Totally Phospholipidic Mesoporous Particles - The Journal of

Article pubs.acs.org/JPCC

Totally Phospholipidic Mesoporous Particles Shaoling Zhang,†,‡ Kohsaku Kawakami,*,†,‡ Lok Kumar Shrestha,† Gladstone Christopher Jayakumar,† Jonathan P. Hill,†,‡ and Katsuhiko Ariga*,†,‡ †

International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan ‡ Japan Science and Technology Agency (JST), Core Research of Evolutional Science and Technology (CREST), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan S Supporting Information *

ABSTRACT: Medical science is one of the areas where mesoporous materials can offer important advances despite the stringent safety requirements for potentially useful materials. Here we report totally phospholipidic mesoporous particles which may be used as a novel drug carrier. This material is anticipated to be safe for human internal use since it is composed solely of phosphatidylcholine (PC) which is a major component of biological membranes and is approved for use in humans by the US Food and Drug Administration (FDA). We have established a simple production methodology of mesoporous phospholipid particles (MPPs) in which PC is dissolved in two-component solvent mixtures, followed by incubation of the resulting solutions at depressed temperatures, which induces liquid−liquid demixing and leads to the agglomeration of PC as spherical particles. A mesoporous form was then obtained by removing ice crystals through freeze-drying of the particles. MPP could accommodate both hydrophilic and hydrophobic guest molecules in the lamellar structure and the mesopores. It might be applied as a novel drug carrier in a complementary or even a replacement technology of liposomes.

■

multiemulsion precipitation16−18 or spray drying.19−21 Porous poly(lactide-co-glycolide) (PLGA) and chitosan particles are usually fabricated by using one of these two methods and porogen/templating materials such as salts, surfactants, or polymer particles must be added to induce pore formation. Thus, depending on the type of porogens, an additional step is required for its removal. Moreover, the spray drying process for solvent evaporation should be conducted at relatively high temperature presenting a significant disadvantage for heat sensitive materials. Spray freeze-drying (SFD) is an alternative method for fabrication of porous particles, in which the solvent is frozen and removed by sublimation, and a porous structure can be obtained simultaneously.10,22−24 Since even heat sensitive biomaterials are stable under these conditions, SFD is favored for the preparation of proteins in a porous particulate form. However, polymeric materials are not free of safety concerns either because of their slow degradation and the possible presence of low molecular weight impurities. Here we report a novel production methodology of mesoporous phospholipid particles (MPPs), which are anticipated to be safe for human internal use, especially as a

INTRODUCTION Mesoporous particles are attractive as materials in a variety of fields of application because of their unique properties.1−7 The confined spaces provided by mesoporous materials offer an unusual environment, which may alter the properties of guest substances including their vapor pressures, crystal forms, or freezing temperatures. In the biomedical field, mesoporous particles may function as drug reservoirs.4−7 Additionally, their low densities can be advantageous for efficient pulmonary drug delivery because they may be delivered deep into the lung.8−11 Research on mesoporous materials is, in many cases, focused on inorganic “hard” materials. However, there exist concerns over the safety of hard materials if they were to be administered internally to humans. Safety requirements are especially severe in the case of pulmonary drug delivery, since lung tissue is extremely sensitive to the presence of foreign bodies. Polymeric materials have been widely used in the design of drug carriers due to their better biocompatibilities with polymeric mesoporous particles often being produced by heterogeneous polymerization using two or more immiscible liquids. This approach uses monomers, which are polymerized using a variety of techniques, leading to porous particles with a wide range of particle sizes (from less than 1 μm to several mm) and surface areas (from 10 to 1000 m2/g).12−15 Porous particles of commercially available polymers are usually prepared by © XXXX American Chemical Society

Received: January 7, 2015 Revised: February 28, 2015

A

DOI: 10.1021/acs.jpcc.5b00159 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

ice for approximately 10 s. Subsequently, the solution was investigated. Preparation of Mesoporous Phospholipid Particles. HSPC was dissolved in cyclohexane/t-BuOH mixture at 50 °C to obtain transparent solutions, followed by incubation of the solutions at a designated temperature for 1 day to induce HSPC precipitation. The precipitates were frozen in liquid nitrogen for 2 h before being transferred to a freeze-dryer. A primary drying was conducted at −20 °C for 24 h, followed by a secondary drying at room temperature for 24 h. SAXS Measurements of HSPC Solutions. SAXS measurements were carried out on 9.6% HSPC in t-BuOH/ cyclohexane =1/2 solution at different concentrations of water using a SAXSess camera (Anton Paar, Austria) attached to a PW3830 sealed-tube anode X-ray generator (PANalytical, The Netherlands) operating at 40 kV and 50 mA. A monochromatic X-ray beam of CuKα radiation (λ = 0.1542 nm) with a welldefined focused line-shape was obtained using a Göbel mirror fitted with a block collimator. The thermostated sample holder unit (TCS 120, Anton Paar) attached in the SAXSess system maintained the temperature of samples with an accuracy of ±0.1 °C. 2D scattering patterns recorded on an image plate (IP) detector (Cyclone, PerkinElmer, USA) were integrated into 1D scattering intensities, I(q), as a function of the magnitude of the scattering vector, q, using the SAXSQuant software (Anton Paar). The scattering vector, q, is related to the total scattering angle, θ, as

