Fabrication of Microgel-in-Liposome Particles with Improved Water

Article pubs.acs.org/Langmuir

Fabrication of Microgel-in-Liposome Particles with Improved Water Retention Eunjung An,† Choon Bok Jeong,† Chaenyung Cha,‡ Do Hoon Kim,† Haekwang Lee,†,‡ Hyunjoon Kong,‡ Junoh Kim,† and Jin Woong Kim*,§ †

Amore-Pacific Co. R&D Center, 314-1, Bora-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-729, South Korea Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology University of Illinois at Urbana−Champaign, 108 RAL, Urbana, Illinois 61801, United States § Department of Applied Chemistry, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Gyeonggi-do 426-791, South Korea ‡

ABSTRACT: Corneocytes represents the main water reservoir of stratum corneum, and that ability intimately arises from their architecture and total composition. Here we describe a novel method for fabricating a microgel-inliposome (M-i-L) structure consisting of a sodium hyaluronate microgel and a lipid membrane envelop in order to mimic corneocyte cell structures. The essence of our approach is to use a lecithin-based microemulsion with a very low interfacial tension between the water droplet and oil continuous phase. Using this emulsion enables us to stabilize a dispersion of microgel particles without phase separation or aggregation. The addition of excess water produced single-core or multicore microgel particles enveloped in a lipid layer. To demonstrate the applicability of this unique vesicle system, we encapsulated a high concentration of natural moisturizing factor (NMF) in the microgel core and investigated how the M-i-L structure affected the water retention in comparison with other control systems. We have observed that our M-i-L particles with the NMF in the core, which mimicked the corneocyte cell structure, showed an excellent ability to retain water in the system. This experimental result inspired us to investigate how corneocyte cells, which feature a lipid-enveloped hydrogel structure, provide such long-lasting hydration to the skin.

1. INTRODUCTION Stratum corneum (SC), a sophisticated outermost skin layer, regulates the loss of water from the skin.1−4 Water molecules in the SC facilitate SC maturation and desquamation through enzyme reactions and play an essential role in maintaining homeostasis at the skin level. Hydration of corneocytes is particularly essential to SC function.5−7 The ability to retain water within corneocytes is closely related to its architecture and total composition.1,3 Mature corneocytes are dead cells covered with a chemically cross-linked protein and lipid shell, usually referred to as the corneocyte lipid envelope, and are filled with a matrix that consists mainly of keratin fibrillae.8,9 The corneocytes are embedded in a lipid matrix to form intercellular lipid lamellae.10,11 The compositions of proteins and lipid components in the SC are critical for establishing normal skin barrier function. The natural moisturizing factor (NMF) is present in high concentrations mostly within corneocytes and can compose 20−30% of the dry weight of the SC.12−15 NMF is composed primarily of amino acids or their derivatives, such as pyrrolidone carboxylic acid, urocanic acid, and lactic acid, which are intensely hydroscopic. The intracellular presence of NMF in the corneocytes allows the skin to retain water.6,7 Thanks to this cellular hygroscopicity, the surface layer of the SC is resistant to desiccation factors, such as heating, drying, and salt exposure. © 2012 American Chemical Society

Corneocyte structures may be mimicked using microgel-inliposome (M-i-L) particles with a length scale similar to that of the corneocyte cells, which are flat in shape with a diameter of ∼30 μm.1 Their ability to hold water in their microgel cores can potentially fortify skin barrier function and be useful for the treatment of barrier-deficient skin, such as atopic dermatitis and psoriasis. Despite the strong need for corneocyte-like biomaterials, until recently, only a few such attempts have been made. M-i-L particles have been fabricated by mechanically adsorbing lipids onto microgel particles16−19 or by directly injecting an aqueous gel-in-oil emulsion onto a lipid monolayer at a planar oil−water interface.20,21 Lipid coatings can be prepared by anchoring lipid molecules to a hydrogel surface.22,23 Another approach to constructing M-i-L particles is to form liposomes and then gelate their interior.24,25 Several methods for fabricating a microgel surrounded by a semipermeable polymer membrane have been reported for use as pulsed drug delivery systems.26−29 These studies are scientifically informative and have advanced the fabrication of M-i-L materials. However, there is a need for practical methods for Received: November 25, 2011 Revised: January 31, 2012 Published: February 1, 2012 4095

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Figure 1. Schematic diagram showing the fabrication of M-i-L particles using a lecithin-based microemulsion. The lecithin-based microemulsion was prepared after a pseudoternary phase study.

