Fabrication of microspheres via solvent volatization induced aggregation of self-assembled nanomicellar structures and their use as a pH-dependent drug release system

Article abstract of DOI:10.1021/la303634y

A series of oleamide derivatives, (C₁₈H₃₄NO) ₂(CH₂)_n [n = 2 (1a), 3 (1b), 4 (1c), or 6 (1d); C₁₈H₃₄NO = oleic amide fragment] and (C ₁₈H₃₄NO)(CH₂)₆NH₂ (2), have been synthesized and their self-assembly is investigated in ethanol/water media. Self-assembly of 1a and 1b in ethanol/water (1/0.1 v/v) solution (5 mg mL^-1) yields microspheres (MSs) with the average diameter ～10 μm via a gradual temperature reduction and solvent volatilization process. Under the same self-assembly conditions, microrods (average diameter ～6 μm and several tens of micrometers in length), micronecklace-like, and shape-irregular microparticles are formed from the self-assembly of 1c, 1d, and 2, respectively. The kinetics of evolution for their self-assemblies by dynamic light scattering technique and in situ observation by optical microscopy reveals that the microstructures formation is from a well-behaved aggregation of nanoscale micelles induced by solvent volatilization. The FT-IR and temperature-dependent ¹H-NMR spectra demonstrate the hydrogen bonding force and π-π stacking, which drove the self-assembly of all oleamide derivatives in ethanol/water. Among the fabricated microstructures, the MSs from 1a exhibit the best dispersity, which thus have been used as a scaffold for the in vitro release of doxorubicin. The results demonstrate a pH-sensitive release process, enhanced release specifically at low pH 5.2.

Full text of DOI:10.1021/la303634y

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Article
Fabrication of Microspheres via Solvent Volatization Induced
Aggregation of Self-Assembled Nanomicellar structures
and Their Use as a pH-Dependent Drug Release System
Lidong Zhang, Young-IL Jeong, Sudan Zheng, Hongsuk Suh, Dae Hwan Kang, and Il Kim
Langmuir, Just Accepted Manuscript • DOI: 10.1021/la303634y • Publication Date (Web): 08 Dec 2012
Downloaded from http://pubs.acs.org on December 11, 2012
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Fabrication of Microspheres via Solvent Volatization
Induced Aggregation of Self-Assembled
Nanomicellar structures and Their Use as a pH-
Dependent Drug Release System
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Lidong Zhang, YoungꢀIl Jeong, Sudan Zheng, Hongsuk Suh, Dae Hwan Kang, Il Kim *
a
The WCU Center for Synthetic Polymer Bioconjugate Hybrid Materials, Department of Polymer
Science and Engineering, Pusan National University, Pusan 609 735, Korea. Fax: (+82) 51ꢀ513ꢀ
720; Tel: (+82) 51 510 2466; *Eꢀmail: ilkim@pusan.ac.kr
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b
National Research and Development Center for Hepatobiliary Cancer, Pusan National University,
Yangsan Hospital, Yangsan 626ꢀ870, Korea
c
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National
University, Pusan 609 735, Korea.
Eꢀmail: hssuh@pusan.ac.kr
ABSTRACT: A series of oleamide derivatives, (C H NO) (CH ) [n = 2 (1a), 3 (1b), 4 (1c), or 6
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2 n
(
1d); C H NO = oleic amide fragment] and (C H NO)(CH ) NH (2), have been synthesized and
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their self–assembly is investigated in ethanol/water media. Selfꢀassembly of 1a and 1b in ethanol/water
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1/0.1 v/v) solution (5 mg mL ) yields microspheres (MSs) with the average diameter ∼10 ꢁm via a
gradual temperature reduction and solvent volatilization process. Under the same selfꢀassembly
conditions, microrods (average diameter ∼6 ꢁm and several tens of micrometers in length), microꢀ
necklaceꢀlike and shapeꢀirregular microparticles are formed from the selfꢀassembly of 1c, 1d and 2,
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respectively. The kinetics of evolution for their selfꢀassemblies by dynamic light scattering technique
and in situ observation by optical microscopy reveals that the microstructures formation is from a wellꢀ
behaved aggregation of nanoscale micelles induced by solvent volatilization. The FT–IR and
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temperature–dependent H–NMR spectra demonstrate the hydrogen bonding force and π–π stacking,
which drove the selfꢀassembly of all oleamide derivatives in ethanol/water. Among the fabricated
microstructures, the MSs from 1a exhibit the best dispersity, which thus have been used as a scaffold for
the in vitro release of doxorubicin. The results demonstrate a pHꢀsensitive release process, enhanced
release specifically at low pH 5.2.
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Keywords: selfꢀassembly; oleamide derivatives; microstructures; doxorubicin; controlled release
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INTRODUCTION
Microspheres with hollow or porous structures have enhancive applications in targeted delivery,
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–4
energy storage, catalysis, and artificial cells. The simple and efficient protocol for the immobilization
of active ingredients such as dyes, inks, cells, drugs, and proteins into the interior cavities is a key factor
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for the achievement of these promising applications. Generally, if the structures such as porous
spheres possessed good dispersity, proper size and ideal surface permeability, an easy access for guest
materials into the interior of the materials can be achieved with higher feasibility. For example,
monodisperse systems permit the reliable and precise dosage of drugs, while carriers < 1 ꢁm in diameter
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avoid capillary blockage or filtration, leading to effective uptake by cells. This size range can also
exploit the leaky nature of tumor blood vessels (380–780 nm), providing a means for uptake by cancer
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,9
cells in vivo. A recent study demonstrates that bacterially drived minicells of size ∼400 nm can be
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effectively used for targeting cancer cells. Larger particles (up to ∼5 ꢁm in diameter) have also been
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administered intratumorally and subcutaneously for cancer drugꢀdelivery applications.
Up to now,
several effective strategies, such as emulsion process, surfaceꢀprotected etching, supramolecular selfꢀ
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assembly, as well as the replication method, have been developed to prepare hollow spheres with porous
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shells.
