Unexpected and Successful “One-Step” Formation of Porous

Article pubs.acs.org/Langmuir

Unexpected and Successful “One-Step” Formation of Porous Polymeric Particles Only by Mixing Organic Solvent and Water under “Low-Energy-Input” Conditions Taku Takami and Yoshihiko Murakami* Department of Organic and Polymer Materials Chemistry, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan ABSTRACT: We found that porous particles were unexpectedly obtained in a “one-step” manner only by mixing an organic solvent and water under “low-energy-input” (i.e., low-homogenization-rate) conditions. This phenomenon was attributable to the unexpected formation of the spontaneously formed water-in-oil (w/o) emulsions in the droplets of o/w emulsions. The unexpected formation resulted in the successful formation of water-in-oil-in-water (w/o/w) emulsions instead of o/w emulsions, although the mixed solution containing both an organic solvent and water were simply emulsified in the presence of block copolymers. The present study clarifies the effects of the various preparation conditions on the morphology of unexpected w/o/w emulsions and resulting particles. The porous particles are expected to be suitable drug carriers for pulmonary delivery. The results obtained in the present study show that a newly developed one-step emulsification can be a powerful and facile technique for preparing porous polymeric particles.

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INTRODUCTION There are many methods for the administration of drugs, including oral, pulmonary, intravenous, subcutaneous, transdermal, transmucosal, and rectal administrations. Of these routes, the pulmonary route has attracted growing interest. Pulmonary administration has various advantages over other routes, insofar as the administration is simple, shows early effects of drugs’ pharmacological actions, and has no risk of drug decomposition.1−8 Among the several types of drug carriers including liposomes,9−13 micelles,14−18 and gels,19 polymeric “particles” are suitable for pulmonary administration owing to their stable drug delivery and their easily controllable drug-release profile.20−23 Some noteworthy properties are necessary for a drug carrier used in conjunction with pulmonary administration. First, biocompatible molecules such as poly(ethylene glycol) (PEG), polylactide (PLA), and poly(lactide-co-glycolide) (PLGA) are used as particle-forming polymers.24−29 In particular, PEG has been a well-known biocompatible modifier of drug carriers30,31 and protein particles32−35 because the presence of PEG both renders the carriers unrecognizable to phagocytes and reduces the aggregation of the carriers. Second, the diameter of the particles for pulmonary drug delivery is controlled within 1−5 μm.36 Third, it is important to modify the surface of the particles. The particles having functional molecules on the surface can bind to the tissue surface and, consequently, tend to be absorbed into tissue. In general, PLGA and PLA are known as molecules where surface modification of the resulting particles is difficult because PLGA and PLA have almost no reactive groups in their molecules. Fourth, particles © 2014 American Chemical Society

having a low density or a large surface area are suitable for pulmonary drug delivery. In these regards, we have reported a novel facile technique for preparing surface-modified nano- and microsized particles for intravenous and pulmonary deliveries via newly developed emulsification/evaporation processes in the presence of block copolymers.37,38 In this method, surface-modified particles were easily obtained by the evaporation of oil-in-water (o/w) emulsions containing both hydrophobic polymers and amphiphilic block copolymers. Interestingly, we successfully obtained the microparticles having “dimpled” surfaces with a “spray dry-based” evaporation process.38 Dimpled microparticles can positively serve as drug carriers for pulmonary delivery because the particles have a large surface area. However, the spray drying of particles is expensive and timeconsuming because the drying process needs to be optimized for each combination of hydrophobic polymer−amphiphilic block copolymer. Consequently, there is demand for methodologies for preparing low-density or large-surface-area particles. In the present paper, it was reported that porous particles were unexpectedly and successfully obtained in a “one-step” manner by mixing an organic solvent and water only under “low-energy-input” (i.e., low-homogenization-rate) conditions. This phenomenon was attributable to the unexpected formation of the spontaneous formation of water-in-oil (w/o) emulsions in the droplets of o/w emulsions. The water-in-oilReceived: October 18, 2013 Revised: March 4, 2014 Published: March 6, 2014 3329