Figure 2. (a) A pseudoternary phase diagram showing the existence area (gray region) of a microemulsion. The mixing ratio of lecithin to ethanol was set to 1 (w/w). (b) The particle formation behavior as a function of the lipid mixture composition. Regions a, b, and c were chosen from the pseudoternary phase diagram. The w/o emulsion in region b contains lecithin (26 wt %), ethanol (26 wt %), dichloromethane (43 wt %), and dried HA microgel (5 wt %). Therefore, after removing volatile solvents, lecithin:HA microgel is approximately 5:1 by weight. The scale bars indicate 10 μm.

fabricating high-performance M-i-L particles that have materials properties similar to those of corneocytes. Here, we describe a flexible and robust method for mimicking the corneocyte cell structure. Our artificial

corneocyte encapsulates a high concentration of NMF in a microgel core enveloped with lipid layers. The biomimetic corneocyte-like microstructures were prepared using a technique that took advantage of the phase change in a lipid4096

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Figure 3. (a) Particle size distribution of the M-i-L particles. This was determined by analyzing the bright-field microscopy image. (b) A fieldemission scanning electron microscopy image of the lyophilized M-i-L particles.

Figure 4. Microscopy images of M-i-L particles. A single-core M-i-L particle: (a) a bright-field microscopy image, (b) a polarized optical microscopy image, (c) a CLSM image. A multicore M-i-L particle: (d) a bright-field microscopy image, (e) a polarized optical microscopy image, (f) a CLSM image. The CLSM images measured after incorporation of FITC-labeled dextran. Lipids were labeled with TRITC. The scale bar indicates 10 μm.

microemulsion provided several advantages. The swollen microgel particles could be stably dispersed in an oil phase without the aid of any other surfactants. Lecithin is known to have surface activity, but it is too lipophilic to spontaneously form a curvature at the interface of the microemulsion. Thus, this study adds ethanol to reduce the rigidity of the lecithin membrane to form the curvature necessary for droplet formation.32,33 The low interfacial tension of microemulsion permitted the microgel particles to be stably dispersed in the oil phase without phase separation or aggregation. When the composition fell outside of the microemulsion region of the phase diagram, the swollen microgel particles became separated from the oil phase, or the oil phase containing lecithin became crystallized, as shown in Figure 2b. The particle sizes of the Mi-L particles prepared in the regime corresponding to formation of the microemulsion were less than 15 μm (Figure 3a). Even after drying, the particles retained their spherical shape due to formation of a well-aligned robust lipid shell (Figure 3b). The M-i-L particles fabricated using the microemulsion separation had both single-core and multicore structures because phase separation occurred randomly. The fraction of single-core particles was 0.84. The vesicle structure was characterized by visualizing the lipid layer surrounding the

based microemulsion. The microemulsion not only provided a stable dispersion of microgel particles containing NMF but also selectively separated the hydrophobic lipid phase from the system in the presence of a large amount of water. The M-i-L particles containing NMF in the core experimentally demonstrated excellent water retention, indicating that the corresponding properties of corneocytes arise from their structures.

2. RESULTS AND DISCUSSION M-i-L particles were fabricated using a new fabrication method developed for this purpose. The method is illustrated in Figure 1. Our method comprised three steps: (i) the formulation of a lecithin base water/oil (w/o) microemulsion made from a mixture of lecithin/dichloromethane/ethanol/water, (ii) dispersion and swelling of hyaluronic acid (HA) microgel particles in the microemulsion, and (iii) removal of solvents and addition of excess water, which led to the spontaneous formation of lipid shells around the HA microgel particles. Lecithin-stabilized water/oil/water (w/o/w) double emulsion drops have been used previously as a template for fabricating liposomes.30,31 The application of this technique for our purposes relied on the use of a lecithin-based microemulsion prepared from a pseudoternary phase (Figure 2a). This 4097

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Figure 5. (a) Mean particle sizes of the HA microgel particles, depending on the formulation step. Particle diameters were directly measured from bright-field microscopy images. (b) Encapsulation efficiency of a water-soluble molecule, caffeine, in the microgel core. RPE is the reverse phase evaporation method.