drug delivery platform, since they are composed solely of phosphatidylcholine (PC), a major component of biological membranes. In our procedure, hydrogenated soybean phosphatidylcholine (HSPC) is dissolved in an appropriate solvent mixture to obtain transparent solutions, followed by incubation of the solutions below the liquid−liquid demixing temperature to induce HSPC precipitation, which is obtained in a spherical morphology because of microphase separation. This process resembles with the spherical crystallization technology proposed by Kawashima et al., in which needle-like crystals of salicylic acid were transformed to spherical agglomeration by utilizing phase separation of the mixed solvent.25 The precipitates are subsequently freeze-dried leading to formation of a mesoporous structure in the particles. MPPs are expected to possess the similar advantages with those of liposomes such as ability to accommodate both hydrophilic and hydrophobic guest molecules, ease of surface decoration, and high biocompatibility. Also expected for MPPs are functions as mesoporous materials including capability for accepting large amount of guest molecules and their physical/chemical stabilization. Difficulty in industrial production because of its nonequilibrium nature had been one of the issues that inhibited wide use of the liposome technology. In contrast, production of MPPs is much simpler than that of liposomes because its fabrication process is governed by thermodynamics, which should be another great advantage of MPP as a drug carrier. A preparation method of MPP is introduced below with its principle and basic properties as a drug carrier.

■

q=

MATERIALS AND METHODS Materials. Hydrogenated soybean phosphatidylcholine, HSPC, was obtained from Nippon Oil & Fats (Tokyo, Japan). The acyl-chains are composed of approximately 89% stearoyl and 10% palmitoyl acids, and therefore, major components of HSPC are distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), stearoylpalmitoylphosphatidylcholine (SPPC), and palmitoylstearoylphosphatidylcholine (PSPC). As an example, chemical structure of DSPC is presented in the Supporting Information. Fluorescein isothiocyanate (FITC)-dextran (average molecular weight: 70 kDa, 0.003−0.020 mol FITC per mol glucose) and glucose were purchased from Sigma-Aldrich (St. Louis, MO) and Nacalai Tesque (Kyoto, Japan), respectively. All solvents were of reagent grade and used as supplied. Construction of the Phase Diagram. Phase behaviors of cyclohexane/t-BuOH mixtures were investigated by using differential scanning calorimetry (DSC). Measurements were performed on a DSC Q2000 (TA Instruments, New Castle, DE), which were periodically calibrated using indium and sapphire. Dry nitrogen was used as the inert gas at a flow rate of 50 mL/min. Typically, a sample (∼20 mg) was hermetically sealed in an aluminum pan. A cooling rate of 5 °C/min was used. The onset temperature of exothermic peak was used to construct the phase diagram. Liquid−liquid demixing temperature was also investigated in the presence of 6.0 wt % HSPC at a cooling rate of 1 °C/min. Examples of DSC curves can be found in Supporting Information. Visualization of Liquid−Liquid Demixing. Optical microscopic image of the liquid−liquid demixing process was obtained using a digital microscope VHX200N equipped with a wide-range zoom lens VH-Z100R (Keyence, Osaka, Japan). Six w/v% HSPC was dissolved in cyclohexane/t-BuOH = 2/1 and the solution was placed on a slide glass, followed by cooling on

4π sin(θ /2) λ

(1)

Measured SAXS intensities were calibrated for transmission by normalizing a zero-q attenuated primary beam intensity to unity and the I(q)s were corrected for background scattering (capillary and water). The scattering intensity, I(q), represents the Fourier transform of the scattering length density difference, Δρ(r), and thus describes the structure of particle in real-space.26 In scattering experiments of micellar solutions or microemulsions, the measured I(q) can be defined by the following expression. I(q) = 4π

∫0

∞

p(r )

sin qr dr qr

(2)

Here p(r) is the pair-distance distribution function giving structural information regarding the scattering particles in realspace. The p(r) function directly represents a histogram of distances within the particle for homogeneous scattering particles.27 Generally, the intraparticle scattering contributions are related to the form factor, P(q), and theoretically corresponds to the Fourier transformation of the p(r). For monodisperse spherical particle systems, the total scattering intensity, I(q), can be expressed as

I(q) = nP(q)S(q)

(3)

where P(q) is the form factor representing intraparticle scattering contributions and theoretically corresponds to the Fourier transformation of the p(r), and S(q) is the structure factor representing the interparticle interfaction and corresponds to the Fourier transformation of the total correlation function. Since our system contains rather large quantities of HSPC, we had to take interparticle interactions in to account during SAXS data evaluation. Therefore, we have analyzed the B