However, controlling the spheres size, dispersity and surface character for restraining burst
release of the active ingredients has proved to be still a hard work.
The surface characteristics are of paramount importance in determining the dispersion behavior and
surfaceꢀchemical function of microspheres (MSs) in various media and thus their practical applications.
The surface characteristics of MSs are mainly determined by their chemical compositions. With specific
functional groups, the surface layer molecules can endow the MSs with improved physical and chemical
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properties (e.g., superhydrophobicity, superhydrophilicity, and amphiphilicity).
However, the asꢀ
obtained porous spheres are usually not provided with the desired characteristics, and further surface
functionalization is often needed. Generally, control of the surface functionality of MSs can be realized
through two main approaches. The first one, especially for polymer MSs, is to incorporate reactive
block comonomers with desired functional groups into the polymer chains during the polymerization
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process. The presence of these reactive comonomers on MS surfaces can alter the surface
characteristics. Another commonly applied approach is chemical modification of the MS surfaces by a
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postꢀtreatment process.
By taking advantage of covalent bonds and hydrogenꢀbonding interactions,
functional agents can anchor onto the surfaces of preformed MSs. However, additional chemical
modification of these structures makes the synthesis procedure multistep and troublesome. Therefore, a
direct and efficient synthesis of MSs is still a challenge, especially when customized surface properties
are expected.
In this work, we present a selfꢀassembly method for direct fabrication of various microstructures
based on a series of supramolecular oleamide derivatives (OADs) (Figure 1) containing two oleamide
chains bridged by methylene chains with different lengths (1a–1d), or one oleamide chain bridged with
(
CH ) ꢀamine (2). The oleic acid was chosen as starting material for designing intended compounds,
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because the oleic acid has acceptable biocompatibility, that is a monoꢀunsaturated omega–9 fatty acid in
various animals and vegetables, and often as an excipient in pharmaceuticals and an emulsifying or
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solubilizing agent in aerosol products. Through a simple modification to oleic acid in hydrophilic
group by diamines, new amphiphilic molecules were obtained with great transformation in solubility
(increase in organic solvents) and configuration (oil to solid). It has been reported that synthetic
amphiphiles with cyclic sugars as hydrophilic moieties of oleic acid derivatives are easy to produce
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chiral supramolecular fibers and ribbons by selfꢀassembly in aqueous media,
and the nanostructures
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can also be generated through a direct selfꢀassembly of the oleic acid on a hydroxylated aluminum
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surface. In our system, the introduction of hydrophilic moieties allows creating microspherical or rodꢀ
like morphologies with highly rough surface structures in ethanol/water media. As far as we know, it is
a new result for the selfꢀassembly of oleamide derivatives into microspherical structures. Compared to
the small micelles coalescing to microspheres by the spontaneous selfꢀassembly of waterꢀsoluble
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nucleotideꢀcalixarene conjugates, the microstructures fabricated by OADs possess highly rough
surfaces, which may be advantageous for the applications, especially in controlled drug delivery, since
drugs, proteins or other biomolecules can be more readily incorporated in microspheres with rough
surface so that further modifications are not needed for effective loading. In order to check this point
MSs fabricated by 1a are tried to utilize as a scaffold of doxorubicin (DOX) and the controlled release
behavior is investigated in vitro under various pH conditions. The processes are also observed by
confocal laser scanning microscopy (CLSM) analysis.
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Figure 1. Molecular structures of the oleamide amphiphiles 1a–1d and 2.
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2. MATERIALS AND METHODS
2
.1 General Methods
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Melting point: CTPꢀMP 300 hotꢀplate apparatus with ASTM 2C thermometer. H NMR: Varian
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Mercury 400 spectrometers (400 MHz). C NMR: Varian Mercury 400 spectrometers (100 MHz).
NMR spectra were determined in CDCl and chemical shifts are expressed in parts per million (δ)
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relative to the central peak of the solvent. UVꢀvis absorption spectra were obtained on UVꢀ1800
Shimadzu UVꢀvis spectrometer. Microanalyses were performed on a Carlo Erba Fisons EA 1108
analyzer in the Korea Basic Science Institute of Busan. Fast atom bombardment (FAB) mass spectra
were obtained using a MSꢀ9 VG updated or ZABꢀHF (VG Analytical U.K.) mass spectrometer with a
commercial FAB source. Ions were produced by a primary beam of xenon atoms of 8 keV, extracted
and accelerated with an 8 kV potential.
The surface structures of the selfꢀassembled samples were investigated using scanning electron
microscope (SEM) on an Sꢀ4800 (Hitachi, Tokyo, Japan). A drop of the sample (0.1 mg) in
ethanol/water (1 mL) was applied onto a clean silicon wafer and then slowly dried in air for the SEM
observation. The size of the nanoparticles was determined by dynamic light scattering (DLS) using a
Zetasizer Nano ZS series (Malvern Instruments) and SEM technique together with ImageJ 2.1.4.6 image
analysis software.
For the release studies Cellu•Sep® T3 tubings [flat width = 25 mm, diameter (dry) = 15.9 mm, and
MWCO 12000–14000 Da], purchased from Membrane Filtration Products, Inc., were used. The release
experiment was carried out in vitro as follows. 10 mg of MSs (obtained from the selfꢀassembly of 1a in
ethanol/water in the ratio of 1/0.1) incorporating around 1.2 mg of DOX were reconstituted in 5 mL of
phosphateꢀbuffered saline (PBS) (0.1 M, pH (7.2. 8.0, 5.2 respectively)), and the ensuing solution was
introduced into the dialysis membrane. Next, the dialysis membrane was placed in a 200 mL bottle with
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5 mL of PBS. This bottle was placed in a shaking incubator with a stirring speed of 100 rpm at 37 °C.
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At specific time intervals, the media were sampled for analysis of drug concentration. Afterwards, the
whole media were replaced with fresh PBS to prevent drug saturation. The concentration of DOX
released into the PBS was measured using the UVꢀvis spectrometer at 483 nm.