Figure 6. (a) Water evaporation profiles of test samples as a function of time. Temperature and relative humidity were precisely controlled to be 25 ± 1 °C and 38 ± 1%, respectively. For all samples, the initial water content was set to 90 wt %. (b) Water holding parameters obtained during the second evaporation stage. The evaporation rate at BWE was obtained by differentiating the mass change with respect to t and then dividing this value again by the mass of water at the starting point of the BWE stage.

phase phase-separated to form a capsule with a robust lipid shell, and a high encapsulation efficiency for water-soluble materials could be achieved. This encapsulation efficiency was equivalent to that obtained using multilamellar vesicles prepared by the reverse-phase evaporation method, 30%.34−36 Comparably, preparation of the M-i-L particles using conventional film hydration methods yielded an encapsulation efficiency of 7.7 ± 5%. In this case, the film hydration method provided no driving force for the lipid molecules to interact with the microgel particles. Our M-i-L particle system will be useful for exploring novel biomedical applications. As a drug carrier, for example, the M-iL particles can perform better than liposomes in terms of enhancing the physical stress tolerance, controlling the mesh properties of the hydrogel network and increasing the drug loading capacity.37 And also, the internal framework composed with cross-linked polymer inside liposomes provides the basic functions of cytoskeleton in order to mimic cell architecture.38 To experimentally demonstrate this, a high concentration of NMF, which is a key bioingredient to effect water retention in corneocytes, was encapsulated in the HA microgel core of the M-i-L particles. NMF could be incorporated to maximum of 15 wt % dry weight of particles, which was close to the concentration present in corneocytes.12−15 The water retention capacity in the M-i-L particles containing NMF was evaluated and is shown in Figure 6a. Croll’s model for the drying colloidal films describes the drying process as being divided into two stages:39,40 free water evaporation during the early stages and bound water

HA microgel using optical microscopy and confocal laser scanning microscopy (CLSM). The results are shown in Figure 4. The cross-polarization observations clearly showed a maltese cross irrespective of the core number. Generation of a maltese cross indicated that the lipid molecules assembled to form a multilamellar phase. A clear fluorescent red ring around the microgel cores was observed in CLSM analysis, which indicated formation of a lipid membrane surrounding the microgel cores. The HA microgel particles assumed different particle sizes depending on the fabrication steps employed (Figure 5a). The initial mean diameter of the dried HA microgels was 1.7 μm. Incorporation into the microemulsion resulted in the absorption of water with swelling to a diameter of 4 μm. After coating with lipids, the mean HA microgel diameter further increased to 5.8 μm. Exposure of the M-i-L particles to a surfactant aqueous solution (1 wt % Triton X-100) resulted in dissolution of the coated lipid layer, which allowed swelling of the microgel particles to a maximum size, which facilitated complete escape from the vesicles. These observations indicate that coating the microgels with a lipid layer affected the equilibrium conditions of the HA hydrogel network. In the fields in which liposomal materials are studied, a central concern is the development of methods for increasing the efficiency of encapsulating water-soluble ingredients in a water-based core. The benefit of our M-i-L particles is that a high fraction of the aqueous phase is entrapped within the vesicles, which increases the loading efficiency. As shown in Figure 5b, the efficiency of encapsulating caffeine in the M-i-L particles was 30 ± 6%. In this case, the drug-containing water 4098

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was used as a model extracellular matrix (ECM) to observe the intramatrix water diffusion. Color maps shown in Figure 7a represent the relative proton signal intensity from the water in the hydrogel. The water-rich region corresponding to the fully swollen gel provided the highest intensity, as can be seen in yellow, whereas the dried regions are shown in dark green. The intensity color map of the collagen hydrogel itself rapidly turned from red to blue during the early drying stages. Collagen covered with the M-i-L containing NMF was uniformly hydrated and retained a constant mass of water on the experimental time scale. The integral of the MRI signal intensity profiles could be used to estimate the water content in the hydrogel at each point in time, It, which was directly related to the drying process. The normalized integral intensity, It/I0, where I0 is the integral of the intensity profile under the initial conditions for each of the samples, is plotted in Figure 7b. The trend of these curves is very similar to the one obtained by gravimetric analysis (Figure 6). The hygroscopic properties of NMF retarded the rate of water evaporation from the hydrogel, but the water content decreased to 87% within 220 min. The water content of the hydrogel treated with M-i-L with NMF remained nearly constant with more than 95% of the initial content. This result experimentally supported that a corneocyte-like particle structure played an essential role in preventing water loss from the hydrogel.