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copolymers to PLGA varied from 1:99 to 90:10. After a portion of the mixed solvent (0.56 mL) was added to water (13.4 mL), the mixture was emulsified at 8000−16 000 rpm for 10 min with a high-speed homogenizer (T-25 ULTRA-TURRAX Digital Homogenizer, IKA). The obtained emulsion (4 mL) was poured into water (12 mL) and mixed magnetically at 100 rpm for 12 h at room temperature for the purpose of evaporating the organic solvent. The resulting solids were collected by centrifugation at 2000 rpm for 5 min and washed three times with distilled water to remove residual block copolymers. Microscopic Observation of the Emulsions and the PLGA Microparticles. The obtained emulsion (2 μL) was poured on a microscope slide glass and sealed with a glass cover. The sample was observed by means of an optical microscope (BX41 microscope plus CCD DP20 digital camera system, Olympus, original magnification ×400). The surface morphology of the PLGA microparticles was observed by means of a scanning electron microscope (SEM, VE-9800, KEYENCE Co., Ltd., Japan). The specimens for SEM observation were prepared by mounting the sample on an aluminum plate and coating a thin platinum film (approximately 10 nm in thickness) on a sample under reduced pressure with MSP-1S ion water (Vacuum Device Inc., Ibaraki, Japan). Static Observation of Spontaneous w/o Emulsification in Organic Solvents. The dichloromethane−toluene mixed solvents containing either the synthesized block copolymers (0.10 g, 2.90 mL) or PLGA (0.99 g, 19.8 mL) were mixed, so that the molar ratio of the block copolymers to PLGA varied from 10:90 to 90:10. After a portion of the mixed solvent (0.6 mL) was gently added to the water (0.6 mL), the mixture was placed under static observation. Determination of the Diameter of the Spontaneously Formed w/o Emulsions. The dichloromethane−toluene mixed solvents containing either the synthesized block copolymers (0.44 g, 12.6 mL) or PLGA (0.48 g, 9.6 mL) were mixed, so that the molar ratio of the block copolymers and PLGA varied from 10:90 to 90:10. After a portion of the mixed solvent (1.5 mL) was added to water (1.5 mL), the solvent was magnetically stirred at 100 rpm for 5 min at room temperature. The diameter of the droplets in the spontaneously formed w/o emulsion in the white turbid organic phase was determined by dynamic light scattering (DLS, Zetasizer Nano-ZS, Malvern Instruments, England).

in-water (w/o/w) emulsions were then successfully obtained instead of o/w emulsions, although the mixed solution containing both an organic solvent and water were simply emulsified in the presence of block copolymers. The results obtained in the present study show that a newly developed onestep emulsification can be a powerful, facile, and cost-effective technique for preparing porous polymeric particles. To our knowledge, this is the first report addressing this interesting phenomenon, where porous particles can be easily prepared by means of a “one-step” emulsification process under the “lowenergy-input” conditions in the presence of block copolymers.

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EXPERIMENTAL SECTION

Materials. Ethylene oxide (Sumitomo Seika Chemicals Co., Ltd., Osaka, Japan) was purified through distillation with CaH2. DL-Lactide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was recrystallized twice from ethyl acetate. 2-Methoxyethanol was distilled with sodium under reduced pressure. Potassium naphthalene was obtained by mixing potassium and naphthalene in anhydrous tetrahydrofuran (THF) for 18 h. PLGA (monomer ratio of lactide/glicolide: 3; molecular weight: 10 000) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All the other reagents were of analytical grade and were used without further purification. Synthesis of Methoxy-Terminated PEG−PLA Block Copolymers (PEG-b-PLA). A methoxy-terminated PEG−PLA block copolymer (PEG-b-PLA) was synthesized by the ring-opening polymerization of both ethylene oxide and DL-lactide in THF according to the previously reported method39,40 with slight modifications (Scheme 1). 2-Methoxyethanol (1 mmol) and

Scheme 1. Synthesis of the Amphiphilic MethoxyTerminated Poly(ethylene glycol)-b-polylactide Block Copolymers (PEG-b-PLA) by Means of Ring-Opening Polymerization from Ethylene Oxide and DL-Lactide

