Fabrication of Microspheres via Solvent Volatization Induced

Subscriber access provided by UNIV OF ARIZONA

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Fabrication of Microspheres via Solvent Volatization Induced Aggregation of Self-Assembled Nanomicellar structures and Their Use as a pHDependent Drug Release System Lidong Zhang,a Young-Il Jeong,b Sudan Zheng,a Hongsuk Suh,c Dae Hwan Kang,b Il Kima* 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) 51-5137720; Tel: (+82) 51 510 2466; *E-mail: [email protected] b

National Research and Development Center for Hepatobiliary Cancer, Pusan National University, Yangsan Hospital, Yangsan 626-870, Korea c

Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Pusan 609 735, Korea. E-mail: [email protected]

ABSTRACT: A series of oleamide derivatives, (C18H34NO)2(CH2)n [n = 2 (1a), 3 (1b), 4 (1c), or 6 (1d); C18H34NO = oleic amide fragment] and (C18H34NO)(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,

ACS Paragon Plus Environment

1

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 44

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 wellbehaved 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.

Keywords: self-assembly; oleamide derivatives; microstructures; doxorubicin; controlled release

1 INTRODUCTION Microspheres with hollow or porous structures have enhancive applications in targeted delivery, energy storage, catalysis, and artificial cells.1–4 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 for the achievement of these promising applications.5–7 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 µm in diameter avoid capillary blockage or filtration, leading to effective uptake by cells.8 This size range can also exploit the leaky nature of tumor blood vessels (380–780 nm), providing a means for uptake by cancer cells in vivo.8,9 A recent study demonstrates that bacterially drived minicells of size ∼400 nm can be effectively used for targeting cancer cells.10 Larger particles (up to ∼5 µm in diameter) have also been administered intratumorally and subcutaneously for cancer drug-delivery applications.11,12 Up to now, several effective strategies, such as emulsion process, surface-protected etching, supramolecular self-


2

Page 3 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

assembly, as well as the replication method, have been developed to prepare hollow spheres with porous shells.13–18 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 surface-chemical 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 properties (e.g., superhydrophobicity, superhydrophilicity, and amphiphilicity).19–21 However, the asobtained 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 process.22 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 post-treatment process.23,24 By taking advantage of covalent bonds and hydrogen-bonding 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 self-assembly 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 (CH2)6-amine (2). The oleic acid was chosen as starting material for designing intended compounds, because the oleic acid has acceptable biocompatibility, that is a mono-unsaturated omega–9 fatty acid in various animals and vegetables, and often as an excipient in pharmaceuticals and an emulsifying or


3

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 44

solubilizing agent in aerosol products.25 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 chiral supramolecular fibers and ribbons by self-assembly in aqueous media,26-33 and the nanostructures can also be generated through a direct self-assembly of the oleic acid on a hydroxylated aluminum surface.34 In our system, the introduction of hydrophilic moieties allows creating microspherical or rodlike morphologies with highly rough surface structures in ethanol/water media. As far as we know, it is a new result for the self-assembly of oleamide derivatives into microspherical structures. Compared to the small micelles coalescing to microspheres by the spontaneous self-assembly of water-soluble nucleotide-calixarene conjugates,35 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.


4

Page 5 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1. Molecular structures of the oleamide amphiphiles 1a–1d and 2.

2. MATERIALS AND METHODS 2.1 General Methods Melting point: CTP-MP 300 hot-plate apparatus with ASTM 2C thermometer. 1H NMR: Varian Mercury 400 spectrometers (400 MHz).

13

C NMR: Varian Mercury 400 spectrometers (100 MHz).

NMR spectra were determined in CDCl3 and chemical shifts are expressed in parts per million (δ) relative to the central peak of the solvent. UV-vis absorption spectra were obtained on UV-1800 Shimadzu UV-vis 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 MS-9 VG updated or ZAB-HF (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 self-assembled samples were investigated using scanning electron microscope (SEM) on an S-4800 (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 self-assembly 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 phosphate-buffered 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


5

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 44

95 mL of PBS. This bottle was placed in a shaking incubator with a stirring speed of 100 rpm at 37 °C. 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 UV-vis spectrometer at 483 nm. 2.2 Materials 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 oleoyl chloride prepared by the chlorination of oleic acid with thionyl chloride.36 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 CHCl3 (20 mL) for 10 min in a 100–mL round bottom flask at room temperature. After transferring the mixture to the ice bath, 3.0 g (0.01 mol) of oleoyl chloride dissolved in purified CHCl3 (10 mL) was added dropwise for 20 min so that the temperature of the reaction mixture cannot 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) 3300, 2936, 2841, 1643, 1563, 1468, 1238, 1123, 940, 722 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.650.78 (m, 6H), 1.12-1.20 (m, 40H), 1.45 (m, 4H), 1.80-1.98 (m, 8H), 2.05-2.19 (m, 4H), 3.20-3.41 (s, 4H), 5.20-5.38 (m, 4H), 6.2 (s, 2H);