Table 1. Parameters Calculated from the Water Evaporation Profiles sample HA microgel NMF M-i-L M-i-L with NMF

slope at FWEa

tc (s)b

mwater at tc (%)c

Revap (×103) at BWE (s−1)d

mwater at t∞ (%)

0.15

420

25.19

2.51

3.87

0.13 0.24 0.13

490 240 370

28.67 33.62 43.68

2.10 1.61 1.31

6.52 6.95 20.42

a

FWE is the free water evaporation stage. bTime to change the stage from FWE to BWE. cmwater is the mass of remaining water. dRevap is the evaporation rate obtained from fitting the mass change data using nonlinear regression methods during the BWE stage.

evaporation during the later stages. During the free water evaporation stage of the studies presented here, the mass of water decreased linearly as a function of time. The evaporation of water from the solutions containing NMF and M-i-L with NMF, determined from the slope of the linear region, were lowest due to the hygroscopic properties of the NMF components. Interestingly, the materials with an M-i-L structure reached the end of the free water evaporation stage faster and included a higher mass fraction of water compared with the other two samples. This implies that water trapped in the M-i-L remained unchanged. Analysis of the bound water evaporation was conducted and the plots of the mass change due to water loss over time were fit using nonlinear regression methods (Figure 6b). The M-i-L particles with NMF showed the lowest evaporation rate during this stage, and the highest fraction of water remained. These results indicated that the M-iL structure provided excellent barrier function with respect to water evaporation from the system. The lipid layer surrounding the microgel particles appeared to restrict the escape of the bound water. Once the NMF was incorporated into the M-i-L, improved water retention could be obtained because the NMF provided hygroscopic properties to the microgel core. The hydration and barrier function of the M-i-L particle system was monitored using a noninvasive analytical procedure to visually quantify water evaporation from the matrix using magnetic resonance imaging (MRI).41,42 A collagen hydrogel

3. CONCLUSIONS In summary, we developed a novel method for fabricating a corneocyte-like particle system consisting of a microgel core and a lipid membrane shell. Incorporation of microgels into microemulsion techniques enabled fabrication of a unique microgel-in-liposome (M-i-L) structure with a high loading efficiency and a surfactant-induced release profile. The M-i-L system containing NMF performed well in our experimental investigation of water retention, possibly by regulating the behavior of the free and bound water in the system. These observations enabled us to gain an insight into figuring out how corneocytes supply such long-lasting water retention capacities in the skin. The system fabricated in this study provides a new

Figure 7. (a) Water evaporation from the collagen hydrogel as a function of time was visualized using MRI. I indicates the collagen itself, II indicates the collagen treated with NMF, and III indicates the collagen treated with M-i-L particles containing NMF. The time scale is minutes. (b) The water content was quantified within the hydrogel based on the MRI images. Plots of the pixel intensity vs count graph were obtained from each MRI image, which were then integrated to obtain the total water content within a hydrogel. 4099

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of caffeine = the total amount of added caffeine − the amount of nonentrapped caffeine in the flow-through liquid. 4.6. Gravimetric Determination of the Evaporation Rate. The water drying rates were determined using gravimetric techniques. Thirty-five microliters of the sample was evenly spread onto a 5 × 5 cm2 artificial leather substrate. The sample was then placed in a desiccator. The desiccators in which the experiment were performed were environmentally controlled with a temperature of 25 ± 1 °C and a relative humidity of 38 ± 1%. The test sample was weighed and automatically collected at intervals of 10 s for 1 h using an electronic scale (XS204V, capacity: 220 g; readability: 0.1 mg), and data were processed using the LAbX Light balance 1.5 program. The experiments were repeated at least three times for each sample. The free water evaporation stage during the early stages was fit using linear regression methods. The time to transition, tc, from the free water evaporation (FWE) stage to the bound water evaporation (BWE) stage was assigned when the linear regression coefficient was below 0.98. The mass change data after tc were fit to an exponential decay, m(t) = m0 + ae−bt, to analyze the bound water evaporation stage. In all cases, the regression coefficient exceeded 0.99. According to the firstorder kinetics,40 which were defined by dm/(A dt) = −qc, the water loss rate was proportional to the water concentration (c). Thus, the evaporation rate at BWE was obtained by differentiating the mass change with respect to t and then dividing this value by the mass of water at the starting point of the BWE stage to compensate for the differences in c between each sample. 4.7. Characterization of the Water Content Using Magnetic Resonance Imaging. MRI was carried out using 600 MHz Varian Unity/Inova nuclear magnetic resonance spectrometer (14.1 T magnet) at room temperature. The maximum strength of the magnetic field gradient was 90 G cm−1. A 0.2 mL of matrix collagen gel was prepared in a test tube (6 × 55 mm), and 0.02 mL of a M-i-L dispersion was placed on top of the prepared matrix collagen gel. The test tubes were then inserted into a sample holder within a radiofrequency coil for the measurements. Vertical cross-sectional images of the hydrogel were taken at various time points. A spin-echo multislice (SEMS) pulse sequence was used to acquire the resonance data, which were then converted into a water density map using the VNMR 6.1C software. The SEMS pulse sequence included a repetition time (TR) of 0.7 s and an echo time (TE) of 10 ms. The field of view (FOV) was 1.5 × 1.5 cm with a slice thickness of 1 mm, and the image matrix was 256 × 256 pixels. After acquiring the images, the colors were added to the images to visualize the water density spectrum using Image J (Free software from National Institutes of Health). The water content within a hydrogel was quantified using the MRI images, and a plot of the pixel intensity vs count was obtained from each MRI image using Image J. The images were then integrated to obtain the total water content within the hydrogel.

method for regulating water evaporation from the skin and may potentially be applied in the fields of cosmetics, dermatology, and pharmaceuticals.