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RESULTS AND DISCUSSION Synthesis and Characterization of PEG-b-PLA. The obtained PEG-b-PLA was characterized by 1H NMR and GPC. The 1H NMR spectra of the PEG-b-PLA shown in Figure 1 indicates that the block copolymers were successfully synthesized. Table 1 summarizes the results of the synthesis of the block copolymers. The number-average molecular weights of PEG and PLA units were determined on the basis of gel permeation chromatography (GPC) and 1H NMR, respectively. The HLB (hydrophile−lipophile balance) values of the block copolymers were determined to be 20 × (Mh/Mw), where Mh and Mw were respective molecular weights of hydrophilic groups and (hydrophilic + hydrophobic) groups of the molecules (Griffin’s method).41 Low and high HLB values were ascribed to lipophilic and hydrophilic surfactants, respectively. A w/o emulsion was obtained using surfactants with HLB values ranging between 3 and 8, whereas an o/w emulsion was obtained using surfactants with HLB values between 9 and 12. The results in Table 1 show that the block copolymers 1 and 2−4 preferentially promoted the formation of w/o and o/w emulsions, respectively. Observation of Particle Morphology. Solvent evaporation is a facile method for obtaining polymeric particles having homogeneously smooth (i.e., nonporous) surfaces. The method involves both an evaporation of organic solvents from o/w emulsions containing hydrophobic polymers (and emulsifiers, if necessary) and a consequent entanglement of

potassium naphthalene (1 mmol) were mixed in THF for 1 h. The purified ethylene oxide (100−130 mmol) was added to the obtained potassium 2-methoxyethoxide solution (total volume: 50 mL). After stirring for 48 h, the THF solution of purified DL-lactide (22−52 mmol) was added to the solution. After the reaction, the resulting block copolymers were precipitated into cold 2-propanol, centrifuged at 10 500 rpm, and lyophilized in benzene. The average molecular weight of the obtained block copolymers was determined by the use of gel permeation chromatography (GPC) (column: TSKgel G3000HHR, TOSOH, Japan; eluent: N,N′-dimethylformamide in the presence of 10 mM LiBr; flow: 1 mL/min; column temperature: 40 °C) and 1H NMR (AL-300, 300 MHz, JEOL Ltd., Tokyo). PEGp-b-PEGq means that the respective Mn of the PEG and PLA blocks would be p and q. Preparation of Nonporous and Porous PLGA Microparticles. The preparation of nonporous and porous PLGA particles involved a block copolymer-assisted emulsification/solvent evaporation method. The dichloromethane−toluene mixed solvents containing either the synthesized block copolymers (0.06−0.18 g, 1.7−4.0 mL) or PLGA (0.28 g, 5.6 mL) were mixed, so that the molar ratio of block 3330

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various concentrations of PEG-b-PLA, one of the factors affecting both emulsification and evaporation. Figure 2 shows the effect of the molar ratio of PEG3600-b-PLA3400 to PLGA on both the inner structure of o/w emulsions and the morphology of the resulting polymeric particles that were prepared at 8000 rpm (lower than in the general preparation condition). We focused on the “low-energy-input” (i.e., lowhomogenization-rate) condition (8000 rpm) despite the possibility that unstable formation of particles would occur because we expected that the effects of several preparation factors on the variation of the morphology of the particles would easily appear under such an low-energy-input condition. Prior to this experiment, we hypothesized that the nonporous particles would be obtained even under the low-homogenization-rate condition, as reported in previous conventional reports42,43 involving the evaporation of o/w emulsions. However, amazingly, the porous microparticles were unexpectedly obtained by mixing an organic solvent and water containing hydrophobic polymers at 8000 rpm when the molar ratio of PEG3600-b-PLA3400 to PLGA varied from 10:90 to 90:10. With careful observations, we found that there were a small amount of hardly noticeable pores even when the amount of coexisting block copolymers was low (i.e., when the molar ratio of PEG3600-b-PLA3400 to PLGA was 1:99 and 3:97), as shown by white circles in Figure 2. Apparently, the pores on the surface of the particles increased, as the amount of amphiphilic block copolymers increased. Furthermore, the diameter of the pores increased, reached the saturation value, and gradually decreased, as the amount of the amphiphilic block copolymers increased. These observation results differed from generally reported facts that nonporous42,43 and porous44 particles are generally obtained by means of the evaporation of organic solvents from hydrophobic polymer-containing o/w and w/o/w emulsions, respectively. By carefully investigating the effects of amphiphilic block copolymers on the morphology of emulsions before evaporation of oil phase, we found that w/ o/w emulsions were unexpectedly obtained in one-step emulsification of the mixed solution containing both an organic solvent and water in the presence of block copolymers, as shown in Figure 2. Spontaneous w/o Emulsification in Organic Solvents. To fully understand the mechanism of the unexpected formation of porous particles, it is necessary to address the following question: why were “w/o/w” emulsions unexpectedly and successfully obtained by means of a “one-step” emulsification of an organic solvent and water?

Figure 1. 1H NMR spectrum of the synthesized PEG-b-PLA.

Table 1. Characterization of the Synthesized PEG−PLA Block Copolymers PEG (including methoxy terminus)

a

a

code

Mn

1 2 3 4

3400 3600 3500 9400

GPC.

b1

PLA

PEG−PLA

Mw/Mna

Mnb

a,b

Mn

1.05 1.04 1.05 1.05

5600 3400 1800 3300

9000 7000 5300 12700

Mw/Mna

HLBc

1.22 1.15 1.10 1.06

7.56 10.3 13.2 14.8

H NMR. cGriffin’s method.

the polymers. Previous reports have shown that nonporous polymeric particles having homogeneously smooth surfaces were obtained with42 or without43 coexistent surfactants under “high-energy-input” (i.e., high-homogenization-rate) conditions in the range of approximately 12 000−30 000 rpm. We investigated the effects of the molar ratio of the PEG-b-PLA to PLGA on the morphology of the resulting particles under

Figure 2. Effect of the molar ratio of PEG3600-b-PLA3400 to PLGA on both the inner structure of o/w emulsions and the morphology of the resulting polymeric particles as prepared at 8000 rpm. White circles exhibit hardly noticeable pores (see text). 3331

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Figure 3 shows the time course of the appearance involving the two-phase solutions that were prepared from an organic

Figure 4. Effect of the amount of the amphiphilic block copolymers on the average diameter of the w/o emulsion in the upper organic phase.

Figure 3. Time course of the appearance of the two-phase solutions prepared from an organic solvent and water in the presence of block copolymers under a static condition.

solvent and water in the presence of block copolymers under a static condition. Even though the two-phase solutions were placed statically, three phenomena were observed: (1) the layer of the organic phase at the top of the two-phase solution gradually became clouded as time passed, (2) the white turbidity in the upper organic phase increased as the amount of block copolymers increased, and (3) the upper organic phase did not become clouded in the absence of the block copolymers. We suspect that the cloudy phase (an organic phase) was induced by the formation of the spontaneous w/o emulsion, where droplets of water dispersed spontaneously in the upper organic phase. The white turbidity in the upper organic phase increased as the amount of block copolymers increased because the scattered reflection intensity increased as the number of w/o emulsions increased. These results suggest both that, in general, the amphiphilic block copolymers are necessary compounds for formation of spontaneous emulsion and that, in this particular study, w/o emulsion was easily formed in the upper organic phase when water and an organic solvent were in contact with each other in the presence of amphiphilic block copolymers. Figure 4 shows the effects of the amount of the amphiphilic block copolymers on the average diameter of the spontaneously formed w/o emulsion in the upper organic phase. We found that the average diameter of the w/o emulsion increased, reached the saturation value, and gradually decreased. As shown in Figure 5, the effects of the ratio of PEG-b-PLA to PLGA on the formation of the spontaneously formed w/o emulsion can be explained as follows: (1) the stability of the spontaneously formed w/o emulsion in the organic phase increased as the amount of PEG-b-PLA increased because PEG-b-PLA acts as a surfactant, and (2) the stability of the spontaneous w/o emulsion in the organic phase decreased as the amount of PLGA decreased because the decrease in the PLGA amount presumably reduced the viscosity in the organic phase.