13

C NMR (100 MHz, CDCl3) δ 174.53, 129.93, 129.62, 39.98,


6

Page 7 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

36.59, 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 Calcd for C38H72N2O2: C, 77.49; H, 12.32; N, 4.76. Found C, 77.33; H, 12.80; N, 4.92; Fab-MS m/z (%) 590.20 (100) [M + H]+. N,N'-1,3-propylenebis(oleamide) (1b). Colorless solid; yield 85%. mp 80 – 83 °C; IR (KBr) 3301, 2927, 1652, 1576, 1475, 1367, 1310, 1153, 731 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.64-0.89 (m, 6H), 1.23-1.30 (m, 40H), 1.46 (m, 4H), 1.80-1.98 (m, 10H), 2.05-2.20 (m, 4H), 3.15-3.31 (s, 4H), 5.20-5.40 (m, 4H), 6.4 (s, 2H); 13C NMR (100 MHz, CDCl3) δ174.00, 129.92, 129.67, 36.83, 35.55, 31.85, 29.72, 29.67, 29.47, 29.26, 29.23, 29.12, 27.17, 25.82, 22.63, 14.06; Anal Calcd for C39H74N2O2: C, 77.68; H, 13.37; N, 5.32; Found C, 77.52; H, 13.82; N, 5.87; Fab-MS m/z (%) 603.58 (100) [M + H]+. N,N'-1,4-butylenebis(oleamide) (1c). Colorless solid; yield 82%. mp 111 – 114 °C; IR (KBr) 3319, 2922, 2849, 1643, 1541, 1474, 1421, 1263, 1194, 954 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.79 (s, 6H), 1.19-1.22 (m, 38H), 1.44 (m, 4H), 1.45-1.53 (m, 4H), 1.90-1.93 (m, 8H), 2.07-2.11 (m, 4H), 3.16-3.17 (s, 4H), 5.24-5.29 (m, 4H), 6.41 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 173.60, 129.87, 129.59, 38.95, 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 C40H76N2O2: C, 77.86; H, 12.41; N, 4.54. Found C, 77.56; H, 12.90; N, 4.88; Fab-MS m/z (%) 617.60 (100) [M + H]+. N,N'-1,6-hexylenebis(oleamide) (1d). Colorless solid; yield 85%. mp 113 – 115 °C; IR (KBr) 3318, 2929, 2850, 1635, 1532, 1469, 1422, 1264, 1214, 691 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.79-0.83 (m, 6H), 1.19-1.26 (m, 44H), 1.42 (m, 4H), 1.54 (m, 4H), 1.92-1.94 (m, 8H), 2.08-2.11 (m, 4H), 3.123.16 (s, 4H), 5.25-5.30 (m, 4H), 6.10 (s, 2H);

13

C NMR (100 MHz, CDCl3) δ 173.35, 129.88, 129.62,

38.85, 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 C42H80N2O2: C, 78.20; H, 12.50; N, 4.34. Found C, 78.45; H, 12.83; N, 4.68; FabMS m/z (%) 645.63 (100) [M + H]+. N-6-aminohexyl(oleamide) (2). Colorless solid; yield 75%. mp 90 – 93 °C; IR (KBr) 3319, 3308, 2942, 2930, 1649, 1563, 1471, 1425, 1284, 1193, 875 cm-1; 1H NMR (400 MHz, CDCl3) δ 0.85-0.90


7

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 44

(m, 3H), 1.15-1.26 (m, 24H), 1.50-1.52 (m, 6H), 1.82-1.98 (m, 4H), 2.15-2.19 (m, 2H), 2.56-2.63 (m, 2H), 3.20-3.23 (m, 2H), 5.30-5.31 (m, 2H); 13C NMR (100 MHz, CDCl3) 171.23, 129.86, 129.56, 38.83, 36.65, 31.84, 29.58, 29.53, 29.24, 25.83, 22.63, 14.11; Anal Calcd for C24H48N2O: C, 75.73; H, 12.71; N, 7.36. Found C, 75.80; H, 12.98; N, 7.87. Fab-MS m/z (%) 381.60 (100) [M + H]+. 2.4 Self-assembling experiment The self-assembly of OADs was performed via a gradual temperature reduction and solvent volatilization process. A suspension of OAD in ethanol/water (concentration: 5 mg mL−1) 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 self-assembled 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 HuCCT1 cells line 293T cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (5% CO2 at 37 °C). The viability of 293T cell line derived from human embryonic kidney cells was evaluated by thiazolyl blue tetrazolium bromide (MTT) cell proliferation assay. 1×104 cells seeded in 96–well plates with 100 µL of medium were incubated overnight in a CO2 incubator (5% CO2 at 37 °C) and then the media was exchanged with 100 µL of serum–free RPMI1640 media. For the cytotoxicity test, MSs from 1a were distributed in serum–free RPMI1640 media and diluted to appropriate concentrations. After 3 days of incubation, 30 µL of MTT (5 mg mL-1) was added in 96–well plates and incubated for 4 h. The formazan crystals formed were dissolved in DMSO, and the absorbance (560 nmtest/630 nm-reference) was determined using an automated computer-linked micro-plate 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


