littrell K.; Thiyagarajan P.; Cookson D.; Fery A.; Wang Q.; Russell T. P. Self-assembly and cross-linking of bionanoparticles at liquid–liquid interfaces. Angew. Chem., Int. Ed. 2005, 44, 2420–2426. 10.1002/anie.200462653. [DOI] [PubMed] [Google Scholar]
  26. Jansen F.; Harting J. From bijels to Pickering emulsions: A lattice Boltzmann study. Phys. Rev. E 2011, 83, 046707. 10.1103/PhysRevE.83.046707. [DOI] [PubMed] [Google Scholar]
  27. Arabjamaloei R.; Shah R. S.; Bryant S.; Trifkovic M. Controlling structure of materials derived from spinodally decomposing liquids. Phys. Fluids 2021, 33, 032020. 10.1063/5.0041193. [DOI] [Google Scholar]
  28. Carmack J.; Millet P. C. Numerical simulations of bijel morphology in thin films with complete surface wetting. J. Chem. Phys. 2015, 143, 154701. 10.1063/1.4932191. [DOI] [PubMed] [Google Scholar]
  29. Cai D.; Clegg P. S. Stabilizing bijels using a mixture of fumed silica nanoparticles. Chem. Commun. 2015, 51, 16984–16987. 10.1039/C5CC07346D. [DOI] [PubMed] [Google Scholar]
  30. Bai L.; Fruehwirth J. W.; Cheng X.; Macosko C. W. Dynamics and rheology of nonpolar bijels. Soft Matter 2015, 11, 5282–5293. 10.1039/C5SM00994D. [DOI] [PubMed] [Google Scholar]
  31. Hijnen N.; Cai D.; Clegg P. S. Bijels stabilized using rod-like particles. Soft Matter 2015, 11, 4351–4355. 10.1039/C5SM00265F. [DOI] [PubMed] [Google Scholar]
  32. Pizzetti F.; Rossetti A.; Marchetti A.; Castiglione F.; Vanoli V.; Coste E.; Veneruso V.; Veglianese P.; Sacchetti A.; Cingolani A.; Rossi F. Bicontinuous structures formed by monomer/water interface stabilization with colloidal nanoparticles. Adv. Mater. Interfaces 2021, 8, 210091. 10.1002/admi.202100991. [DOI] [Google Scholar]
  33. Gören M. Z.; Onat F. Ethosuximide: from bench to bedside. CNS Drug Rev. 2007, 13, 224–239. 10.1111/j.1527-3458.2007.00009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Blair H. A. Dimethyl fumarate: a review in moderate to severe plaque psoriasis. Drugs 2018, 78, 123–130. 10.1007/s40265-017-0854-6. [DOI] [PubMed] [Google Scholar]
  35. Burness C. B.; Deeks E. D. Dimethyl fumarate: a review of its use in patients with relapsing-remitting multiple sclerosis. CNS Drugs 2014, 28, 373–387. 10.1007/s40263-014-0155-5. [DOI] [PubMed] [Google Scholar]
  36. Santoro M.; Marchetti P.; Rossi F.; Perale G.; Castiglione F.; Mle A.; Masi M. Smart approach to evaluate drug diffusivity in injectable agar-carbomer hydrogels for drug delivery. J. Phys. Chem. B 2011, 115, 2503–2510. 10.1021/jp1111394. [DOI] [PubMed] [Google Scholar]
  37. Kimmich R.Principles of Soft-Matter Dynamics. Springer, London, 2012. [Google Scholar]
  38. Jenkins J. E.; Hibbs M. R.; Alam T. M. Identification of multiple diffusion rates in mixed solvent anion exchange membranes using high resolution MAS NMR. ACS Macro Lett. 2012, 1, 910–914. 10.1021/mz300124j. [DOI] [PubMed] [Google Scholar]
  39. Perale G.; Rossi F.; Santoro M.; Marchetti P.; Mele A.; Castiglione F.; Raffa E.; Masi M. Drug release from hydrogel: a new understanding of transport phenomena. J. Biomed. Nanotechnol. 2011, 7, 476–481. 10.1166/jbn.2011.1302. [DOI] [PubMed] [Google Scholar]
  40. Ferro M.; Castiglione F.; Punta C.; Melone L.; Panzeri W.; Rossi B.; Trotta F.; Mele A. Anomalous diffusion of Ibuprofen in cyclodextrin nanosponges hydrogels: an HRMAS NMR study. Beilstein J. Org. Chem. 2014, 10, 2580–2593. 10.3762/bjoc.10.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kumar R.; Prakash K. H.; Cheang P.; Khor K. A. Temperature driven morphological changes of chemically precipitated hydroxyapatite nanoparticles. Langmuir 2004, 20, 5196–5200. 10.1021/la049304f. [DOI] [PubMed] [Google Scholar]