2
.2 Materials
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Oleic acid (99%), thionyl chloride (99%), pyridine (99.5%), ethylenediamine (EDA; 99%), 1,3–
diaminopropane (99%), 1,4–diaminobutane (99%), and hexamethylenediamine (99%) were obtained
from Sigma Aldrich Chemical Company (St Louis, MO), and all of them were used without further
purification. 1,2–Dichloroethane (DCE), dimethylformamide (DMF), tetrahydrofuran (THF), toluene
and ethanol were distilled before using. Dextran from Leuconostoc spp (average molecular weight
approximately 6000), triethylamine, DOX, hexamethylene diamine, and thiazolyl blue tetrazolium
bromide (MTT) were also purchased from Sigma Chemical Company.
2
.3 Synthesis of oleamide derivatives
The monochain derivatives of oleic acid (1a–1d and 2 in Figure 1) were synthesized starting from
36
oleoyl chloride prepared by the chlorination of oleic acid with thionyl chloride. For example, for the
preparation of N,N'–ethylenebis(oleamide) (1a), 1.2 g (0.02 mol) of EDA was mixed with 0.79 g (0.01
mol) of pyridine in CHCl (20 mL) for 10 min in a 100–mL round bottom flask at room temperature.
3
After transferring the mixture to the ice bath, 3.0 g (0.01 mol) of oleoyl chloride dissolved in purified
CHCl (10 mL) was added dropwise for 20 min so that the temperature of the reaction mixture cannot
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exceed 5 °C. The resulting solution was stirred for 5 h at room temperature and then poured to ice–water
to remove pyridine salt. The white solid obtained by filtration was washed three times by distilled water
and dried under vacuum for 24 h at 50 °C. 1a: Colorless solid; yield 95%. mp 110 – 113 °C; IR (KBr)
ꢀ1
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3
0
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300, 2936, 2841, 1643, 1563, 1468, 1238, 1123, 940, 722 cm ; H NMR (400 MHz, CDCl ) δ 0.65ꢀ
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.78 (m, 6H), 1.12ꢀ1.20 (m, 40H), 1.45 (m, 4H), 1.80ꢀ1.98 (m, 8H), 2.05ꢀ2.19 (m, 4H), 3.20ꢀ3.41 (s,
1
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H), 5.20ꢀ5.38 (m, 4H), 6.2 (s, 2H); C NMR (100 MHz, CDCl ) δ 174.53, 129.93, 129.62, 39.98,
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6.59, 31.85, 29.71, 29.66, 29.61, 29.48, 29.36, 29.26, 29.14, 27.17, 27.15, 25.71, 22.62, 14.05; Anal
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Calcd for C H N O : C, 77.49; H, 12.32; N, 4.76. Found C, 77.33; H, 12.80; N, 4.92; FabꢀMS m/z (%)
3
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72
2
2
+
590.20 (100) [M + H] .
N,N'ꢀ1,3ꢀpropylenebis(oleamide) (1b). Colorless solid; yield 85%. mp 80 – 83 °C; IR (KBr) 3301,
ꢀ1 1
2
1
927, 1652, 1576, 1475, 1367, 1310, 1153, 731 cm ; H NMR (400 MHz, CDCl ) δ 0.64ꢀ0.89 (m, 6H),
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.23ꢀ1.30 (m, 40H), 1.46 (m, 4H), 1.80ꢀ1.98 (m, 10H), 2.05ꢀ2.20 (m, 4H), 3.15ꢀ3.31 (s, 4H), 5.20ꢀ5.40
13
(m, 4H), 6.4 (s, 2H); C NMR (100 MHz, CDCl ) δ174.00, 129.92, 129.67, 36.83, 35.55, 31.85, 29.72,
3
29.67, 29.47, 29.26, 29.23, 29.12, 27.17, 25.82, 22.63, 14.06; Anal Calcd for C H N O : C, 77.68; H,
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2
+
1
3.37; N, 5.32; Found C, 77.52; H, 13.82; N, 5.87; FabꢀMS m/z (%) 603.58 (100) [M + H] .
N,N'ꢀ1,4ꢀbutylenebis(oleamide) (1c). Colorless solid; yield 82%. mp 111 – 114 °C; IR (KBr) 3319,
ꢀ1 1
2922, 2849, 1643, 1541, 1474, 1421, 1263, 1194, 954 cm ; H NMR (400 MHz, CDCl ) δ 0.79 (s, 6H),
3
1
.19ꢀ1.22 (m, 38H), 1.44 (m, 4H), 1.45ꢀ1.53 (m, 4H), 1.90ꢀ1.93 (m, 8H), 2.07ꢀ2.11 (m, 4H), 3.16ꢀ3.17
1
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(s, 4H), 5.24ꢀ5.29 (m, 4H), 6.41 (s, 2H); C NMR (100 MHz, CDCl ) δ 173.60, 129.87, 129.59, 38.95,
3
36.62, 31.82, 29.68, 29.44, 29.28, 29.26, 29.23, 29.12, 27.14, 27.11, 26.87, 25.81, 22.59, 14.02; Anal
Calcd for C H N O : C, 77.86; H, 12.41; N, 4.54. Found C, 77.56; H, 12.90; N, 4.88; FabꢀMS m/z (%)
4
0
76
2
2
+
6
17.60 (100) [M + H] .
N,N'ꢀ1,6ꢀhexylenebis(oleamide) (1d). Colorless solid; yield 85%. mp 113 – 115 °C; IR (KBr) 3318,
ꢀ1
1
2
929, 2850, 1635, 1532, 1469, 1422, 1264, 1214, 691 cm ; H NMR (400 MHz, CDCl ) δ 0.79ꢀ0.83
3
(m, 6H), 1.19ꢀ1.26 (m, 44H), 1.42 (m, 4H), 1.54 (m, 4H), 1.92ꢀ1.94 (m, 8H), 2.08ꢀ2.11 (m, 4H), 3.12ꢀ
1
3
3
.16 (s, 4H), 5.25ꢀ5.30 (m, 4H), 6.10 (s, 2H); C NMR (100 MHz, CDCl ) δ 173.35, 129.88, 129.62,
3
3
8.85, 36.69, 31.83, 29.69, 29.66, 29.63, 29.58, 29.45, 29.40, 29.31, 29.24, 29.12, 27.14, 25.81, 22.60,
14.03; Anal Calcd for C H N O : C, 78.20; H, 12.50; N, 4.34. Found C, 78.45; H, 12.83; N, 4.68; Fabꢀ
4
2
80
2
2
+
MS m/z (%) 645.63 (100) [M + H] .