Article

The Journal of Physical Chemistry C experimental scattering data using the generalized indirect Fourier transformation (GIFT) method,28 which was used to determine both the form factor and the structure factor without any model assumption for the form factor. Nevertheless, appropriate assumptions for the interparticle interaction potentials and the closure relation for the structure factor are required. In the calculation, we used an averaged structure factor model of hard-sphere (HS) interactions. Physical Characterization of MPPs. Morphology of the particles was investigated by using scanning electron microscopy (SEM) (S8000, Hitachi, Tokyo, Japan) after sputtercoating using a platinum coater (E-1030 ion sputter, Hitachi, Tokyo, Japan). Primal particle size was determined by image analysis of the SEM pictures using Mac-View ver. Four (Mountech, Tokyo, Japan). Three hundred particles were selected randomly from the image to obtain the Heywood diameter. Volume-mean diameter was used for the analysis. Small angle X-ray scattering (SAXS) patterns of MPPs were obtained on a Rigaku Nano Viewer (Rigaku Denki, Tokyo, Japan) using Cu Kα radiation with 1.540 Å wavelength. The voltage and the current were 40 kV and 30 mA, respectively. FT-IR studies were carried out on a Jasco 6200 equipped with a temperature-variable ATR accessory. All spectra were recorded with 2 cm−1 resolution. Specific surface areas and pore volumes were determined by using the nitrogen adsorption method on a Belsorp mini (Bel Japan, Osaka, Japan). Approximately 100 mg samples were dried at 25 °C for 2 h under vacuum and the dead volume of the sample cell was measured at room temperature prior to measurement. Nitrogen adsorption/desorption measurements were performed using sample cells and an empty reference cell immersed in liquid nitrogen. Each measurement was repeated at least twice to obtain the mean surface area calculated by the BET (Brunauer−Emmett−Teller) method. Pore size distribution and pore volume were calculated using BJH (Barrett− Joyner−Halenda) models. Incorporation of Hydrophilic Guest Molecules in MPP and Release Study. MMP was prepared from 9.6 wt % HSPC solution in cyclohexane/t-BuOH = 2/1 at a precipitation temperature at 0 °C. Various amount of 40% w/v glucose solutions or 4.8 wt % of various concentration of glucose solutions were added to HSPC solutions prior to precipitation. For the release study of FITC−dextran, MMP was prepared from 8.0 wt % HSPC solution in cyclohexane/t-BuOH = 2/1 at a precipitation temperature at 0 °C. Then, 200 or 400 μL of aqueous FITC−dextran solution (1 mg/mL) was added to the HSPC solution prior to precipitation to obtain MPPs that contain 0.025 or 0.05 wt % FTTC−dextran. A release study was conducted by dispersing 200 or 100 mg of each FITC−dextrancontaining MPP in 100 mL of phosphate buffer at room temperature under stirring with a magnetic stirrer (n = 2). Aliquots were taken at predetermined time intervals, and filtered using a syringe filter with a pore size of 0.45 μm. Quantification of FITC−dextran was performed by fluorescence analysis with excitation and emission wavelengths at 492 and 518 nm, respectively.

Figure 1. Phase diagram of cyclohexane/t-BuOH mixture and a schematic representation of the preparation process of MPPs. Solid cyclohexane and t-BuOH are expressed as C(s) and B(s), respectively. Demixing temperature of 6.0% HSPC solution is indicated.

incorporation of the other component. A eutectic point was found at t-BuOH/cyclohexane = 1/1 with a freezing temperature of approximately −39 °C. All precipitation procedures in this study were conducted in the liquid region. The liquid− liquid demixing temperature of a 6.0 wt % HSPC solution is also indicated in Figure 1. The demixing temperature is not sensitive to cyclohexane/t-BuOH ratio, and is below 25 °C for all the mixed solvents used in this study. HSPC concentration did not have a significant influence on demixing temperature in the concentration range from 3.0 to 9.6 wt %. For the cyclohexane/t-BuOH = 2/1 solution, the demixing temperature was 17.2, 18.1, and 17.8 °C for 3.0, 6.0, and 9.6 wt % HSPC, respectively. HSPC is soluble at 9.6 wt % in t-BuOH/cyclohexane =1/2 solution at 50 °C, whereas solubilities of HSPC at 25, 0, and −20 °C are 0.99, 0.30, and 0.052 wt %, respectively (Supporting Information, Table S1). Figure 2a shows an optical microscopic image of the HSPC solution during the cooling process, where spherical liquid droplets that should be rich in t-BuOH appeared immediately. These droplets can be assigned as being the result of liquid−liquid demixing. Parts b− d of Figures 2 show SEM images of the HSPC lyophilates after precipitation at three different temperatures. Morphology of the HSPC lyophilates depends on the precipitation temperature; a flake structure was obtained from the precipitates at 25 °C, while porous and dense particles were respectively obtained from the precipitates at 0 and −20 °C. The particle sizes were quite homogeneous being of mean diameters 15.8 and 8.2 μm, respectively, for particles obtained by precipitation at 0 and −20 °C. The largest surface area was obtained for the porous particles precipitated at 0 °C, followed by the flake lyophilates and the dense particles. Pore volume of the porous particles obtained from the precipitates at 0 °C was 0.26 cm3/g, which is more than double that of the other lyophilates. The liquid− liquid demixing temperature was ca. 17.8 °C for the 9.6 wt % HSPC solution. Thus, precipitation at 25 °C is simply due to a decrease in the solubility, while liquid−liquid demixing was induced before the precipitation below 0 °C. Differences in the lyophilate morphologies can be explained by the involvement of the liquid−liquid demixing process that should lead to precipitation of HSPC in a spherical morphology.

■

RESULTS AND DISCUSSION Formation Mechanism of MPP. Figure 1 shows the phase diagram of the cyclohexane/t-BuOH mixture system. The onset freezing temperatures of pure cyclohexane and t-BuOH according to thermal analysis are approximately 5.3 and 7.5 °C, respectively, and that of mixtures is lowered by C


Article


Figure 2. (a) Optical microscopic image of the liquid−liquid demixing process. (b−d) Scanning electron microscopic images of lyophilates precipitated from 9.6 wt % HSPC in cyclohexane/t-BuOH = 2:1 solution at +25 (b), 0 (c), and −20 °C (d). S and D represent specific surface area and diameter, respectively. S is the average value of two or three samples, and D is that of the sample shown. Standard errors of the S value were within 15%.

Figure 3. SAXS and FT-IR patterns of the lyophilates presented in Figure 2b−d. The precipitation temperatures are −20, 0, and +25 °C (top to bottom). The oval in the FT-IR spectra indicates PO stretching region, which is sensitive to structural variation in the headgroups of phospholipids (see text).

Unit Structure of MPP: Lipid Bilayers. SAXS patterns and FT-IR spectra of the HSPC lyophilates are shown in Figure 3. The particles obtained by precipitation at 0 and −20 °C show an X-ray scattering peak at the same position, 1.03 nm−1, which indicates the presence of a periodic structure with an interval of 6.1 nm. This distance corresponds to the bilayer thickness of phospholipids.29−31 In contrast, only a small shoulder peak was found at 1.03 nm−1 in the scattering pattern of the HSPC flakes obtained from precipitates at 25 °C. The major peak was reduced in intensity and shifted to 1.15 nm−1, corresponding to a periodic distance of 5.5 nm. Thus, the unit structure of the flake lyophilate is different from those of the particles, which are assumed to consist of lipid bilayers. Characteristic infrared absorption peaks for phosphatidylcholines appear at 1741 cm−1 (CO, carbonyl groups), 1260 cm−1 (PO), and 1064 cm−1 (P−O−C). However, the 1260 cm−1 band shifts to 1242 cm−1 for the particulate lyophilates. This

band is sensitive to structural changes such as, for example, formation of hydrogen-bonding due to the hydration of phosphate groups.32−34 Since this shift was maintained even after heating of the samples to 80 °C (data not shown), it is likely not due to adsorption of water but because of formation of a hydrogen-bond network between HSPC headgroups within the periodic bilayer structure. Thus, the particles obtained by precipitation at 0 and −20 °C are composed of lipid bilayers. Since the bilayer structure of the HSPC flakes obtained from precipitates at 25 °C was disordered, the liquid− liquid demixing process seems to be important for formation of the bilayer structure as well as for precipitation in a spherical morphology. Effect of Phospholipid Concentration on the Pore Size. Ice-templating is a powerful method for creating macroporous structures in materials.35−37 In our study, mesopores are also likely to be formed due to penetration of D


Article


concentration. Given that the peak pore size was above 100 nm for the MPP obtained from 3.0 wt % solution, the pore size decreased with increasing HSPC concentration, most likely because of the decrease in the relative quantity of solvent. Specific surface area was largest for the 6.0 wt % concentration at 43 m2/g. Unexpected small surface area for the 9.6 wt % concentration indicates that excessively small pores (mesopores and/or smaller ones) might collapse due to soft nature of the materials. The effect of variation of solvent ratio was also investigated at a fixed HSPC concentration of 6.0 wt % and precipitation temperature of 0 °C. The cyclohexane/t-BuOH ratio was varied from 2/1 to 1/4 revealing that its variation has no significant effect on the morphology of the particles obtained (Supporting Information), which indicates that the droplet size after the demixing and the solvent composition in the droplet size are not greatly influenced by the initial solvent composition. Incorporation of Guest Molecules. MPP can accommodate both hydrophilic and hydrophobic guest molecules. Hydrophobic molecules are incorporated by dissolving them in the HSPC organic solution prior to the precipitation process. A case study where prednisolone was loaded as a model drug is presented in the Supporting Information. As same as the liposomes, hydrophobic molecules are expected to be incorporated in the acyl-chain region of the lipid bilayers. In addition, the mesopores are also expected as reservoirs for drug molecules, where stable amorphous form may be obtained.7 Investigation on this matter is in proceeding in our laboratory and will be presented in the forthcoming paper. The HSPC solution before the freeze-drying can incorporate an aqueous phase in the form of a microemulsion. In the presence of water, HSPC does not precipitate at 25 °C because it is stabilized at the water/oil interfaces. Effect of water on the microstructure of HSPC in t-BuOH/cyclohexane =2/1 solution was studied by using SAXS. Parts a and b of Figure 6 show the experimental scattering intensities, I(q), as a function of q, and the pair-distance distribution functions, p(r), derived by the GIFT method, respectively. The I(q) vs q curve of HSPC solution suggests that HSPC forms an aggregate structure in absence of water. The overall scattering intensity increased upon addition of water followed by the appearance of small SAXS peaks in the I(q) curve (q ∼ 1.3 for 10% H2O, and q ∼ 1.0 for 20% H2O). These observations are the indication of bigger and interactive aggregate structures in the presence of water. The water induced microstructure transformations can be read out from the shape of the p(r) curve, which essentially indicates the shape of scattering objects and that the value of r at which p(r) reaches zero at higher-r values gives an estimate of the maximum dimension, Dmax.38 In the absence of water, HSPC forms ellipsoidal prolate type aggregates (average size 3.3 nm) at 50 °C. Addition of water increases the overall size of HSPC aggregates; the position of the maximum in the lower-r region of the p(r) function shifts to the right-hand side and Dmax increases to 4.8 nm (for 10% H2O system), and 7.0 nm (for 20% H2O system). Moreover, the shape of the p(r) curve (with a pronounced peak in the lower-r with linear decay in the higher-r side) in the water containing system resembles that usually observed for rod-like aggregates.39 Water-induced growth of HSPC aggregate structures can be explained in terms of critical packing and the formation of water pool in the core of aggregates. In reverse micellar solutions or water-in-oil microemulsion systems, water tends to soluble at the micellar core swelling the aggregate structure as a result overall size