8

Page 9 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

eight wells. The viability of a human cholangiocellular carcinoma cell line, HuCC-T1, was also evaluated by employing similar method. 3. RESULTS AND DISCUSSION 3.1 Synthesis and Self-assembly of OADs The oleic acid and diamines were firstly purified to remove impurities like moisture. The oleic acid was then reacted with excess thionyl chloride to produce oleoyl chloride.36 Further condensation reactions of oleoyl chloride with various diamine compounds containing different methylene groups in pyridine/CHCl3 mixture gave oleamide amphiphiles 1a–1d in high yield (>82%). For comparison of different self-assembling 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 self-assembling 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 structure through intermolecular translation-related hydrogen bonding.23,24 The π–π interaction and the intermolecular hydrogen bonds might have a synergistic effect on the formation of stable aggregations.37 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 intermolecular hydrogen bonds and π–π stacking (red broken line).38 Some practical measurements such as FT–IR and 1H–NMR spectra have also been provided for understanding the intermolecular interaction and packing order (vide infra).


9

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 44

Figure 2. The minimum energy structures of amphiphilic oleamide derivatives, 1a (a), 1b (b), 1c (c), 1d (d) and 2 (e), obtained using a GGA/PBE/DNP level of theory with the DMol3 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 self-assembling conditions (solvent, temperature and kinetics) and the hydrophilic moieties also strongly influence the aggregation behavior of OADs.39–41 When the oleic acid was modified by glucopyranosylamine to form a glucopyranosylamide lipid, the self-assembly of this lipid in water allows generating a tubular structure.39 Similarly, by changing the hydrophilic moieties to D-glucose, phenolic glycose or phenyl glucose, nanofibers or nanotubles could be produced via a self-assembling process respectively in water.42–45 In our study, ethanol/water mixture instead of pure water was used as a solvent for the self-assembly of OADs. A class of new morphology of the microspherical structure with highly rough surface structures was obtained, which is completely different from the selfassembled structures based on other OADs reported previously.42–45 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 selfassembling microsphere morphologies. ACS Paragon Plus Environment

10

Page 11 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The self-assembly processes can be separated into two stages: i.e. the temperature-reduction-induced nucleation into nanoscale micelles, followed by the solvent-volatization-induced growth into microstructures. In the first stage, the kinetics of evolution for the temperature-reduction-induced selfassembly of 1a was studied by DLS (Figure 3a). Initially formed micelles around 100 nm in diameter at 70 °C increase slowly with a decrease in the system temperature, and finally grows up to around 400 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 solvent-volatization-induced growth, we traced this process with SEM technique. After performing the temperature-reduction process, the self-assembling 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 pre-formed micelles. After complete solvent evaporation, the microstructures grow to ~10 µm in diameter. Moreover, the in situ observation of the microstructure formation of 1a under an optical microscopy displays that plenty of small particles (~1 µm 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 self-


11

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 44

assembly of 1a and (b) effect of solvent volatilization on the size of formed microspheres. The detailed surface structures of self-assembled 1a were studied by SEM observation in Figure 4, demonstrating a well-defined MSs with tunability in size, surface characteristics and dispersity. A small increase in the water fraction of the self-assembling media results in a decrease in size of formed MSs from 1a (named MS1a). For getting more insights into the water effect, the MS1a self-assembled 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 self-assembly of the amphiphilic mono-chain derivatives of stearic acid in pure 1,2-dichloroethane yields MSs with the diameter up to around 40 µm, most probably due to the fast evaporation of 1,2-dichloroethane.36 Employing pure water for the self-assembly of various amphiphilic mono-chain derivatives,

the

nanostructures were observed instead of microstructures.37 These results prove that solvents with high volatility such as ethanol and 1,2-dichloroethane 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 self-assembling 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).


12

Page 13 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 4. SEM images of the self-assembled products from 1a at a concentration of 5 mg mL−1.

Figure 5. Effect of ethanol to water ratio on the dispersity of microspheres from 1a at a concentration of 5 mg mL−1.