Nꢀ6ꢀaminohexyl(oleamide) (2). Colorless solid; yield 75%. mp 90 – 93 °C; IR (KBr) 3319, 3308,
ꢀ1 1
2
942, 2930, 1649, 1563, 1471, 1425, 1284, 1193, 875 cm ; H NMR (400 MHz, CDCl ) δ 0.85ꢀ0.90
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(
m, 3H), 1.15ꢀ1.26 (m, 24H), 1.50ꢀ1.52 (m, 6H), 1.82ꢀ1.98 (m, 4H), 2.15ꢀ2.19 (m, 2H), 2.56ꢀ2.63 (m,
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H), 3.20ꢀ3.23 (m, 2H), 5.30ꢀ5.31 (m, 2H); C NMR (100 MHz, CDCl ) 171.23, 129.86, 129.56, 38.83,
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36.65, 31.84, 29.58, 29.53, 29.24, 25.83, 22.63, 14.11; Anal Calcd for C H N O: C, 75.73; H, 12.71;
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+
N, 7.36. Found C, 75.80; H, 12.98; N, 7.87. FabꢀMS m/z (%) 381.60 (100) [M + H] .
2
.4 Self-assembling experiment
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The selfꢀassembly of OADs was performed via a gradual temperature reduction and solvent
−1
volatilization process. A suspension of OAD in ethanol/water (concentration: 5 mg mL ) in a 10–mL
vial was heated to reflux (∼70 °C) until all the solids were completely dissolved to form a colorless,
clear solution. The solution was allowed to cool gradually to room temperature. The sealed vial
containing the selfꢀassembled sample was then opened to the air for the concentration of the sample by
evaporation in a static condition, where the microstructures were produced by the aggregation of
nanoscale micelles.
2
.5 Cytotoxicity assay of empty MSs from 1a to human embryonic kidney 293T cells and HuCC-
T1 cells line
2
93T cells were maintained in RPMIꢀ1640 medium supplemented with 10% fetal bovine serum (5%
CO at 37 °C). The viability of 293T cell line derived from human embryonic kidney cells was
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evaluated by thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay. 1×10 cells seeded in
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6–well plates with 100 ꢁL of medium were incubated overnight in a CO incubator (5% CO at 37 °C)
2 2
and then the media was exchanged with 100 ꢁL of serum–free RPMI1640 media. For the cytotoxicity
test, MSs from 1a were distributed in serum–free RPMI1640 media and diluted to appropriate
ꢀ1
concentrations. After 3 days of incubation, 30 ꢁL of MTT (5 mg mL ) was added in 96–well plates and
incubated for 4 h. The formazan crystals formed were dissolved in DMSO, and the absorbance (560 nmꢀ
test/630 nmꢀreference) was determined using an automated computerꢀlinked microꢀplate reader
(Molecular Device Co., San Diego, CA, U.S.A.). The results were expressed as a percentage of
absorbance compared to that in the control cells. Each measurement was obtained as the mean value of
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eight wells.
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The viability of a human cholangiocellular carcinoma cell line, HuCCꢀT1, was also evaluated by
employing similar method.
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. RESULTS AND DISCUSSION
.1 Synthesis and Self-assembly of OADs
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The oleic acid and diamines were firstly purified to remove impurities like moisture. The oleic acid
36
was then reacted with excess thionyl chloride to produce oleoyl chloride. Further condensation
reactions of oleoyl chloride with various diamine compounds containing different methylene groups in
pyridine/CHCl mixture gave oleamide amphiphiles 1a–1d in high yield (>82%). For comparison of
3
different selfꢀassembling behaviors, monochain compound 2 was also synthesized.
Amphiphilic OADs 1a–1d of the current study have a central soft alkyl segment, two secondary
amide functional groups, and two flexible hydrocarbon chains with one double bond. The unique
structural feature of OADs makes them an ideal candidate for selfꢀassembling through directed
intermolecular hydrogen bonds and π–π stacking. The double bond segment has a strong tendency to
aggregate through π–π overlap. The structural feature allows OADs to form a supramolecular ordered
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3,24
structure through intermolecular translationꢀrelated hydrogen bonding.
The π–π interaction and the
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intermolecular hydrogen bonds might have a synergistic effect on the formation of stable aggregations.
Theoretical packing structures for all OADs are shown in Figure 2 calculated and predicted using
Polymorph Predictor of Materials Studio 4.3, and the detailed calculation and discussion processes can
be found in supporting information (SI). The predicted structures in Figure 2(f–J) indicate obvious
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intermolecular hydrogen bonds and π–π stacking (red broken line). Some practical measurements such
1
as FT–IR and H–NMR spectra have also been provided for understanding the intermolecular
interaction and packing order (vide infra).
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Figure 2. The minimum energy structures of amphiphilic oleamide derivatives, 1a (a), 1b (b), 1c (c), 1d
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(d) and 2 (e), obtained using a GGA/PBE/DNP level of theory with the DMol DFT code of Materials
Studio 4.3, and the predicted packing orders of them, 1a (f), 1b (g), 1c (h), 1d (i) and 2 (j), calculated
using Polymorph Predictor of Materials Studio 4.3 (see SI).
The selfꢀassembling conditions (solvent, temperature and kinetics) and the hydrophilic moieties also
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strongly influence the aggregation behavior of OADs.