solvents into the molecular assemblies composed of HSPC. Pore size is expected to be regulated by controlling growth of the ice crystals with the simplest way to control the crystal size being by varying the solvent/solute ratio. The effect of HSPC concentration on the particle morphology was investigated at the precipitation temperature of 0 °C at a cyclohexane/t-BuOH ratio of 2/1. At all concentrations, mesoporous spherical particles of similar dimensions were obtained (Figures 4, 5).

Figure 4. (a−d) SEM images of MPP prepared at 0 °C at HSPC concentrations of (a, b) 3.0 wt % and (c, d) 6.0 wt % in cyclohexane/tBuOH = 2:1 solution.

Figure 5. Pore size distribution for the particles with different HSPC concentrations determined by BJH (Barrett−Joyner−Halenda) plot based on nitrogen adsorption analysis. The diameters, surface areas, and pore volumes are presented in the table. The particle size indicated is of the sample shown in the image, and other data are averaged values of two or three samples. Standard errors of the surface area and pore volume were within 18%.

Calculation of the pore size distribution in the particles revealed that mesopores were effectively formed above the 6.0 wt % E


Article


Figure 6. (a) Normalized X-ray scattering intensity, I(q), of 9.6% HSPC in t-BuOH/cyclohexane =2/1 solution at different concentrations of water (0, 10, and 20%) on absolute scales. Solid and broken lines represent the GIFT fit and calculated form factor for n particles existing in unit volume, nP(q), respectively. (b) Structure of HSPC aggregates in real-space: pair-distance distribution functions, p(r), of 9.6 wt % HSPC in t-BuOH/ cyclohexane =2/1 solution at different concentrations of water (0, 10, and 20 vol %). Arrows shown at higher r values of the p(r) curves indicate the maximum dimension of micelle, Dmax. Inflection points appearing after the maximum of the p(r) curve at lower r values (broken line) qualitatively indicate the cross-sectional radius of elongated aggregates.

Figure 7. (a) SEM image of MPP prepared at 0 °C at HSPC concentration of 7.8 wt % in cyclohexane/t-BuOH = 2/1 solution including 2.4 vol % water. (b) SEM image of MMP prepared at 0 °C at HSPC concentration of 9.6 wt % in cyclohexane/t-BuOH = 2/1. 4.8 vol % of 80 w/v% glucose solution was added to HSPC solutions prior to precipitation. (c) Lamellar repeating distance of glucose-containing MPPs that contain glucose determined by the SAXS measurements. The horizontal axis indicates the glucose concentration against the resultant MPP weight. Glucose amount was controlled by changing amount of the glucose solution added or by changing glucose concentration of the added solutions.

glucose added (Figure 7c), indicating that most of the glucose molecules were distributed to the interlayer regions within the lamellar structure. When liposome is used as a carrier for hydrophilic molecules, special efforts such as pH-gradient is required for achieving effective drug-loading, because the aqueous core of the liposome, where the hydrophilic molecules can be loaded spontaneously, is very small. MPP can obviously accommodate hydrophilic molecules more effectively than the liposome, because the large hydrophilic interlayer regions in the lamellar phase are available for the hydrophilic guest molecules. Figure 8 shows the release of FITC−dextran from MPP in aqueous media, in which the biphasic release pattern was investigated. It is highly likely that guest molecules distributed in two different environments lead to observation of fast (from mesopores) and slow (from interlayer spaces) release in our dissolution study since increases in the loading amount of FITC−dextran resulted in an increase in the fraction undergoing fast-release. Unlike the case of glucose, the accommodation efficiency of FITC−dextran may not be high, presumably because of its large molecular size. The

increases. Additionally, water can make hydrogen bonding with hydrophilic moiety of the surfactant resulting in an increase in the effective cross-sectional area of hydrophilic headgroup. Increase in the headgroup size of surfactant decreases the critical packing parameter favoring sphere-to-rod type transition in nonaqueous media.40,41 Addition of small amount of water to the HSPC solution does not alter the liquid−liquid demixing temperature significantly. Thus, MPPs can be prepared in the same manner with that from the nonaqueous system. MPPs prepared from the microemulsion solution possessed a more open porous structure as presented in Figure 7a, presumably because of influence on the crystal growth of the solvent. Hydrophilic guest molecules can be incorporated in the MPPs by dissolving them in the aqueous phase of the HSPC microemulsion prior to precipitation. Guest molecules can be assumed to be distributed in the mesopores and in hydrophilic interlayer regions within the lamellar structure. SAXS measurements revealed that the lamellar repeating distance of the resultant MPPs exhibited linear relationship with the amount of the F


Article


in MPP in a simple procedure. In this paper, a prototype MPP is introduced with some basic characteristics as a drug carrier. Further modification of the characteristics such as particle size, pore structure, and surface property should widen its potential as a novel drug delivery platform.