Under the optimal self-assembly condition of 1a, the self-assembling behaviors of 1b, 1c, 1d and 2 were also investigated. The concentration of oleamide compounds and the ratio of ethanol to water were fixed at 5 mg mL−1 and 1/0.1, respectively. The compound 1b yields very uniform MSs with the average diameter ∼10 µm (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 self-assembly behavior has been observed. Instead of forming MSs, microrods with the average diameter ∼6 µm 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 ACS Paragon Plus Environment

13

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 44

concentration of 1c and the ratio of ethanol to water, well-dispersed and uniform MSs could not be achieved.

Figure 6. SEM images of the self-assembled products from 1b and 1c in ethanol/water (1/0.1 v/v) at the compound concentration of 5 mg mL−1: (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 self-assembly in ethanol/water media, self-assembly of 1d with six methylenes in the hydrophilic head has also been investigated at the same conditions. Different from the self-assembly of 1c, 1d yields micro-necklace-like structures. Figure 7a shows a well-ordered array of such micronecklaces from 1d. Based on the detailed observation by SEM in Figure 7b and 7c, the necklace-like morphology was spontaneously assembled by interconnecting MSs each other, meaning that the selfassembly of 1d was first to give MSs and then further solvent volatilization drived the MSs to arrange and link one another. The self-assembling 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 self-assembly behavior, we have synthesized monochain compound 2, in which one of the primary amines in hexane-1,6-diamine was


14

Page 15 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

substituted by oleic acyl group. The self-assembly was carried out under the same conditions employed in the self-assembly 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 microrod-like or microspherical structures are not formed.

Figure 7. SEM images of the self-assembled products from 1d and 2 in ethanol/water (1/0.1 v/v) at the compound concentration of 5 mg mL−1: (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 translation-related hydrogen bonding (see also Figure 2).23,24,36 Therefore, the formation process of microstructures can be considered as head-tohead jointing of molecules and tail-tail-tail packing to aggregate into micellar structure during the temperature-reduction. 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


15

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 44

speculated that the MSs from the self-assembly of 1a and 1b were generated through a layer-by-layer 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 rod-like structure. Interestingly, the micronecklaces are not spontaneously assembled from 1d micelles, alternatively, the micelles are firstly aggregated to microparticles structure, and then they self-assemble in a coaxial direction. During the self-assembly of compound 2, solvent volatilization drives the micelles to form anomalous microparticles, followed by continuous linkage among them to form bigger microparticles.

Figure 8. The plausible procedures of the formation of microstructures by the self-assembly of various oleamide derivatives in ethanol/water media.

1

H–NMR and FT–IR spectra investigation

For demonstrating the hydrogen bonding interaction and π–π stacking in the self-assembly, the 1a 1H spectra were measured via a temperature-dependent process in D4-methanol solution. The initial temperature for 1H–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 D4-methanol was employed as


16

Page 17 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

solvent, since the self-assembling behavior of 1a in D4-methanol was similar to that in ethanol/water, also giving microspherical structures as shown in Figure S1(in SI). Seen from the full view of the 1H 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 D4-methanol. The Figure 9a shows the chemical shift change of Ha/Hb with detailed data in Table 1, where the chemical shift 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 π-π stacking took place at double bond position. Another obvious chemical shift change was found in Hc as shown in Figure 9b, where the chemical shift shifted 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 Hd, He and Hf positions (Figure 9c and Table 1). The results above indicate that the intermolecular/intramolecular interactions such as hydrogen bonding and π-π stacking drive the self-assembly of 1a in deed. Unfortunately, the signals indicating the 1H chemical shift variation of N-H framework was not appeared in 1a 1H-NMR spectra. Since the intermolecular/intramolecular hydrogen bonding interaction via NH frameworks is of great significance for the self-assembling behaviors, instead, the FT-IR spectra (Figure 10) were used to demonstrate the existence of its hydrogen bonding interaction with detailed descriptions given below.

Figure 9. (Magnified view) Temperature dependence of the 1a 1H spectrum in D4-methanol solution, indicating greatly chemical shifts variation as the temperature decreased from 70 to 20 ºC.


17

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 44

Table 1. Chemical shift values of Ha to Hf for compound 1a at appointed temperatures. Temperature T (ºC) 70 60 50 40 30 20

Ha/Hb 5.300 5.275 5.274 5.245 5.203 5.117

Chemical shift values δ (ppm) Hc Hd He 3.242 2.131 1.988 3.214 2.110 1.968 3.212 2.105 1.967 3.181 2.079 1.943 3.137 2.039 1.897 3.039 1.947 1.811