When the oleic acid was modified by
glucopyranosylamine to form a glucopyranosylamide lipid, the selfꢀassembly of this lipid in water
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allows generating a tubular structure. Similarly, by changing the hydrophilic moieties to Dꢀglucose,
phenolic glycose or phenyl glucose, nanofibers or nanotubles could be produced via a selfꢀassembling
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2–45
process respectively in water.
In our study, ethanol/water mixture instead of pure water was used as
a solvent for the selfꢀassembly of OADs. A class of new morphology of the microspherical structure
with highly rough surface structures was obtained, which is completely different from the selfꢀ
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assembled structures based on other OADs reported previously.
It was found that the alteration of
the ratio of ethanol to water has a huge influence on the size, surface structure and dispersity of the selfꢀ
assembling microsphere morphologies.
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The selfꢀassembly processes can be separated into two stages: i.e. the temperatureꢀreductionꢀinduced
nucleation into nanoscale micelles, followed by the solventꢀvolatizationꢀinduced growth into
microstructures. In the first stage, the kinetics of evolution for the temperatureꢀreductionꢀinduced selfꢀ
assembly of 1a was studied by DLS (Figure 3a). Initially formed micelles around 100 nm in diameter at
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nm at 20 °C. It means that the main aggregation growth of 1a into microstructures takes place in the
second stage. For the demonstration of the solventꢀvolatizationꢀinduced growth, we traced this process
with SEM technique. After performing the temperatureꢀreduction process, the selfꢀassembling system
was opened to the air for solvent volatilization at room temperature. At specific time intervals, the
solution was taken for SEM observation and analyzed by ImageJ software. An obvious aggregation
process happened at the range of 40 to 20% solvent remained, since there is an obvious increase in the
diameter of microstructures (Figure 3b), indicating the solvent volatilization induces the aggregation of
preꢀformed micelles. After complete solvent evaporation, the microstructures grow to ~10 ꢁm in
diameter. Moreover, the in situ observation of the microstructure formation of 1a under an optical
microscopy displays that plenty of small particles (~1 ꢁm in diameter) can be observed after about one
minute has been passed since a drop of solution applied on glass sheet. As the time goes by, the small
particles start to rapidly aggregate to bigger ones induced by solvent volatilization (the video not shown
here).
Figure 3. (a) Effect of solution temperature decrease on the size of nanoparticles formed by the selfꢀ
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assembly of 1a and (b) effect of solvent volatilization on the size of formed microspheres.
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The detailed surface structures of selfꢀassembled 1a were studied by SEM observation in Figure 4,
demonstrating a wellꢀdefined MSs with tunability in size, surface characteristics and dispersity. A small
increase in the water fraction of the selfꢀassembling media results in a decrease in size of formed MSs
from 1a (named MS1a). For getting more insights into the water effect, the MS1a selfꢀassembled in six
different ethanol/water ratios were collected after natural solvent volatilization and then were analyzed
by SEM and ImageJ software. As shown in Figure 5, the average diameter of MS1a decreases with
increasing water fraction, probably because relatively slower evaporation of water makes the
aggregation growth of micelles slow. We have previously reported that the selfꢀassembly of the
amphiphilic monoꢀchain derivatives of stearic acid in pure 1,2ꢀdichloroethane yields MSs with the
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diameter up to around 40 ꢁm, most probably due to the fast evaporation of 1,2ꢀdichloroethane.
Employing pure water for the selfꢀassembly of various amphiphilic monoꢀchain derivatives, the
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nanostructures were observed instead of microstructures. These results prove that solvents with high
volatility such as ethanol and 1,2ꢀdichloroethane provide the compounds with better environment for
molecular aggregation into microstructures. However, the dispersity of MS1a became better by adding a
small amount of water in selfꢀassembling media (compare Figure 4a with 4b), since the conglutination
phenomenon is restricted in ethanol/water media. The surface of the MS1a also becomes rougher with
the addition of water to ethanol, especially, under the condition of Vethanol/Vwater = 1/0.6 (Figure 4f).
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Figure 4. SEM images of the selfꢀassembled products from 1a at a concentration of 5 mg mL .
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Figure 5. Effect of ethanol to water ratio on the dispersity of microspheres from 1a at a concentration of
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Under the optimal selfꢀassembly condition of 1a, the selfꢀassembling behaviors of 1b, 1c, 1d and 2
were also investigated. The concentration of oleamide compounds and the ratio of ethanol to water were
−1
fixed at 5 mg mL and 1/0.1, respectively. The compound 1b yields very uniform MSs with the average
diameter ∼10 ꢁm (Figure 6a and 6b). Note that compound 1a has 2 methylene groups between 2 amide
groups and the compound 1b has 3 methylene groups between them. Due to this structural similarity
both compounds yields MSs with uniform size, even though MSs by 1b reveals denser surface than that
MSs by 1a. In case of the compound 1c, with 4 methylenes in the hydrophilic head, completely different
selfꢀassembly behavior has been observed. Instead of forming MSs, microrods with the average
diameter ∼6 ꢁm and several tens of micrometers in length were generated, which were probably formed
by the aggregation of initially generated MSs in one direction [see Figure 6(c–f)]. Changing the
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concentration of 1c and the ratio of ethanol to water, wellꢀdispersed and uniform MSs could not be
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compound concentration of 5 mg mL : (a, b) from 1b and (c–f) from 1c.
In order to get deeper insight into the effect of the methylene chain length (flexibility) in hydrophilic
moieties of OADs on selfꢀassembly in ethanol/water media, selfꢀassembly of 1d with six methylenes in
the hydrophilic head has also been investigated at the same conditions. Different from the selfꢀassembly
of 1c, 1d yields microꢀnecklaceꢀlike structures. Figure 7a shows a wellꢀordered array of such microꢀ
necklaces from 1d. Based on the detailed observation by SEM in Figure 7b and 7c, the necklaceꢀlike
morphology was spontaneously assembled by interconnecting MSs each other, meaning that the selfꢀ
assembly of 1d was first to give MSs and then further solvent volatilization drived the MSs to arrange
and link one another. The selfꢀassembling results of 1c and 1d demonstrate that the monodisperse
microspheres structure is limited as the flexibility of hydrophilic head increases by increasing the
methylene chain length.