  42. Marino A.; Arai S.; Hou Y.; Pellegrino M.; Mazzolai B.; Mattoli V.; Suzuki M.; Ciofani G.. Assessment of the effects of a wireless neural stimulation mediated by piezoelectric nanoparticles. In: Santamaria F., Peralta X. (eds) Use of Nanoparticles in Neuroscience. Neuromethods 2018, 135Humana Press, New York, NY. [Google Scholar]
  43. Pelta M. D.; Barjat H.; Morris G. A.; Davis A. L.; Hammond S. J. Pulse sequences for high-resolution diffusion-ordered spectroscopy (HR-DOSY). Magn. Reson. Chem. 1998, 36, 706–714. 10.1002/(SICI)1097-458X(199810)36:10<706::AID-OMR363>3.0.CO;2-W. [DOI] [Google Scholar]
  44. Zubkov M.; Dennis G. R.; Stait-Gardner T.; Torres A. M.; Willis S. A.; Zheng G.; Price W. S. Physical characterization using diffusion NMR spectroscopy. Magn. Reson. Chem. 2017, 55, 414–424. 10.1002/mrc.4530. [DOI] [PubMed] [Google Scholar]
  45. Groves P. Diffusion ordered spectroscopy (DOSY) as applied to polymers. Polym. Chem. 2017, 8, 6700–6708. 10.1039/C7PY01577A. [DOI] [Google Scholar]
  46. Fiorati A.; Negrini N. C.; Baschenis E.; Altomare L.; Faré S.; Schieroni A. G.; Piovani D.; Mendichi R.; Ferro M.; Castiglione F.; Mele A.; Punta C.; Melone L. TEMPO-nanocellulose/Ca2+ hydrogels: Ibuprofen drug diffusion and in vitro cytocompatibility. Materials 2020, 13, 183–199. 10.3390/ma13010183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Phillies G. Diffusion on a molecular scale as observed using PGSE NMR. Concepts Magn. Reson., Part A 2015, 44, 1–15. 10.1002/cmr.a.21325. [DOI] [Google Scholar]
  48. Castiglione F.; Casalegno M.; Ferro M.; Rossi F.; Raos G.; Mele A. Evidence of superdiffusive nanoscale motion in anionic polymeric hydrogels: Analysis of PGSE- NMR data and comparison with drug release properties. J. Controlled Release 2019, 305, 110–119. 10.1016/j.jconrel.2019.05.027. [DOI] [PubMed] [Google Scholar]
  49. Holz M.; Heil S. R.; Sacco A. Temperature-dependent self-di†usion coefficients of water and six selected molecular liquids for calibration in accurate 1H NMR PFG measurements. Phys. Chem. Chem. Phys. 2000, 2, 4740–4742. 10.1039/b005319h. [DOI] [Google Scholar]
  50. McDevitt K. M.; Thorson T. J.; Botvinick E. L.; Mumm D. R.; Mohraz A. Microstructural characteristics of bijel-templated porous materials. Materialia 2019, 7, 100393. 10.1016/j.mtla.2019.100393. [DOI] [Google Scholar]
  51. Shen L.; Chen Z. Critical review of the impact of tortuosity on diffusion. Chem. Eng. Sci. 2007, 62, 3748–3755. 10.1016/j.ces.2007.03.041. [DOI] [Google Scholar]
  52. Rottreau T. J.; Parlett C. M. A.; Lee A. F.; Evans R. Diffusion NMR characterization of catalytic silica supports: a tortuous path. J. Phys. Chem. C 2017, 121, 16250–16256. 10.1021/acs.jpcc.7b02929. [DOI] [Google Scholar]
  53. Ritger P. L.; Peppas N. A. A simple equation for description of solute release II Fickian and anomalous release from swellable devices. J. Controlled Release 1987, 5, 37–42. 10.1016/0168-3659(87)90035-6. [DOI] [PubMed] [Google Scholar]
  54. Korsmeyer R. W.; Gurny R.; Doelker E.; Buri P.; Peppas N. A. Mechanisms of solute release from porous hydrophilic polymers. Int. J. Pharm. 1983, 15, 25–35. 10.1016/0378-5173(83)90064-9. [DOI] [PubMed] [Google Scholar]
  55. Peppas N. A.; Narasimhan B. Mathematical models in drug delivery: How modeling has shaped the way we design new drug delivery systems. J. Controlled Release 2014, 190, 75–81. 10.1016/j.jconrel.2014.06.041. [DOI] [PubMed] [Google Scholar]
  56. Singh B.; Kumar R. Designing biocompatible sterile organogel–bigel formulations for drug delivery applications using green protocol. New J. Chem. 2019, 43, 3059–3070. 10.1039/C8NJ05480K. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao2c04834_si_001.pdf (437.9KB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES