Letter pubs.acs.org/Langmuir
Meso-Ordered PEG-Based Particles Maria Wallin,*,† Annika Altskar̈ ,‡ Lars Nordstierna,† and Martin Andersson† †
Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Göteborg, Sweden The Swedish Institute for Food and Biotechnology (SIK), SE-402 29 Göteborg, Sweden
‡
S Supporting Information *
ABSTRACT: We report on the formation of meso-ordered hydrogel particles by cross-linking poly(ethylene glycol) diacrylate (PEG-DA) in the presence of surfactants in a confined environment. The results demonstrated that wellordered mesoporous hydrogel particles having a pore size of about 5 nm could be formed. It is suggested that these mesoordered hydrogel particles might have unique drug-delivery capabilities.
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INTRODUCTION Hydrogels have exceptional characteristics in terms of mechanical properties, water uptake ability and biocompatibility, which are of interest in tissue engineering, biomedical implantation, biosensing, and bionanotechnology.1−5 One particularly interesting group of hydrogels aimed toward biomedical applications is meso-ordered hydrogels due to their well-defined geometry and small pore size.6−9 The geometry is mainly controlled by the use of amphiphiles, such as surfactants and block copolymers, and their concentration and the size of the pores are directly related to the length of the hydrophobic part of the amphiphile.10,11 Meso-ordered polymeric hydrogels formed using amphiphiles as a template have so far only been obtained as bulk materials and to date never as discrete nanosized particles. Meso-ordered hydrogel particles are considered to be highly useful as drug delivery vehicles due to their high porosity, large surface area and well-defined pore size distribution and geometry.12 The purpose of this study was to synthesize poly(ethylene glycol) (PEG)-diacrylate hydrogels in the confined environment of a water-in-oil (w/o) emulsion to form meso-ordered particles.
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Figure 1. Chemical structure of PEG-DA. PEG-DA particles were prepared by mixing 1.2 g of the emulsifier (Sorbitane monooleate, Span 80) with 20 mL hexane, which was stirred at 12 500 rpm using a homogenizer (SilentCruser M, Heidolph, Schwabach, Germany). This was followed by slow addition (∼1 min) of 30 mL PEG precursor mixture, containing 5 wt % PEG-DA (MW = 1500 g/mol), deionized water, and photoinitiator (2-hydroxy-2methylpropiophenone) at 1 wt % with respect to PEG, under constant stirring, forming a w/o emulsion. The mixture was stirred using the homogenizer for 1 h under UV light to ensure complete cross-linking of the PEG-DA. The particles were then purified by liquid−liquid extraction (deionized water:hexane, 1:3) four times, followed by removal of the water by freeze drying (Scanvac, Lynge, Denmark) and then redispersed in milli-q water.16 After washing, the particles did not contain any residuals from Span 80, which was verified by NMR as shown in Supporting Information Figure S4. Prior to particle preparation, PEG-DA was synthesized as previously described.6,13,14 The microstructure of fully swollen PEG-DA hydrogel particles were analyzed using transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), polarized light microscopy (PLM) and nuclear magnetic resonance spectroscopy (NMR); for detailed information, see the Supporting Information. Electron micrographs were taken on fully swollen hydrogel particles and bulk hydrogels (used as reference), by preparing replicas according to the mica sandwich and the freeze-etching techniques. TEM was also performed on hydrogel particles that were embedded in agarose. For preparation of bulk hydrogels, PEG precursor mixture was prepared by dissolved dried 0.20 g PEG-DA in 0.80 g deionized water. This was followed by addition of a photoinitiator, 2-hydroxy-2-
EXPERIMENTAL SECTION
The following chemicals were used in the synthesis of diacrylateterminated PEG: 2-hydroxy-2-methylpropiophenone, acryloyl chloride, triethylamine, diol-terminated PEG (Mn ≈ 1 500 g/mol), ethanol, dichloromethane, diethyl ether, anhydrous tetrahydrofuran, xylene, hexane, and sorbitan monooleate (Span 80) were all purchased from Sigma-Aldrich and used as received. Diacrylate-terminated PEG was synthesized by reacting diol-terminated PEG (Mn = 1500 g/mol) with a 2.5 molar excess of acryloyl chloride and the base catalyst trietylamine in tetrahydrofuran overnight at room temperature, as described previously.6,13−15 The product was then purified by filtration, liquid−liquid extraction in dichloromethane and precipitation in diethyl ether. The functionalization was confirmed using H1 NMR, as described earlier.13 The chemical structure of poly(ethylene glycol) diacrylate (PEG-DA) is shown in Figure 1. © 2014 American Chemical Society
Received: April 17, 2014 Published: December 19, 2014 13
DOI: 10.1021/la504710a Langmuir 2015, 31, 13−16
Letter
Langmuir methylpropiophenone, at 1 wt % with respect to PEG. The precursor mixture was then placed between two glass slides separated by 2 mm Teflon spacers followed by UV treatment for 5 min. Finally, all samples were swollen in water overnight. Furthermore, bulk hydrogels containing 20 wt % of Span 80 with respect to the wt % of PEG-DA (corresponding to the ratios used in the preparation of the particles) were prepared following the same procedure as described above.
TEM on freeze-etched PEG-DA bulk hydrogels showed that the structure was porous with holes ranging from a few nanometers to several microns, as can be seen in Figure 3. No ordered mesoporous structures could be observed in the bulk hydrogels.
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RESULTS AND DISCUSSION Results showed that particles were successfully formed when Span 80 was used as the structure directing agent (emulsifier). However, no bulk hydrogel could be prepared using the same amphiphile. Most likely, Span 80 (hydrophilic lipophilic balance number, HLB = 4.317,18) is too hydrophobic to enable interaction with PEG-DA in a pure aqueous environment, as in the preparation of bulk hydrogels. From here on, results on hydrogel particles will be discussed and compared to bulk hydrogels formed without Span 80. TEM micrographs of the particle samples revealed that these contained spherical well-dispersed polymer particles, as is shown in Figure 2. The average size of the particles was
Figure 3. TEM micrographs on PEG-DA bulk hydrogels prepared using the freeze-etching technique.
SAXS measurements showed that the formed hydrogel particles were ordered at a meso-range, which was observed as three distinct peaks in the diffractogram at q = 0.65 nm−1, q = 0.92 nm−1, and q = 1.20 nm−1 (see Figure 4a). The ordering of
Figure 4. SAXS diffractograms shown as the arbitrary intensity as a function of the q-value (nm−1) of a hydrogel bulk and hydrogel particles are shown in panel a. The *’s demonstrate the peak corresponding to the distance between cross-link junctions within the hydrogels, and the arrows show Bragg peaks corresponding to a hexagonal meso-ordered structure. In panel b, a PLM micrograph of the hydrogel particles is presented showing that polarized light was scattered by the particles confirming the presence of long-range order.
Figure 2. TEM micrographs of PEG-DA particles. Panels a and b show micrographs prepared according to the mica sandwich technique and panels c and d show micrographs of agarose embedded particles. In panel b, cross-link junction distances and Pt grains are highlighted. In panel d, the 5 nm mesopores can be seen.
the structure, such as hexagonal, cubic, and lamellar, are retrieved from the relative positions of the Bragg peaks in SAXS diffractograms.20 In the SAXS data obtained from the particles, the relative peak position for the three sharp peaks demonstrates that the particles might have a distorted hexagonal structure21,22 A distortion in the cylindrical packing results in that the cross-section of the cylinders is no longer perfectly circular. The deformation of the cylinders increases with increasing deviation from the typical degree of 120° for hexagonal structures. In diffractograms obtained from both PEG-DA bulk hydrogels and particles (Figure 4), a relatively broad peak marked with a star (*) is visible. This peak represents the distance between cross-link junctions between the polymer segments.6,13 The different positions of this peak; for bulk hydrogels (q = 1.2 nm−1) and particles (q = 1.7 nm−1) demonstrates that the cross-link junction distance was shorter (3.7 nm) within the hydrogel particles compared to the bulk materials (5.2 nm). This difference is probably due to the fact
measured using dynamic light scattering (DLS) to be 267 ± 21 nm. DLS and TEM were performed on dried and redispersed particles, which indicate that the system is highly stable for storage in dry state. The particles were observed to be mesoporous with a pore size of ∼5 nm according to Figure 2d. At high magnifications, cross-link junctions were possible to observe, which were visualized either as dark regions that are elevated from the hydrogel surface or as brighter regions (holes in TEM micrographs) when taken on replicas prepared following the mica sandwich technique, as is illustrated in Figure 2b. The distance between cross-link junctions was estimated to be approximately 3 to 5 nm in average. The black spots seen in the micrographs prepared according to the mica sandwich technique represent contamination from platinum (Ptgrain size ∼ 2 nm).19 14
DOI: 10.1021/la504710a Langmuir 2015, 31, 13−16
Letter
Langmuir
choosing Span 80 was, however, its ability to form kinetically stable w/o emulsions.24 Probably, the emulsion droplets reduce the flexibility of the polymer segments before cross-linking, resulting in the hydrophilic polymer segments being forced to interact with the rather hydrophobic emulsifiers. These spatial restrictions in the system are accordingly hypothesized to facilitate the formation of ordered hydrogel particles having a structure similar to hexagonal.
that the polymer segments are more restricted within the emulsion droplets compared to the flexibility in solution resulting in the formed particles becaming more densely crosslinked. It has previously been shown that the cross-link junction distance decreases with increasing polymer concentration due to restrictions of the polymer segments,6 which agrees well with our observations. By comparing TEM micrographs for hydrogel particles and bulk hydrogels, it was confirmed that the structure of the particles was more dense as compare to the bulk hydrogels, which were observed to be more open and to contain large pores. The pore size and volume fraction for the ordered hydrogel particles were calculated to be 4.6 nm and 0.76, respectively, using SAXS data (see Supporting Information for details). In those calculations the particles were assumed to be hexagonally ordered. A weak peak is observed at about 0.22 nm−1 for the bulk sample; however, no possible explanation for this peak can be found. PLM measurements showed (Figure 4b) that the particles were birefringent, hence confirmed the SAXS results with respect to the presence of a long-range meso order. From PLM it could also be concluded that all particles were anisotropic, since birefringence was observed. PLM also indicated that the synthesis procedure resulted in homogeneous material, since all parts of the sample provided the same birefringence. The ordering of the structure, hexagonal or lamellar, could not be retrieved from the PLM analysis; however, it is believed that the particles have a distorted hexagonal order because of the apparent SAXS results. PLM analysis of the bulk hydrogel did not reveal any birefringence. In 2H NMR spectra obtained from samples dispersed in D2O (see Figure S4 in the Supporting Information), no splitting of the D2O peak could be observed. Theoretically, a split of the peek is observed for D2O within an anisotropically ordered material, e.g., hexagonal or lamellar, compared to the surrounding and isotropic bulk D2O environment. However, the diffusive transport of D2O molecules between the pores and the surrounding isotropic environment is, for our system, much faster than the time scale of the NMR experiment. Since the size of the particles in this study was measured using DLS to be 267 ± 21 nm, it is reasonable that they are too small to give rise to such split, which accordingly explains the lack of peak splitting. Our group has previously shown that meso-ordered lamellar structured PEG-DA (MW = 1500 g/mol) bulk hydrogels were possible to synthesize when a triblock copolymer (Pluronic P123, poly(ethylene glycol)20−poly(propylene glycol)70−poly(ethylene glycol)20, MW = 5800 g/mol) was used as structure directing agent.6 No other structures were possible to form, which was attributed to the relatively long length of the PEGDA segments. However, when shorter PEG-DA segments (MW = 258 or 575 g/mol) are combined with smaller templating molecules (Brij-56 surfactant, MW = 682 g/mol) also other types of meso-ordered bulk hydrogels have been formed.7−9 The surfactant used in this present study, Span 80, is smaller than Brij-56, which accordingly would facilitate the formation of ordered hydrogels, even though rather large PEG-DA macromolecules are used (MW = 1500 g/mol). Also the hydrophilicity of the structure-directing agent has shown to determine whether meso-ordered hydrophilic hydrogels, like PEG-DA hydrogels, are formed.6 More hydrophobic molecules such as Span 80 (HLB = 4.317,18) as compared to Brij-56 (HLB = 12.923) are considered to be a disadvantage in the formation of ordered hydrogels due to phase separation. The reason for
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CONCLUSION In summary, highly ordered mesoporous hydrogel particles were successfully prepared using a w/o emulsion-based technique. It was shown that the distance between cross-link junctions was shorter within the particles than within bulk hydrogels, which was concluded to be because PEG-DA segments experience more restrictions during the cross-linking procedure when these are dispersed in an emulsion as compared to in pure water. This hypothesis was confirmed from TEM micrographs, which clearly showed that the polymer particles were denser than the pure bulk hydrogels. The crosslink distance within the particles was measured to be about 3 to 5 nm. From calculations, the pore size and the pore volume fraction of fully swollen particles, which were assumed to have a hexagonal structure, were 4.6 nm and 0.76, respectively. These findings demonstrate that it is possible to synthesis wellordered hydrogel particles, which might be designated for drugdelivery applications.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental procedures and additional characterization. Also, calculations for the volume fraction and pore size of the hydrogel particles are included. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support was obtained from the Swedish Research Council, VR 2008-3660, and the Materials Area of Advance, Chalmers University of Technology. SAXS measurements on PEG-DA hydrogels were performed at the Stanford Radiation Light source (SSRL). The author thanks John Polpe at SSRL for assistance with SAXS measurements on beamline 1-4 and Kristin Engberg and Curtis W. Frank at Stanford University for valuable discussions. The authors thank Sylvio Hass for assistance with SAXS measurements on the PEG-DA particles, which were carried out at Maxlab (Lund, Sweden) on beamline I911. 2H NMR spectra were recorded at the Swedish NMR Centre.
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