To further investigate the effect of hydrophilic head group on the selfꢀassembly behavior, we have
synthesized monochain compound 2, in which one of the primary amines in hexaneꢀ1,6ꢀdiamine was
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substituted by oleic acyl group. The selfꢀassembly was carried out under the same conditions employed
in the selfꢀassembly of 1d. As confirmed by SEM images in Figure 7e and 7f, the microparticle
structures are predominantly observed and the conglutination behaviors among initially generated
particles were still existed; however, uniform microrodꢀlike or microspherical structures are not formed.
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compound concentration of 5 mg mL : (a–d) from 1d and (e, f) from 2.
Based on the results above, a possible mechanism is proposed for the formation of various
microstructures as shown in Figure 8. Because the OAD has a special structure such as a central soft
alkyl segment with two secondary amide functional groups which are hydrophilic, and two flexible
alkyl chains with double bond which are hydrophobic, the unique structural feature allows OAD to form
a supramolecular ordered structure through intermolecular translationꢀrelated hydrogen bonding (see
23,24,36
also Figure 2).
Therefore, the formation process of microstructures can be considered as headꢀtoꢀ
head jointing of molecules and tailꢀtailꢀtail packing to aggregate into micellar structure during the
temperatureꢀreduction. Succeeding solvent volatilization drives the formed micelles to continuously
congregate as a specific manner according to the type of molecular structure. Accordingly, it can be
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speculated that the MSs from the selfꢀassembly of 1a and 1b were generated through a layerꢀbyꢀlayer
aggregation of micelles by an anisotropic micelles assembling process, while in case of 1c, micelles
assembly mainly takes place in a coaxial direction to create rodꢀlike structure. Interestingly, the microꢀ
necklaces are not spontaneously assembled from 1d micelles, alternatively, the micelles are firstly
aggregated to microparticles structure, and then they selfꢀassemble in a coaxial direction. During the
selfꢀassembly of compound 2, solvent volatilization drives the micelles to form anomalous
microparticles, followed by continuous linkage among them to form bigger microparticles.
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Figure 8. The plausible procedures of the formation of microstructures by the selfꢀassembly of various
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H–NMR and FT–IR spectra investigation
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For demonstrating the hydrogen bonding interaction and π–π stacking in the selfꢀassembly, the 1a H
spectra were measured via a temperatureꢀdependent process in D ꢀmethanol solution. The initial
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temperature for H–NMR measurement of 1a solution was 70 ºC, and next measurements were carried
out as the temperature decreased from 70 to 20 ºC (Figure 2). The D ꢀmethanol was employed as
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solvent, since the selfꢀassembling behavior of 1a in D ꢀmethanol was similar to that in ethanol/water,
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also giving microspherical structures as shown in Figure S1(in SI).
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Seen from the full view of the H spectra of 1a in Figure S2 measured at different temperatures, the
chemical shifts for 1a vary strongly with the temperature decrease, especially when compared to the
initial temperature dependence observed for the corresponding resonances in D ꢀmethanol. The Figure
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a shows the chemical shift change of H /H with detailed data in Table 1, where the chemical shift
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value was around 5.300 ppm at 70 ºC, shifting to 5.117 ppm while the temperature was decreased to 20
ºC, and indicating possible interaction through πꢀπ stacking took place at double bond position. Another
obvious chemical shift change was found in H as shown in Figure 9b, where the chemical shift shifted
c
from 3.242 ppm at 70 ºC to 3.039 ppm at 20 ºC (Table 1). Similar phenomenon on chemical shift
variation was also found in H , H and H positions (Figure 9c and Table 1). The results above indicate
d
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that the intermolecular/intramolecular interactions such as hydrogen bonding and πꢀπ stacking drive the
1
selfꢀassembly of 1a in deed. Unfortunately, the signals indicating the H chemical shift variation of NꢀH
1
framework was not appeared in 1a HꢀNMR spectra. Since the intermolecular/intramolecular hydrogen
bonding interaction via NH frameworks is of great significance for the selfꢀassembling behaviors,
instead, the FTꢀIR spectra (Figure 10) were used to demonstrate the existence of its hydrogen bonding
interaction with detailed descriptions given below.
1
Figure 9. (Magnified view) Temperature dependence of the 1a H spectrum in D ꢀmethanol solution,
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indicating greatly chemical shifts variation as the temperature decreased from 70 to 20 ºC.
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1
1
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2
2
2
2
2
2
2
2
2
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3
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3
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Table 1. Chemical shift values of H to H for compound 1a at appointed temperatures.
a
f
Temperature
T (ºC)
Chemical shift values δ (ppm)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H /H
a
Hc
Hd
He
Hf
b
7
6
5
4
30
20
0
0
0
0
5.300
5.275
5.274
5.245
5.203
5.117
3.242
3.214
3.212
3.181
3.137
3.039
2.131
2.110
2.105
2.079
2.039
1.947
1.988
1.968
1.967
1.943
1.897
1.811
0.857
0.827
0.822
0.801
0.785
0.675
1
The Hꢀchemical shifts variation shows a nonꢀlinear behavior:
ꢀ5
2
2
H /H : δ [ppm] = ꢀ8.3751x10 T + 0.0109 T + 4.9414; (R = 0.9876)
a
b
ꢀ5
2
2
H : δ [ppm] = ꢀ9.2501x10 T + 0.0120 T + 2.8463; (R = 0.9859)
c
ꢀ5
2
2
H : δ [ppm] = ꢀ8.8393x10 T + 0.0113 T + 1.7663; (R = 0.9868)
d
ꢀ5
2
2
H : δ [ppm] = ꢀ9.1071x10 T + 0.0114 T + 1.6269; (R = 0.9881)
e
ꢀ5
2
2
H : δ [ppm] = ꢀ7.9286x10 T + 0.0102 T + 0.5212; (R = 0.9536)
f
For a stable structure in solution, the hydrogen bond donor protons caused chemical shift variation
can often be interpreted in terms of (intraꢀ or intermolecular) hydrogen bond dynamics, which typically
provide a positive dependence with temperature, while the high temperature coefficients was observed
46
in selfꢀassembling system. Since the selfꢀassembled aggregation of 1a in D ꢀmethanol was driven by
4
πꢀπ stacking and hydrogen bonding force as discussed above, the similar interaction force was also
deemed to exist in the selfꢀassembly of 1a in ethanol/water and the packing structure.
The FTꢀIR spectroscopy as a powerful tool for investigating hydrogen bonding interaction has been
proverbially applied in the analysis of morphology of selfꢀassembling organic compounds. According to
the FTꢀIR spectroscopy, the characteristic IR signals of the 1a and 1d show blue shift after assembling
into special morphologies. As an example, Figure 10a shows FTꢀIR spectra of 1a before and after selfꢀ
assembly and Table 2 summarized the results of assignments, which demonstrate that the peaks at 3300
ꢀ1
and 1563 cm for 1a before selfꢀassembly that are assigned to ν (NHCO) and ν(NH) stretching, shift to
as
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ꢀ1
ꢀ1
3
294 and 1556 cm respectively, for the selfꢀassembled microspheres. Especially, the peak at 722 cm
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for 1a before selfꢀassembly assigned to C–H stretching vibrations of double bond (C=C–H) shifts to 714
ꢀ1
cm for the selfꢀassembled morphologies, indicating the double bond of 1a has participated in the selfꢀ
assembling process via π–π stacking and other weak interactions. The similar blue shifts have also been
observed from the absorption bands of the asymmetric (υ ) and symmetric (υ ) ꢀCH stretching
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
as
s
2
3
6
vibrations, indicating the presence of an allꢀtransꢀzigzag form alkyl chain, which can be explained by
that the frequencies of the alkyl chain antisymmetric and symmetric stretching bands are very sensitive
to the conformation of the hydrocarbon chain. The eigenvalue of the carboxyl group also shifts from
ꢀ1
ꢀ1
1
643 cm to 1635 cm . These results testify the existence of strong hydrogenꢀbonding interactions
between O=C and HꢀN as well as NꢀH and NꢀH bonding which play critical role in the formation of
selfꢀassembled nanomicellar structures and highly ordered microsphere aggregate. Therefore, it
becomes clear that the selfꢀassembly of 1a occurs by means of hydrogenꢀbonding between the primaryꢀ
amino/secondaryꢀamino groups and π–π stacking between double bonds, even though van der Waals
interaction, coordination interaction, and other weak interactions cannot be neglected completely. The
similar results have also been found in FTꢀIR spectra of 1d in Figure 10b and Table 2, further revealing
the existence of hydrogen bonding force and π–π stacking in the selfꢀassembling process.
Figure 10. The IR spectra of 1a and 1d before and after selfꢀassembly.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Table 2. The FTꢀIR band assignments of 1a and 1d before and after selfꢀassembly.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
Absorption peak of sample (cm )
Compounds
Assignments
Before
After
selfꢀassembly
selfꢀassembly
 ν (NHCO)
3300
2936
2841
1643
1563
1468
722
3294
2928
2840
1635
1556
1460
714
as
 ν (CH )
as
2
1
a
 δ
ν(C=O)
ν(NH), ν(CN)
s 2
(CH )



δ(CH2)
δ(C=CꢀH)

νas(NHCO)
3318
2929
2850
1635
1532
1469
691
3310
2921
2849
1627
1531
1452
682

ν (CH )
as
2
1
d
 δ
ν(C=O)
 ν(NH), ν(CN)
δ(CH2)
δ(C=CꢀH)
(CH )
s 2


3
.3 Doxorubicin release from MS1a in vitro
Microspherical structures from selfꢀassembly of low mass molecules (LMM) as a device for the
47
controlled drug release are rarely reported, relative to plentiful investigations on constructed release
4
8
system based on polymer–based sphere structures, since the selfꢀassembly of LMM generally create
nanoꢀ/microtubular or beltꢀlike structures, which are not proper to use for constructing drug release
39,42ꢀ45
systems.
Using MSs as a device for constant release is highly desirable for many drug delivery
applications. Wellꢀcontrolling the condition such as concentration, pH and temperature, it appears that
nearly linear release may be achieved at a certain size of the spheres. For example, MSs between 10 and
49
5
0 ꢁm would provide zero–order piroxicam release over a 4– to 8–day duration. Woo et al. formulated
a leuprolide delivery system using MSs with an average diameter of 51.7 ꢁm achieving nearꢀlinear
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5
0
peptide release for 135 days following a 15–day period of “diffusion–controlled release”. The effects
1
2
3
4
5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
1
of MS size on drug release have also been investigated by Bezemer et al. Yang et al. reported a pHꢀ
5
2
sensitive drug delivery systems constructed by MSs with DOX as a model drug. In their system, the
cumulative release of DOX got up to 90% within 25 h at pH = 1 while 30% at pH = 10 and only 15% at
pH = 7 within the same period, indicating a promising controllability in drug release. Many other
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
researchers also demonstrated that controlled release is possible with acrylamideꢀbased MSs as carriers
53,54
which are both pHꢀ and temperatureꢀsensitive.
Temperature sensitivity arises from the lower critical
solution temperature (LCST), which may be pHꢀdependent. Below LCST the microsphere is hydrophilic
due to hydrogen bonding with water, and above the LCST the microsphere becomes hydrophobic due to
5
5
the disruption of hydrogen bonds. Since the LCST is pHꢀdependent, Kim et al. employed the swelling
effect to ensure that a drug was released in the colon at a higher pH instead of the lower stomach pH. In
the hydrophilic state, the loaded drug was rapidly released and in the hydrophobic state, drug release
was very slow. Using similar responsive microspheres, stimulated hypo cortisone release at pH 4 and
5
3
halted release at pH 7.4 have also been reported.