■

CONCLUSIONS We have established a simple procedure for preparation of mesoporous particles consisting solely of phospholipids as a novel solid material. In this procedure, HSPC was dissolved in the solvent mixture composed of cyclohexane and t-BuOH, followed by incubation of the solutions at depressed temperatures to induce liquid−liquid demixing. HSPC precipitate was spontaneously agglomerated as spherical particles, and a mesoporous form was obtained by removing ice crystals through freeze-drying. MPP could accommodate both hydrophilic and hydrophobic guest molecules in the lamellar structure and the mesopores. MPP is anticipated to be safe for human internal use since it is composed solely of PC. It might be applied as a novel drug carrier in a complementary or even a replacement technology of liposomes.

Figure 8. Release profile of FITC−dextran from MPP. The FITC− dextran contents in the MPP were 0.05 (square) and 0.025 (circles) wt %, respectively. Inset image shows the MPP particle that contains 0.05 wt % of FITC−dextran.

accommodation efficiency of the hydrophilic molecules in the water-in-oil microemulsion is sensitive to the size of the microemulsion droplets because this process is dominated by the bending energy of the amphiphilic membranes.42 Nevertheless, size analysis of our HSPC microemulsion suggests that it has acceptable dimensions for incorporation of various types of hydrophilic molecules including peptides and nucleotides. It should be noted that MPP swells upon dispersion in the aqueous media. The MPP composed of only HSPC changes its shape in a time scale of hour, but the time scale of the swelling behavior significantly depends on composition of MPP. This subject will be extensively discussed in a forthcoming paper. Potential of MPP as a Drug Carrier. Phospholipids are regarded as extremely safe material for biomedical use.43,44 MPP can accommodate both hydrophilic and hydrophobic drugs by dissolving them in the HSPC solution or microemulsion prior to the precipitation. In this procedure, the drug molecules are mainly loaded in the bilayer region. Also, as done for other mesoporous carriers, post loading of the drug molecule to the mesopore region is also possible. Liposomes have played an important role in biomedical fields as one of the most important platform technologies for drug delivery. However, liposome materials are usually applied as suspensions since their structures are broken by removal of water.45,46 MPP has strong potential as a novel platform for drug delivery applications as a complementary or even a replacement technology of existing liposome materials. It should be noted that the majority of pharmaceutical products are manufactured as solid dosage forms and we anticipate a greater range of applications for MPPs over the current liposome materials. MPP may be used for any administration routes except intravenous injection where size of the carrier must be smaller than the MPPs introduced in this paper by more than an order of magnitude. The most promising application may be as a carrier for dry powder inhalation. Lipid-based carriers are already recognized as safe for the pulmonary route in the clinical studies.47 Typically, MPP has a bulk density of approximately 0.02 g/cm3. Thus, its aerodynamic diameter is 1−2 μm, which is suitable for pulmonary drug delivery since effective deposition deep in the lung is expected. Pulmonary route is expected for systemic drug delivery as well as treatment of pulmonary disease, since it enables systemic absorption of large molecules that cannot be delivered via oral route. As demonstrated for FITC−dextran, large molecules can be loaded

■

ASSOCIATED CONTENT

* Supporting Information S

Chemical structure of DSPC, description of solubility measurements of HSPC in the organic solutions, effects of solvent composition on the MPP morphology, and an example of incorporation of hydrophobic drug in MPP. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*(K.K.) Present Address: World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. Telephone: +81-29-860-4424. Fax: +81-29-860-4708. E-mail: [email protected]. *(K.A.) Present Address: World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. Telephone: +81-29-860-4597. Fax: +81-29-860-4832. E-mail: [email protected]. Notes

The authors declare no competing financial interests.

■

ACKNOWLEDGMENTS This work was in part supported by World Premier International Research Center (WPI) Initiative on Materials Nanoarchitectonics, MEXT, Japan, and JST, CREST.

■

REFERENCES

(1) Ariga, K.; Vinu, A.; Yamauchi, Y.; Ji, Q.; Hill, J. P. Nanoarchitectonics for Mesoporous Materials. Bull. Chem. Soc. Jpn. 2012, 85, 1−32. (2) Gu, D.; Schuth, F. Synthesis of Non-siliceous Mesoporous Oxides. Chem. Soc. Rev. 2014, 43, 313−344. (3) Li, W.; Wu, Z. X.; Wang, J. X.; Elzatahry, A. A.; Zhao, D. Y. A Perspective on Mesoporous TiO2 Materials. Chem. Mater. 2014, 26, 287−298. (4) Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Y. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers. Adv. Drug Delivery Rev. 2008, 60, 1278−1288. G