Figure 5. Summarized effect of the ratio of PEG-b-PLA to PLGA on the formation of spontaneous w/o emulsion.

Regarding the combined effects of these two factors, the stability of the spontaneous w/o emulsion decreased, reached the saturation value, and gradually increased, as the ratio of PEG-b-PLA to PLGA increased. Our assumption was consistent with the experimental results shown in Figure 4. Furthermore, the results in Figures 2 and 4 show that the molar ratio of PEG-3600-b-PLA3400 to PLGA exhibit the same effects on both the average diameter of the w/o emulsion in the upper organic phase and the morphology of the polymeric particles. These facts suggest that a spontaneous formation of w/o emulsions is attributable to an unexpected formation of porous particles. Morphology of Unexpectedly Obtained w/o/w Emulsions and of the Resulting Particles. Figure 6 shows the micrograph images of the unexpectedly obtained w/o/w emulsion and SEM images of the resulting particles under various molar ratios of PEG-b-PLA to PLGA at 8000 rpm. We observed what appeared to be unstable and stable w/o/w emulsions when the amphiphilic block copolymers having low and high HLB values were present, respectively. Porous particles were obtained almost in all the experimental conditions (in particular, porous particles were successfully obtained when the amphiphilic block copolymers having high HLB values were used). These results suggest that the composition of the block copolymers did not affect the formation of the spontaneous unexpected w/o emulsions. In addition, as the amount of the amphiphilic block copolymers 3332

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Figure 6. Micrograph images of unexpectedly obtained w/o/w emulsion and SEM images of the resulting particles under various molar ratio of PEG-b-PLA to PLGA.

Figure 7. Micrograph images of the obtained w/o/w emulsions and SEM images of the resulting particles under various preparation conditions.

γ = γ∞ + K /M

increased, both the inner-droplet number of the w/o emulsions and the porosity of the resulting particles increased. These results strongly suggest that the morphology of the spontaneously formed w/o emulsions, w/o/w emulsions, and the resulting particles were possibly controlled by the amount and the composition of PEG-b-PLA having various HLB values. An interfacial tension of the system composed of poly(ethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG−PPO−PEG) block copolymer and poly(vinyl acetate) depended on the molecular weight of PEG.45,46 The amount of the block copolymer that adsorbed on the interface increased as the molecular weight of PEG increased, thereby decreasing the surface tension of the system. The phenomenon was reported to be expressed by the linear equation45

(1)

where γ is a surface tension of the system, γ∞ is a surface tension of the system when M is infinite, and K is a positive or negative constant (e.g., positive in a PEG block copolymer system). The equation shows that a surface tension of the system decreases as the molecular weight of PEG in a block copolymer increased. As shown in Figure 6, we found that both the inner-droplet number of the w/o emulsions and the porosity of the resulting particles increased as HLB values (i.e., a portion of PEG) increased. The phenomenon was attributable presumably to an enhanced formation of spontaneous emulsification due to a decreased surface tension, γ, as expressed by eq 1. 3333

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Figure 8. Summary of the effect of the preparation conditions on the morphology of both the emulsions and the resulting particles.

Figure 7 shows the micrographs of the obtained w/o/w emulsions and SEM images of the resulting particles under various preparation conditions (i.e., the homogenization rate and various molar ratios of PEG3600-b-PLA3400 to PLGA). The diameters of the w/o/w emulsion and the finally obtained particles decreased as the homogenization rate increased. Interestingly, we found that the “o/w” (not the w/o/w) emulsions and the resulting “nonporous” (not porous) particles were obtained when the homogenization rate was 16 000 rpm (that was in the range of the generally reported homogenization rate, approximately 12 000−30 000 rpm42,43). The collapse of the w/o emulsion droplets in the w/o/w emulsions occurred under high-homogenization-rate conditions. Therefore, the stable o/w emulsions were obtained instead of the w/o/w emulsion when the homogenization rate was 16 000 rpm. These results strongly suggest porous polymeric particles were easily obtained only under the “low-energy-input” preparation

conditions; i.e., the homogenization rate was lower than previously (and generally) reported rates. A stochastically maximal diameter of emulsion droplets even in a turbulence, Dmax, is statistically given by the equation47,48 Dmax ∝ ε−2/5(ρ /γ )−3/5