In our system, we mainly investigate the effect of pH on the DOX release from pHꢀsensitive MS1a
with ~10 ꢁm in diameter. Added control over drug delivery can be achieved by employing pHꢀtriggered
release. Therefore by the application of pHꢀsensitive MSs, the release could be carried out to various
56ꢀ60
biological environments or to specific organs.
As a device for controlling drug release, the materials
should show acceptable biocompatibility. Therefore, the cytotoxicity of MS1a was determined by the
viability of 293T and HuCCꢀT1 cell lines evaluated by thiazolyl blue tetrazolium bromide (MTT) cell
proliferation assay. The absorbance of a formazan crystal at 560 nm could reflect the number of living
cells. The cell only group was used as control in each experiment, the absorbance of which was similar
among six parallel experiments. Compared to control, the viability of 293T and HuCCꢀT1 cells was
higher than 88% in a wide concentration range in Figure 11, showing that the MS1a has no acute and
intrinsic cytotoxicity against normal cells.
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2
3
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8
9
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1
1
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0
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0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 11. Cytotoxicity of MS1a determined by the viability of 293T (a) and HuCCꢀT1 (b) cells
evaluated by thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay.
A simple process was employed to load DOX molecules into MS1a (see experimental segment) and
5
2
calculated by reported method, the loading capacity of the DOX incorporated into the MS1a was
ꢀ1
determined to be 120 ꢁg mg and the encapsulation efficiency was around 70%. A pHꢀdependent
release behavior was tested at 37 °C in vitro. As shown in Figure 12, the release rate of DOX from
DOXꢀMS1a is highly dependent on pH condition. At pH 5.2, the 3–day cumulative release of DOX
from DOXꢀMS1a goes up to 95%, while only 60% at pH 7.2, indicating obvious pH sensitivity in DOX
release. The results are comparable to the previous investigations by using MSs as a device for DOX
5
2,61
release.
Further evidences could be collected using fluorescence images of DOX–sensitive human
cholangiocellular carcinoma (HuCCꢀT1) cells (Figure 13). DOXꢀMS1a–treated HuCCꢀT1 cells were
observed with confocal laser scanning microscopy (CLSM) (Multiphoton Microscope Leica TCS SP2
AOBS, Leica Microsystems GmbH, Wetzlar, Germany) due to the strong red fluorescence of DOX. In
line with the results obtained from in vitro DOX release tests, the cells exhibit much stronger
fluorescence at acidic pH than basic or neutral conditions.
The structural change of the DOXꢀloaded MS1a during the DOX release test under pH 5.2 was
observed by SEM technique as shown in Figure S3(aꢀc), demonstrating that the DOXꢀloaded MS1a
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were unstable in pH 5.2 solution. Before release test, the DOXꢀloaded MS1a have integral
microstructures (Figure S3a), but after DOX release test for 14 h under pH 5.2, part of MS1a started to
break or become smaller (Figure S3b), and after 60 h, almost all microspheres have broken into
nanoscale particles (Figure S3c). However, the MS1a are quite stable at pH 7.2, while a significant
destabilization in the micelle core took place as pH decreases to 5.2 or increases to 8.0. Therefore, faster
DOX release from MS1a in pH 5.2 is mostly due to the great instability of DOXꢀloaded MS1a and
easily broken in acidic solution.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 12. The profiles of DOX release from MS1a in vitro in various pHs at 37 °C.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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4
4
4
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5
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5
5
5
5
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 13. Fluorescence images of DOXꢀsensitive HuCCꢀT1 cells. HuCCꢀT1 cells were exposed to
ꢀ1
DOXꢀMS1a (equivalent concentration of DOX 1 ꢁg mL ) for 1 hour. Fluorescence images of cells
were observed by confocal laser scanning microscopy; the scale bar is 20 ꢁm.
4
. CONCLUSIONS
A series of oleamide derivatives, (OA) (CH ) (n = 2, 3, 4, or 6; OA = oleic amide fragment) and
2
2 n
(OA)(CH ) NH , modulating the length of central methylene chains between 2 oleamide groups were
2
6
2
synthesized and their self–assembly was investigated in ethanol/water media. All compounds were
subjected to twoꢀstage selfꢀassembly processes comprised of the temperatureꢀreductionꢀinduced
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nucleation and the solventꢀvolatizationꢀinduced growth in various ratios of ethanol/water mixture. The
hydrogen bonding interaction and π–π stacking drove the selfꢀassembly of all oleamide derivatives in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
ethanol/water The FT–IR and temperature–dependent H–NMR spectra demonstrate the existence of the
hydrogen bonding force and π–π stacking, which drove the selfꢀassembly of all oleamide derivatives in
−
1
ethanol/water. Selfꢀassembly of 1a and 1b at 5 mg mL in ethanol/water (1/0.1 v/v) mixture yielded
highly uniform MSs with diameter around 10 ꢁm. Under the same conditions, 1c and 1d gave microꢀ
rods and necklaceꢀlike structures with the diameter ∼6 ꢁm and several tens of micrometers in length,
which were probably formed by the aggregation of nanoꢀsized micelles initially generated by
temperature decrease in a coaxial direction. The selfꢀassembly of 2 yielded anomalous microꢀparticles
that are hard to define as MSs or microꢀrods. The uniform MS1a fabricated by 1a, which was proven to
have reasonable biocompatibility, was chosen as a device for controlling release of DOX. The in vitro
drug release and CLSM observation illuminated that the DOX release from MS1a was a pHꢀdepended
process, much faster release specifically at acidic condition, because of the easy break of MS1a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Acknowledgments
This work was supported by grantsꢀinꢀaid for the World Class University Program (No. R32–2008–
000–10174–0), the Fusion Research Program for Green Technologies through the National Research
Foundation of Korea from MEST (No. 2012M3C1A1054502), and Korea Healthcare Technology R &
D Project from the Ministry for Health Welfare & Family Affairs (A091047).
Supporting Information
This information is available free of charge via the Internet at http://pubs.acs.org/.
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