Hf 0.857 0.827 0.822 0.801 0.785 0.675

The 1H-chemical shifts variation shows a non-linear behavior: Ha/Hb: δ [ppm] = -8.3751x10-5 T 2 + 0.0109 T + 4.9414; (R2 = 0.9876) Hc: δ [ppm] = -9.2501x10-5 T 2 + 0.0120 T + 2.8463; (R2 = 0.9859) Hd: δ [ppm] = -8.8393x10-5 T 2 + 0.0113 T + 1.7663; (R2 = 0.9868) He: δ [ppm] = -9.1071x10-5 T 2 + 0.0114 T + 1.6269; (R2 = 0.9881) Hf: δ [ppm] = -7.9286x10-5 T 2 + 0.0102 T + 0.5212; (R2 = 0.9536) For a stable structure in solution, the hydrogen bond donor protons caused chemical shift variation can often be interpreted in terms of (intra- or intermolecular) hydrogen bond dynamics, which typically provide a positive dependence with temperature, while the high temperature coefficients was observed in self-assembling system.46 Since the self-assembled aggregation of 1a in D4-methanol was driven by π-π stacking and hydrogen bonding force as discussed above, the similar interaction force was also deemed to exist in the self-assembly of 1a in ethanol/water and the packing structure. The FT-IR spectroscopy as a powerful tool for investigating hydrogen bonding interaction has been proverbially applied in the analysis of morphology of self-assembling organic compounds. According to the FT-IR spectroscopy, the characteristic IR signals of the 1a and 1d show blue shift after assembling into special morphologies. As an example, Figure 10a shows FT-IR spectra of 1a before and after selfassembly and Table 2 summarized the results of assignments, which demonstrate that the peaks at 3300 and 1563 cm-1 for 1a before self-assembly that are assigned to νas(NHCO) and ν(NH) stretching, shift to


18

Page 19 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

3294 and 1556 cm-1 respectively, for the self-assembled microspheres. Especially, the peak at 722 cm-1 for 1a before self-assembly assigned to C–H stretching vibrations of double bond (C=C–H) shifts to 714 cm-1 for the self-assembled morphologies, indicating the double bond of 1a has participated in the selfassembling process via π–π stacking and other weak interactions. The similar blue shifts have also been observed from the absorption bands of the asymmetric (υas) and symmetric (υs) -CH2 stretching vibrations, indicating the presence of an all-trans-zigzag form alkyl chain,36 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 1643 cm-1 to 1635 cm-1. These results testify the existence of strong hydrogen-bonding interactions between O=C and H-N as well as N-H and N-H bonding which play critical role in the formation of self-assembled nanomicellar structures and highly ordered microsphere aggregate. Therefore, it becomes clear that the self-assembly of 1a occurs by means of hydrogen-bonding between the primaryamino/secondary-amino 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 FT-IR spectra of 1d in Figure 10b and Table 2, further revealing the existence of hydrogen bonding force and π–π stacking in the self-assembling process.

Figure 10. The IR spectra of 1a and 1d before and after self-assembly.


19

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 44

Table 2. The FT-IR band assignments of 1a and 1d before and after self-assembly. Absorption peak of sample (cm-1) Compounds

1a

1d

Assignments

Before self-assembly

After self-assembly

νas(NHCO) νas(CH2) δs(CH2) ν(C=O) ν(NH), ν(CN) δ(CH2) δ(C=C-H) νas(NHCO) νas(CH2) δs(CH2) ν(C=O) ν(NH), ν(CN) δ(CH2) δ(C=C-H)

3300 2936 2841 1643 1563 1468 722 3318 2929 2850 1635 1532 1469 691

3294 2928 2840 1635 1556 1460 714 3310 2921 2849 1627 1531 1452 682

3.3 Doxorubicin release from MS1a in vitro Microspherical structures from self-assembly of low mass molecules (LMM) as a device for the controlled drug release are rarely reported,47 relative to plentiful investigations on constructed release system based on polymer–based sphere structures,48 since the self-assembly of LMM generally create nano-/microtubular or belt-like structures, which are not proper to use for constructing drug release systems.39,42-45 Using MSs as a device for constant release is highly desirable for many drug delivery applications. Well-controlling 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 50 µm would provide zero–order piroxicam release over a 4– to 8–day duration.49 Woo et al. formulated a leuprolide delivery system using MSs with an average diameter of 51.7 µm achieving near-linear


20

Page 21 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

peptide release for 135 days following a 15–day period of “diffusion–controlled release”.50 The effects of MS size on drug release have also been investigated by Bezemer et al.51 Yang et al. reported a pHsensitive drug delivery systems constructed by MSs with DOX as a model drug.52 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 researchers also demonstrated that controlled release is possible with acrylamide-based MSs as carriers which are both pH- and temperature-sensitive.53,54 Temperature sensitivity arises from the lower critical solution temperature (LCST), which may be pH-dependent. Below LCST the microsphere is hydrophilic due to hydrogen bonding with water, and above the LCST the microsphere becomes hydrophobic due to the disruption of hydrogen bonds. Since the LCST is pH-dependent, Kim et al.55 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 halted release at pH 7.4 have also been reported.53 In our system, we mainly investigate the effect of pH on the DOX release from pH-sensitive MS1a with ~10 µm in diameter. Added control over drug delivery can be achieved by employing pH-triggered release. Therefore by the application of pH-sensitive MSs, the release could be carried out to various biological environments or to specific organs.56-60 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 HuCC-T1 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 HuCC-T1 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.


21

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 44

Figure 11. Cytotoxicity of MS1a determined by the viability of 293T (a) and HuCC-T1 (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 calculated by reported method,52 the loading capacity of the DOX incorporated into the MS1a was determined to be 120 µg mg-1 and the encapsulation efficiency was around 70%. A pH-dependent release behavior was tested at 37 °C in vitro. As shown in Figure 12, the release rate of DOX from DOX-MS1a is highly dependent on pH condition. At pH 5.2, the 3–day cumulative release of DOX from DOX-MS1a 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 release.52,61 Further evidences could be collected using fluorescence images of DOX–sensitive human cholangiocellular carcinoma (HuCC-T1) cells (Figure 13). DOX-MS1a–treated HuCC-T1 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 DOX-loaded MS1a during the DOX release test under pH 5.2 was observed by SEM technique as shown in Figure S3(a-c), demonstrating that the DOX-loaded MS1a


22

Page 23 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

were unstable in pH 5.2 solution. Before release test, the DOX-loaded 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 DOX-loaded MS1a and easily broken in acidic solution.

Figure 12. The profiles of DOX release from MS1a in vitro in various pHs at 37 °C.


23

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 44

Figure 13. Fluorescence images of DOX-sensitive HuCC-T1 cells. HuCC-T1 cells were exposed to DOX-MS1a (equivalent concentration of DOX 1 µg mL-1) for 1 hour. Fluorescence images of cells were observed by confocal laser scanning microscopy; the scale bar is 20 µm.

4. CONCLUSIONS A series of oleamide derivatives, (OA)2(CH2)n (n = 2, 3, 4, or 6; OA = oleic amide fragment) and (OA)(CH2)6NH2, modulating the length of central methylene chains between 2 oleamide groups were synthesized and their self–assembly was investigated in ethanol/water media. All compounds were subjected to two-stage self-assembly processes comprised of the temperature-reduction-induced


24

Page 25 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

nucleation and the solvent-volatization-induced growth in various ratios of ethanol/water mixture. The hydrogen bonding interaction and π–π stacking drove the self-assembly of all oleamide derivatives in ethanol/water The FT–IR and temperature–dependent 1H–NMR spectra demonstrate the existence of the hydrogen bonding force and π–π stacking, which drove the self-assembly of all oleamide derivatives in ethanol/water. Self-assembly of 1a and 1b at 5 mg mL−1 in ethanol/water (1/0.1 v/v) mixture yielded highly uniform MSs with diameter around 10 µm. Under the same conditions, 1c and 1d gave microrods and necklace-like structures with the diameter ∼6 µm and several tens of micrometers in length, which were probably formed by the aggregation of nano-sized micelles initially generated by temperature decrease in a coaxial direction. The self-assembly of 2 yielded anomalous micro-particles that are hard to define as MSs or micro-rods. 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 pH-depended process, much faster release specifically at acidic condition, because of the easy break of MS1a. Acknowledgments This work was supported by grants-in-aid 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/. REFERENCES (1) Zhu, Y. F.; Shi, J. L.; Shen, W. H.; Dong, X. P.; Feng, J. W.; Ruan, M. L.; Li, Y. S. Angew. Chem. Int. Ed. 2005, 44, 5083–5087. (2) Ikeda, S.; Ishino, S.; Harada, T.; Okamoto, N.; Sakata, T.; Mori, H.; Kuwabata, S.; Torimoto, T.;


25

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 44

Matsumura, M. Angew. Chem. Int. Ed. 2006, 45, 7063–7066. (3) Su, J. F.; Wang, L. X.; Ren, L.; Huang, Z. J. Appl. Polym. Sci. 2007, 103, 1295–1302. (4) Yu, A. M.; Wang, Y. J.; Barlow, E.; Caruso, F. Adv. Mater. 2005, 17, 1737–1741. (5) Emami, J.; Hamishehkar, H.; Najafabadi, A. R.; Gilani, K.; Minaiyan, M.; Mahdavi, H.; Nokhodchi, A. J. Pharm. Sci. 2009, 98, 1712–1731. (6) Poe, S. L.; Kobaslija, M.; McQuade, D. T. J. Am. Chem. Soc. 2007, 129, 9216–9221. (7) Sakai, S.; Hashimoto, I.; Kawakami, K. Biotechnol. Bioeng. 2008, 99, 235–243. (8) Brigger, I.; Dubernet, C.; Couvreur, P. Adv. Drug. Delivery Rev. 2002, 54, 631–651. (9) Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Proc. Natl. Acad. Sci. USA 1998, 95, 4607–4612. (10) MacDiarmid, J. A.; Mugridge, N. B.; Weiss, J. C.; Phillips, L.; Burn, A. L.; Paulin, R. P.; Haasdyk, J. E.; Dickson, K. A.; Brahmbhatt, V. N.; Pattison, S. T.; James, A. C.; Al Bakri, G.; Straw, R. C.; Stillman, B.; Graham, R. M.; Brahmbhatt, H. Cancer. Cell. 2007, 11, 431–445. (11) Hill, H. C.; Conway, T. F.; Sabel, M. S.; Jong, Y. S.; Mathiowitz, E.; Bankert, R. B.; Egilmez, N. K. Cancer. Res. 2002, 62, 7254–7263. (12) Dass, C. R.; Choong, P. F. M. Peptides 2006, 27, 3020–3028. (13) Jang, J.; Lee, K. Chem. Commun. 2002, 1098–1099. (14) Ao, Z.; Yang, Z.; Wang, J. F.; Zhang, G. Z.; Ngai, T. Langmuir 2009, 25, 2572–2574. (15) Zhang, Q.; Zhang, T. R.; Ge, J. P.; Yin, Y. D. Nano Lett. 2008, 8, 2867–2871. (16) Zhang, L. J.; Wan, M. X. Adv. Funct. Mater. 2003, 13, 815–820.


26

Page 27 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(17) Zhao, Y.; Jiang, L. Adv. Mater. 2009, 21, 3621–3638. (18) Kim, M.; Yoon, S. B.; Sohn, K.; Kim, J. Y.; Shin, C. H.; Hyeon, T.; Yu, J. S. Microporous. Mesoporous. Mater. 2003, 63, 1–9. (19) Liu, X. M.; Du, X.; He, J. H. Chem. Phys. Chem. 2008, 9, 305–309. (20) Jiang, L.; Zhao, Y.; Zhai, J. Angew. Chem. Int. Ed. 2004, 43, 4338–4341. (21) Sun, T. L.; Wang, G. J.; Feng, L.; Liu, B. Q.; Ma, Y. M.; Jiang, L.; Zhu, D. B. Angew. Chem. Int. Ed. 2004, 43, 357–360. (22) Shi, L.; Bi, W. G.; Chen, H.; Tang, T. J. Polym. Sci. Polym. Chem. Ed. 2007, 45, 4477–4486. (23) Hutchings, L. R.; Narrianen, A. P.; Thompson, R. L.; Clarke, N.; Ansari, L. Polym. Int. 2008, 57, 163–170. (24) Jung, M. O.; Ryu, J. H.; Park, J. G.; Jun, J. B.; Suh, K. D. J. Appl. Polym. Sci. 2006, 100, 1349– 1356. (25) Sivakumar, S.; Bansal, V.; Cortez, C.; Chong, S. F.; Zelikin, A. N.; Caruso, F. Adv. Mater. 2009, 21, 1820–1824. (26) Fuhrhop, J. H.; Helfrich, W. Chem. Rev. 1993, 93, 1565–1582. (27) Estroff, L. A.; Hamilton, A. D. Chem. Rev. 2004, 104, 1201–1217. (28) Fuhrhop, J.-H.; Wang, T. Chem. Rev. 2004, 104, 2901–2937. (29) Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 2812–2818. (30) Nakazawa, I.; Masuda, M.; Okada, Y.; Hanada, T.; Yase, K.; Asai, M.; Shimizu, T. Langmuir 1999, 15, 4757–4764.


27

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 44

(31) Masuda, M.; Shimizu, T. Carbohydr. Res. 2000, 326, 56–66. (32) Shimizu, T. Macromol. Rapid Commun. 2002, 23, 311–331. (33) Smitz, E.; Engmerts, J. B. F. N.; Kellogg, R. M.; Doren, H. A. V. Liq. Cryst. 1997, 23, 481–488. (34) Liascukiene, I.; Aissaoui, N.; Asadauskas, S. J.; Landoulsi, J.; Lambert, J. –F. Langmuir 2012, 28, 5116−5124. (35) Grazia, M. L. C.; G, Granata.; Raffaella, L. N.; Graziella, M.; Corrada, G. Langmuir 2008, 24, 6194–6200. (36) Zhang, L. D.; Li, H. Q.; Ha, C. –S.; Suh, H. –S.; Kim, I. Langmuir 2010, 26, 17890–17895. (37) Shimizu, T.; Masuda, M.; Minamikawa, H. Chem. Rev. 2005, 105, 1401–1443. (38) Miao, J. J.; Zhu, L. Chem. Mater. 2010, 22, 197–206. (39) Kamiya, S.; Minamikawa, H.; Jung, J. H.; Yang, B.; Masuda, M.; Shimizu, T. Langmuir 2005, 21, 743–750. (40) Kulkarni, V. S.; Anderson, W. H.; Brown, R. E.; Biophys. J. 1995, 69, 1976–1986. (41) Kulkarni, V. S.; Boggs, J. M.; Brown, R. E. Biophys. J. 1999, 77, 319-330 (42) John, G.; Masuda, M.; Okada, Y.; Yase, K.; Shimizu, T. Adv. Mater. 2001, 13, 715–718. (43) John, G.; Jung, J. H.; Masuda, M.; Shimizu, T. Langmuir 2004, 20, 2060–2065. (44) John, G.; Jung, J. H.; Minamikawa, H.; Yoshida, K.; Shimizu, T. Chem. Eur. J. 2002, 8, 5494– 5500. (45) Jung, J. H.; John, G.; Yoshida, K.; Shimizu, T. J. Am. Chem. Soc. 2002, 124, 10674–10675. (46) Davy. S.; Pieter. M. S. H.; Johan, V. H.; Eric, P.; Bruno, K.; Jose, C. M. Chem. Eur. J. 2009, 15,


28

Page 29 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

12653–12662. (47) Chen, W. Y.; Yang, Y. J.; Rinadi, C.; Zhou, D.; Shen, A. Q. Lab. Chip, 2009, 9, 2947–2951. (48) Freiberg, S.; Zhu, X. X. Int. J. Pharm. 2004, 282, 1–18. (49) Liggins, R. T.; Amours, S. D’.; Demetrick, J. S.; Machan, L. S.; Burt, H. M. Biomaterials 2000, 21, 1959–1969. (50) Woo, B. H.; Kostanski, J. W.; Gebrekidan, S.; Dani, B. A.; Tahanoo, B. C.; DeLuca, P. P. J. Control. Rel. 2001, 75, 307–315. (51) Bezemer, J. M.; Radersma, R.; Grijpma, D. W.; Dijkstra, P. J.; van Blitterswijk, C. A.; Feijen, J. J. Control. Rel. 2000, 67, 249–260. (52) Yang, X. Y.; Chen, L. T.; Huang, B.; Bai, F.; Yang, X. L. Polymer 2009, 50, 3556–3563. (53) Kim, E. J.; Cho, S. H.; Yuk, S. H. Biomaterials 2001, 22, 2495–2499. (54) Fang, S. -J.; Kawaguchi, H. Colloid. Polym. Sci. 2002, 280, 984–989. (55) Kim, Y. H.; Bae, Y. H.; Kim, S. W. J. Control. Release. 1994, 28, 143–152. (56) Bilia, A.; Carelli, V.; Colo, G. D.; Nannipieri, E. Int. J. Pharm. 1996, 130, 83–92. (57) Cifti, K.; Kas, H. S.; Hincal, A. A.; Ercan, T. M.; Guven, O.; Ruacan, S. Int. J. Pharm. 1996, 131, 73–82. (58) Gupta, K. C.; Kumar, M. N. V. R. J. Mater. Sci. Mater. Med. 2001, 12, 753–759. (59) Jeong, Y. -I.; Prasad, Y. R.; Ohno, T.; Yoshikawa, Y.; Shibata, N.; Kato, S.; Takeuchi, K.; Takada, K. J. Pharm. Pharmacol. 2001, 53, 1079–1085. (60) Lynn, D.; Amiji, M.; Langer, R. Angew. Chem. Int. Ed. 2001, 40, 1707–1710.


29

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 44

(61) Leroux, J. C.; Roux, E.; Garrec, D. L.; Hong, K.; Drummond. D. C. J. Control. Release. 2001, 72, 71–84.

Table of Content Graphic


30

Page 31 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

149x101mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

97x74mm (300 x 300 DPI)


Page 32 of 44

Page 33 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

192x255mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

202x106mm (96 x 96 DPI)


Page 34 of 44

Page 35 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

219x160mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

225x193mm (96 x 96 DPI)


Page 36 of 44

Page 37 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

214x137mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

214x137mm (96 x 96 DPI)


Page 38 of 44

Page 39 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

180x201mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

291x172mm (96 x 96 DPI)


Page 40 of 44

Page 41 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

236x111mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

148x114mm (96 x 96 DPI)


Page 42 of 44

Page 43 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

141x129mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

198x318mm (96 x 96 DPI)


Page 44 of 44

Fabrication of Microspheres via Solvent Volatization Induced

Recommend Documents