(2)

where ε is an energy density, ρ is a density of a continuous phase, and γ is a surface tension of the system. The equation shows that the diameter of emulsions (thereby resulting particles) decreased as γ decreased and/or ε or ρ increased. As shown in Figure 7, the diameters of the w/o/w emulsion and the finally obtained particles decreased as the homogenization rate increased because increased ε resulted in decreased Dmax. Furthermore, a molecular mobility of highmolecular-weight surfactants (PEG−PLAs) used in this study is lower than that of general low-molecular-weight surfactants so that PEG−PLAs tend to be less adsorbable to an interface of the system. The less adsorbability of PEG−PLA resulted in a 3334

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decreased γ, subsequently increasing Dmax. This suggests that larger emulsions tend to be formed in the presence of polymeric surfactants. Figure 8 summarizes our findings concerning the unexpected formation of the porous particles that were successfully prepared by means of a “one-step” emulsification technique under “low-energy-input” conditions.

advanced spray-dried moxifloxacin and ofloxacin dipalmitoylphosphatidylcholine (DPPC) microparticulate/nanoparticulate powders for pulmonary inhalation aerosol delivery. Int. J. Nanomed. 2013, 8, 3489− 3505. (5) Weber, S.; Zimmer, A.; Pardeike, J. Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) for pulmonary application: A review of the state of the art. Eur. J. Pharm. Biopharm. 2013, DOI: 10.1016/j.ejpb.2013.08.013. (6) Kim, H.; Park, H. T.; Tae, Y. M.; Kong, W. H.; Sung, D. K.; Hwang, B. W.; Kim, K. S.; Kim, Y. K.; Hahn, S. K. Bioimaging and pulmonary applications of self-assembled Flt1 peptide-hyaluronic acid conjugate nanoparticles. Biomaterials 2013, 34, 8478−8490. (7) Smaldone, G.; Berkland, C.; Gonda, I.; Mitchell, J.; Usmani, O. S.; Clark, A. Ask the experts: the benefits and challenges of pulmonary drug delivery. Ther. Delivery 2013, 4, 905−913. (8) Goel, A.; Baboota, S.; Sahni, J. K.; Ali, J. Exploring targeted pulmonary delivery for treatment of lung cancer. Int. J. Pharm. Invest. 2013, 3, 8−14. (9) Ikehara, Y.; Shiuchi, N.; Kabata-Ikehara, S.; Nakanichi, H.; Yokoyama, N.; Takagi, H.; Nagata, T.; Koide, Y.; Kuzushima, K.; Takahashi, T.; Tsujimura, K.; Kojima, K. Effective induction of antitumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cells. Cancer Lett. 2008, 260, 137−145. (10) Bonicelli, M. G.; Giansanti, L.; Ierino, M.; Mancini, G. Interaction of cationic liposomes with cell membrane models. J. Colloid Interface Sci. 2011, 355, 1−8. (11) Guan, T.; Miao, Y.; Xu, L.; Yang, S.; Wang, J.; He, H.; Tang, X.; Cai, C.; Xu, H. Injectable nimodipine-loaded nanoliposomes: preparation, lyophilization and characteristics. Int. J. Pharm. 2011, 410, 180−187. (12) Nasr, M.; Taha, I.; Hathout, R. M. Suitability of liposomal carriers for systemic delivery of risedronate using the pulmonary route. Drug Delivery 2013, 20, 311−318. (13) Murata, M.; Tahara, K.; Takeuchi, H. Real-time in vivo imaging of surface-modified liposomes to evaluate their behavior after pulmonary administration. Eur. J. Pharm. Biopharm. 2013, DOI: 10.1016/j.ejpb.2013.09.006. (14) Kataoka, K.; Harada, A.; Nagasaki, Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Delivery Rev. 2001, 47, 113−131. (15) Kedar, U.; Phutane, P.; Shidhaye, S.; Kadam, V. Advances in polymeric micelles for drug delivery and tumor targeting. Nanomedicine 2010, 6, 714−729. (16) Uchida, Y.; Murakami, Y. Trilayered polymeric micelle: a newly developed macromolecular assembly that can incorporate hydrophilic compounds. Colloids Surf., B 2010, 79, 198−204. (17) Uchida, Y.; Murakami, Y. Successful preferential formation of a novel macromolecular assembly–trilayered polymeric micelle–that can incorporate hydrophilic compounds: the optimization of factors affecting the micelle formation from amphiphilic block copolymers. Colloids Surf., B 2011, 84, 346−353. (18) Uchida, Y.; Fukuda, K.; Murakami, Y. The hydrogel containing a novel vesicle-like soft crosslinker, a “trilayered” polymeric micelle, shows characteristic rheological properties. J. Polym. Sci., Part B: Polym. Phys. 2013, 51, 124−131. (19) Kedar, U.; Phutane, P.; Shidhaye, S.; Kadam, V. Advances in polymeric micelles for drug delivery and tumor targeting. Nanomedicine 2010, 6, 714−729. (20) Patel, B.; Gupta, V.; Ahsan, F. PEG-PLGA based large porous particles for pulmonary delivery of a highly soluble drug, low molecular weight heparin. J. Controlled Release 2012, 162, 310−320. (21) Sinsuebpol, C.; Chatchawalsaisin, J.; Kulvanich, P. Preparation and in vivo absorption evaluation of spray dried powders containing salmon calcitonin loaded chitosan nanoparticles for pulmonary delivery. Drug Des., Dev. Ther. 2013, 28, 861−873. (22) Sinha, B.; Mukherjee, B.; Pattnaik, G. Poly-lactide-co-glycolide nanoparticles containing voriconazole for pulmonary delivery: in vitro and in vivo study. Nanomedicine 2013, 9, 94−104.