Article

The Journal of Physical Chemistry C (5) Li, Z.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2590−2605. (6) Mamaeva, V.; Sahlgren, C.; Lindén, M. Mesoporous Silica Nanoparticles in Medicine–Recent Advances. Adv. Drug Delivery Rev. 2013, 65, 689−702. (7) Xu, W.; Riikonen, J.; Lehto, V. P. Mesoporous Systems for Poorly Soluble Drugs. Int. J. Pharm. 2013, 453, 181−197. (8) Edwards, D. A.; Hanes, J.; Caponetti, G.; Hrkach, J.; Ben-Jebria, A.; Eskew, M. L.; Mintzes, J.; Deaver, D.; Lotan, N.; Langer, R. Large Porous Particles for Pulmonary Drug Delivery. Science 1997, 276, 1868−1871. (9) Chan, H. K. Dry Powder Aerosol Delivery Systems: Current and Future Research Directions. J. Aerosol. Med. 2006, 19, 21−27. (10) Chow, A. H. L.; Tong, H. H. Y.; Chattopadhyay, P.; Shekunov, B. Y. Particle Engineering for Pulmonary Drug Delivery. Pharm. Res. 2007, 24, 411−437. (11) Weers, J. G.; Bell, J.; Chan, H. K.; Cipolla, D.; Dunbar, C.; Hickey, A. J.; Smith, I. J. Pulmonary Formulations: What Remains to be Done? J. Aerosol Med. Pulmonary Drug Delivery 2010, 23, S5−S23. (12) Li, W. H.; Stöver, H. D. H. Porous Monodisperse Poly(divinylbenzene) Microspheres by Precipitation Polymerization. J. Polym. Sci., Part A: Polym. Chem. 1998, 36, 1543−1551. (13) Macintyre, F. S.; Sherrington, D. C.; Tetley, L. Synthesis of Ultrahigh Surface Area Monodisperse Porous Polymer Nanospheres. Macromolecules 2006, 39, 5381−5384. (14) Liu, Q.; Wang, L.; Xiao, A.; Yu, H.; Tan, Q.; Ding, J.; Ren, G. Unexpected Behavior of 1-Chlorodecane as a Novel Porogen in Preparation of High Porosity Poly(divinylbenzene) Microspheres. J. Phys. Chem. C 2008, 112, 13171−13174. (15) Gokmen, M. T.; Du Prez, F. E. Porous polymer particlesA Comprehensive Guide to Synthesis, Characterization, Functionalization and Applications. Prog. Polym. Sci. 2012, 37, 365−405. (16) Ungaro, F.; De Rosa, G.; Miro, A.; Quaglia, F.; La Rotonda, M. I. Insulin-loaded PLGA/cyclodextrin Large Porous Particles with Improved Aerosolization Properties: In vivo Deposition and Hypoglycaemic Activity after Delivery to Rat Lungs. J. Controlled Release 2009, 135, 25−34. (17) Yang, Y.; Bajaj, N.; Xu, P.; Ohn, K.; Tsifansky, M. D.; Yeo, Y. Development of Highly porous Large PLGA Microparticles for Pulmonary Drug Delivery. Biomaterials 2009, 30, 1947−1953. (18) Kim, H.; Lee, J.; Kim, T. H.; Lee, E. S.; Oh, K. T.; Lee, D. H.; Park, E. S.; Bae, Y. H.; Lee, K. C.; Youn, Y. S. Albumin-coated Porous Hollow Poly(lactic-co-glycolic acid) Microparticles Bound with Palmityl-acylated Exendin-4 as a Long-acting Inhalation Delivery System for the Treatment of Diabetes. Pharm. Res. 2011, 28, 2008− 2019. (19) Iskandar, F.; Nandiyanto, A. B. D.; Widiyastuti, W.; Young, L. S.; Okuyama, K.; Gradon, L. Production of Morphology-controllable Porous Hyaluronic Acid Particles Using a Spray-drying Method. Acta Biomater. 2009, 5, 1027−1034. (20) Cruz, L.; Fattal, E.; Tasso, L.; Freitas, G. C.; Carregaro, A. B.; Guterres, S. S.; Pohlmann, A. R.; Tsapis, N. Formulation and in vivo Evaluation of Sodium Alendronate Spray-Dried Microparticles Intended for Lung Delivery. J. Controlled Release 2011, 152, 370−375. (21) Naniyanto, A. B. D.; Okuyama, K. Progress in Developing Spray-drying Methods for the Production of Controlled Morphology Particles: From the Nanometer to Submicrometer Size Ranges. Adv. Powder Technol. 2011, 22, 1−19. (22) Engstrom, J. D.; Simpson, D. T.; Lai, E. S.; Williams, R. O., III; Johnston, K. P. Morphology of Protein Particles Produced by Spray Freezing of Concentrated Solutions. Eur. J. Pharm. Biopharm. 2007, 65, 149−162. (23) Saluja, V.; Amorij, J. P.; Kapteyn, J. C.; de Boer, A. H.; Frijlink, H. W.; Hinrichs, W. L. J. A Comparison between Spray Drying and Spray Freeze Drying to Produce an Influenza Subunit Vaccine Powder for Inhalation. J. Controlled Release 2010, 144, 127−133.

(24) Mohri, K.; Okuda, T.; Mori, A.; Danjo, K.; Okamoto, H. Optimized Pulmonary Gene Transfection in Mice by Spray-freeze Dried Powder Inhalation. J. Controlled Release 2010, 144, 221−226. (25) Kawashima, Y.; Okumura, M.; Takenaka, H. Spherical Crystallization: Direct Spherical Agglomeration of Salicylic Acid Crystals during Crystallization. Science 1982, 216, 1127−1128. (26) Glatter, O. Data Evaluation in Small Angle Scattering: Calculation of the Radial Electron Density Distribution by Means of Indirect Fourier Transformation. Acta Phys. Austriaca 1977, 47, 83− 102. (27) Glatter, O. A New Method for the Evaluation of Small-angle Scattering data. J. Appl. Crystallogr. 1977, 10, 415−421. (28) Fritz, G.; Bergmann, A.; Glatter, O. Evaluation of Small-angle Scattering Data of Charged Particles Using the Generalized Indirect Fourier Transformation Technique. J. Chem. Phys. 2000, 113, 9733− 9740. (29) McManus, J. J.; Rädler, J. O.; Dawson, K. A. Phase Behavior of DPPC in a DNA-calcium Zwitterionic Lipid Complex Studied by Small Angle X-ray Scattering. Langmuir 2003, 19, 9630−9637. (30) Wang, T.; Wang, N.; Wang, T.; Sun, W.; Li, T. Preparation of Submicron Liposomes Exhibiting Efficient Entrapment of Drugs by Freeze-drying Water-in-oil Emulsions. Chem. Phys. Lipids 2011, 164, 151−157. (31) Liu, H.; Wu, J.; Guo, H.; Zhou, X.; Xu, G. The Preliminary Study of Hydration of Phosphocholine by FT-IR. Mikrochim. Acta [Wien] 1988, I, 361−364. (32) Nzai, J. M.; Proctor, A. Soy Lecithin Phospholipid Determination by Fourier Transform Infrared Spectroscopy and the Acid Digest/arseno-molybdate Method: A Comparative Study. J. Am. Oil Chem. Soc. 1999, 76, 61−66. (33) Whittinghill, J. M.; Norton, J.; Proctor, A. Stability Determination of Soy Lecithin-based Emulsions by Fourier Transform Infrared Spectroscopy. J. Am. Oil Chem. Soc. 2000, 77, 37−42. (34) Pohle, W.; Gauger, D. R.; Fritzsche, H.; Rattay, B.; Selle, C.; Binder, H.; Bö hlig, H. FTIR-spectroscopic Characterization of Pholsphocholine-headgroup Model Compounds. J. Mol. Struct. 2001, 563−564, 463−467. (35) Mukai, S. R.; Nishihara, H.; Shichi, S.; Tamon, H. Preparation of Porous TiO2 Cryogel Fibers through Unidirectional Freezing of Hydrogel Followed by Freeze-drying. Chem. Mater. 2004, 16, 4987− 4991. (36) Lu, H.; Ko, Y. G.; Kawazoe, N.; Chen, G. Cartilage Tissue Engineering Using Funnel-like Collagen Sponges Prepared with Embossing Ice Particulate Templates. Biomaterials 2010, 31, 5825− 5835. (37) Wang, X.; Sumboja, A.; Khoo, E.; Yan, C.; Lee, P. S. Cryogel Synthesis of Hierarchical Interconnected Macro-/mesoporous Co3O4 with Superb Electrochemical Energy Storage. J. Phys. Chem. C 2012, 116, 4930−4935. (38) Shrestha, L. K.; Shrestha, R. G.; Aramaki, K.; Yoshikawa, G.; Ariga, K. Demonstration of Solvent-Induced One-Dimensional Nonionic Reverse Micelle Growth. J. Phys. Chem. Lett. 2013, 4, 2585−2590. (39) Shrestha, L. K.; Sato, T.; Dulle, M.; Glatter, O.; Aramaki, K. Effect of Lipophilic Tail Architecture and Solvent Engineering on the Structure of Trehalose-Based Nonionic Surfactant Reverse Micelles. J. Phys. Chem. B 2010, 114, 12008−12017. (40) Willard, D. M.; Riter, R. E.; Levinger, N. E. Dynamics of Polar Solvation in Lecithin/Water/Cyclohexane Reverse Micelles. J. Am. Chem. Soc. 1998, 120, 4151−4160. (41) Tung, S.-H.; Huang, Y.-E.; Raghavan, S. R. Contrasting Effects of Temperature on the Rheology of Normal and Reverse Wormlike Micelles. Langmuir 2007, 23, 372−376. (42) Kawakami, K.; Harada, M.; Adachi, M.; Shioi, A. Mechanism of Protein Solubilization in Sodium Bis(2-ethylhexyl) Sulfosuccinate Water-in-Oil Microemulsion. Colloids Surf., A 1996, 109, 217−233. (43) Alving, C. R. Antibodies to Lipids and Liposomes: Immunology and Safety. J. Liposome Res. 2006, 16, 157−166. H


Article

The Journal of Physical Chemistry C (44) Lim, S. B.; Banerjee, A.; Onyuksel, H. Improvement of Drug Safety by the Use of Lipid-based Nanocarriers, J. Controlled Release 163, 34−45. (45) Miyajima, K. Role of Saccharides for the Freeze-thawing and Freeze Drying of Liposome. Adv. Drug Delivery Rev. 1997, 24, 151− 159. (46) Chen, C.; Han, D.; Cai, C.; Tang, X. An Overview of Liposome Lyophilization and Its Future Potential. J. Controlled Release 2010, 142, 299−311. (47) Cipolla, D.; Shekunov, B.; Blanchard, J.; Hickey, A. Lipid-based Carriers for Pulmonary Products: Preclinical Development and Case Studies in Humans. Adv. Drug Delivery Rev. 2014, 75, 53−80.

I


Totally Phospholipidic Mesoporous Particles - The Journal of

Recommend Documents