4. EXPERIMENTAL METHODS 4.1. Synthesis of Sodium Hyaluronate Microgels. Sodium hyaluronate microgel particles were synthesized according to a modified Segura method.43 Twenty grams PEG 30 dipolyhydroxystearate was dissolved in heptanes. Five grams of sodium hyaluronate and 6.6 g of poly(ethylene glycol) diglycidyl ether (PEGDA, MW = 526 Da) were dissolved in a 0.1 N NaOH aqueous solution and subsequently emulsified in a heptane solution. The reaction was carried out at 60 °C for 1 h with mechanical stirring under a nitrogen atmosphere and neutralized with acetic acid. The reaction mixture was precipitated in acetone and dried in vacuo. The average particle diameter in water was 8 μm. 4.2. Pseudoternary Phase Diagram Study. A pseudoternary phase diagram study was conducted to determine the composition that would yield a microemulsion. The microemulsions were prepared using lecithin, absolute alcohol, dichloromethane, and doubly distilled water. The lecithin was hydrogenated soybean phosphatidylcholine (Lipoid S100-3, Lipoid GmbH, Germany) containing more than 94% PC. The experiments were carried out at 25 ± 1 °C, and the surfactant/cosurfactant ratio was fixed to 1 (w/w). The mixture of lecithin, oil phase, and the surfactant/cosurfactant mixture were slowly titrated with water. The transparent fluid formulation was defined as the microemulsion region. 4.3. Fabrication of the Microgel-in-Liposome Particles. Half a gram of dried microgel particles was dispersed in 10 mL microemulsions. In this step, the microgel particles abruptly absorbed water, which was preadded to the microemulsion. Excess water was added to the continuous phase, and dichloromethane and ethanol were removed under reduced pressure applied using a rotary evaporator. During evaporation, the temperature was kept at 55 °C. The samples were centrifuged for 10 min at 2000 rpm, the supernatant was removed, and the particles were redispersed in distilled water. Centrifugation and resuspension were repeated three times. Finally, the microparticles were redispersed in 50 mL of distilled water and were used for further studies. The NMF composition was prepared according to the procedures reported in the literature.14,15 The chemical composition of NMF used for this study was serine (18.2%), glycine (9.1%), arginine (3.2%), glutamic acid (2.3%), tyrosine (0.5%), alanine (6.6%), pyrrolidone carboxylic acid (12%), urea (7%), sodium lactate (5%), and deionized water (to 100%). 4.4. Confocal Laser Scanning Microscopy Measurements. A small amount of TRITC-labeled DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, Invitrogen) and FITC (fluorescein isothiocyanate)-labeled dextran (Aldrich) were added to the microemulsion as tracers to detect the lecithin and hydrophilic molecules incorporated into the particles. Experiments were performed using a Zeiss LSM 510 confocal microscope. A helium neon laser with a 543 nm excitation beam for observing TRICT fluorescence was used, and the emission filters excluded fluorescence of wavelength below 570 nm. FITC were visualized after excitation with an argon laser (488 nm) combined with a 515/30 nm emission filter. 4.5. Determination of the Drug Loading Efficiency. Ultrafiltration centrifugation methods are commonly used to evaluate drug loading efficiency in liposomes.44,45 Briefly, the M-i-L particles containing caffeine as a model drug were added to Amicon Ultra-15 centrifugal filter devices (30 000 NMWL) and centrifuged at 3000g for 10 min at 4 °C. Caffeine in the flow-through liquid was collected and analyzed using high-performance liquid chromatography (HPLC) methods with a methanol/water (3/7, v/v) mobile phase on a Zorbax Rx C18 column (4.6 mm × 15 cm) at flow rate of 0.8 mL/min. Caffeine was detected using a Perkin-Elmer 785A UV−vis detector at 272 nm. The caffeine encapsulation efficiency (%) was obtained by determining the entrapped amount of caffeine: the entrapped amount

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-400-5499; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the research fund of Hanyang University (HY-2011-N).

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REFERENCES

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dx.doi.org/10.1021/la2046349 | Langmuir 2012, 28, 4095−4101