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CONCLUSIONS To date, porous particles have been obtained by means of the evaporation of organic solvents from “two-steps”-formed w/o/ w emulsions where hydrophobic polymers dissolve in the oil phase. However, in the present paper, we found that porous particles were unexpectedly and successfully obtained in a “onestep” manner only by mixing an organic solvent and water under “low-energy-input” (i.e., at low-homogenization-rate) conditions with a proper selection of polymeric ingredients. This outcome is attributable to the unexpected formation of the spontaneous formation of “w/o” emulsions in the organic solvent. Based on various SEM images, unfortunately, the obtained particles were polydisperse with diameters ranging to values above 10 μm, which is larger than values suitable for the particles for pulmonary delivery, 1−5 μm. This drawback is resulted from the low-energy-input required to our newly developed one-step process. However, in some cases, we confirmed that the relatively low-disperse porous particles with diameters only in 1−5 μm were obtained (for example, in the case that PEG3600-b-PEG3400:PLGA was 50:50 at 12 000 rpm, as shown in Figure 7). Other necessary works in the future investigations are evaluations of (1) the effect of the block copolymer/hydrophobic polymer molar ratio on the spontaneous formation of w/o emulsion with a statistical difference, (2) pore properties (such as a pore size and a pore volume), and (3) a particle density. Monodisperse particles are necessary in order to achieve these evaluations, and their optimal preparation is now underway. The porous particles might be suitable drug carriers for pulmonary delivery. The results obtained in the present study show that a one-step emulsification can be a powerful and facile technique for the preparation of porous polymeric particles.

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AUTHOR INFORMATION

Corresponding Author

*Fax +81 42 388 7387; e-mail [email protected] (Y.M.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS PEG, poly(ethylene glycol); PLA, polylactide; PLGA, poly(lactide-co-glycolide); o/w, oil-in-water; w/o, water-in-oil; w/ o/w, water-in-oil-in-water.

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REFERENCES

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dx.doi.org/10.1021/la500324j | Langmuir 2014, 30, 3329−3336