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Photoinduced Diffusion Through Polymer Networks Yuan Meng, Christopher R. Fenoli, Alan Aguirre-Soto, Christopher N. Bowman, and Mitchell Anthamatten* Improving the spatiotemporal control of molecular transport within polymer materials is increasingly desired for applications in areas such as drug delivery,[1,2] biological imaging,[3] and the fabrication of advanced optical components.[4–6] In addition to controlling molecular release, stimulated diffusion through networks could be used to deploy active molecules to surfaces for biorecognition[7] or to create chemical patterns for additive manufacturing.[8] Here, a new strategy to precisely control mass transport through bulk polymer media is demonstrated, whereby chemical species are bonded to a polymer network, but become mobile upon irradiation with light, affording spatio-temporal resolution. Photosensitive compounds are incorporated within the network and lead to active intermediates (e.g., radicals) upon light exposure. These radicals then react with selected moieties (e.g., those capable of additionfragmentation) throughout the network to enable a fraction of network bonds to rapidly undergo bond exchange, leading to concentration gradient-driven diffusion of the released functional groups. Modulated exposure can be activated by adjusting its wavelength, intensity and location, and the light source can be turned off, causing a rapid cessation of the molecular response and bond exchange. In the following, we show initial evidence that photomediated addition-fragmentation chemistry is able to precisely control mass transport. We demonstrate: i) diffusion of covalently bound functional species that become mobile upon irradiation with light; ii) photoinduced extraction of small molecule species from a solvent-swollen network; and iii) localized photoinduced transport of species across a material interface. The allyl sulfide moiety was selected as the addition-fragmentation moiety to incorporate throughout the network because it is synthetically accessible in a variety of monomer structures and it has a demonstrated ability to undergo radicalmediated addition-fragmentation with excellent exchange efficiency and stability.[9,10] Chemical networks containing allyl sulfide or trithiocarbonate functional groups in the backbone undergo efficient bond reshuffling in the presence of free radicals via addition-fragmentation chain transfer (AFCT).[11–13] This ability to reorganize network structures can bring about photoplasticity of polymer networks[14,15] and solvent-assisted Y. Meng, Prof. M. Anthamatten Department of Chemical Engineering University of Rochester 206 Gavett Hall, Rochester, New York 14627, USA E-mail: [email protected] C. R. Fenoli, A. Aguirre-Soto, Prof. C. Bowman Department of Chemical and Biological Engineering University of Colorado UCB 596, Boulder, Colorado 80303, USA
DOI: 10.1002/adma.201402097
Adv. Mater. 2014, DOI: 10.1002/adma.201402097
reformation of network structures;[16] it has facilitated further advances involving photoinduced actuation,[17] self-healing,[18,19] mechano-photopatterning,[20] and localized functionalization of hydrogel networks.[21] In this work, allyl sulfide functional groups are used to connect pendant arms to a covalent network. Upon photomediated radical generation, the additionfragmentation process, shown in Figure 1, generates mobile free radical species that diffuse and subsequently react elsewhere in the network. Rapid termination of diffusible radicals is likely responsible for ceasing the cascade of reaction events when radical generation is stopped.[11] Thus, continuous irradiation and adequate photoinitiator supply are both necessary to drive diffusion. This mechanism then motivates and justifies the idea of photoinduced diffusion, which, in principle, can be spatio-temporally controlled. We prepared a thiol-acrylate Michael addition network with pendant arms that can release and diffuse upon UV irradiation. The base-catalyzed thiol-Michael “click” reaction between thiol and acrylate functional groups is employed to create welldefined polymer networks via a rapid, non-radical mechanism, ensuring preservation of both the allyl sulfide and photoinitiator moieties.[22] As shown in Figure 2, the polymer film was formed from a stoichiometrically balanced (i.e., a 1:1 overall ratio of thiol:acrylate groups) formulation of pentaerythritol tetra(3-mercaptopropionate), (PETMP), (1); tetraethylene glycol diacrylate (TEGDA) (2); and an asymmetric allyl sulfide containing an acrylate group and fluorophore (3). The mixture was loaded with 2 wt% of a photoinitiator that absorbs across the UV spectrum (2,2-dimethoxy-2-phenylacetophenone (DMPA)). Here, tetra-functional 1 and difunctional 2 build up the framework of the covalent network, while 3 provides the pendant arms attached to the photolabile moiety. Fourier transform infrared (FTIR) spectroscopy indicated that the resulting film was nearly fully cured, and DSC revealed a low glass-transition temperature of −27 °C, offering molecular matrix mobility at ambient temperature (see Figure S2, Supporting Information). In the fully cured network, fluorescent moieties having excitation and emission wavelengths at 365 and 500 nm, respectively, are covalently linked to the network through dynamic bonds. UV irradiation of the network plays two important roles: it enables fluorophore release and diffusion through additionfragmentation, and it leads to fluorescence emission. During irradiation, we presume that the formation of reactive termination products such as disulfides can be neglected. Termination products can only be formed at a maximum level approaching the initiator concentration, which is much less than the reactive allyl sulfide concentration used in all the gels evaluated here. Photoinduced diffusion of fluorescent arms through the bulk network was demonstrated using fluorescence recovery after photobleaching (FRAP). FRAP is most often used to study diffusion through biological media, but FRAP has also
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Figure 1. Scheme showing photomediated AFCT reaction cascade within a polymer network containing arms bearing pendant group R attached via an allyl sulfide linkage. The wavy arrow indicates that the R-S• is unattached to the network, mobile, and capable of diffusing through the network prior to adding to another allyl sulfide in a different location.
recently proven useful to characterize diffusion through covalent and transient polymer networks.[23,24] This technique involves applying intense irradiation to a small volume of material, causing photobleaching of fluorophores. The subsequent diffusion of unbleached fluorophores into the bleached region — fluorescence recovery — is then monitored to study diffusive transport. A FRAP experiment was performed on a 300 µm-thick thiol-acrylate Michael addition film using a customized setup, and the results are shown in Figure 3. A circular region of the film with a diameter of 48 µm was photobleached using a UV laser (35 mW cm−2) at 356 nm for 40 s. Fluorescence recovery was subsequently monitored for 20 minutes using bright-field microscopy while irradiating the whole film with a much lower intensity (0.6 mW cm−2) of longer wavelength light at 365 nm. In a conventional FRAP experiment, fluorescence recovery occurs right after photobleaching (typically over millisecond times) because dye molecules are unconstrained. However, during our experiment, no fluorescence recovery was observed, even after long periods of time (>30 min), in the absence of UV irradiation. Without UV light, active fluorophores outside the bleached region remain covalently bound to the network and are unable to diffuse. Only with UV light, fluorescence arms are mobile, and fluorescence recovery is nearly complete after about five minutes.
The observed fluorescence recovery during UV-irradiation was analyzed to determine the diffusion coefficient of arms moving through the thiol-Michael network, DAN. Images were acquired every 6 s during recovery, and analysis was conducted to determine how the average grayscale pixel intensity varies radially with time. The results are shown in Figure 3. Immediately following photobleaching (t = 0), the center-most region (0 < r < 48 µm) was uniformly bleached and exhibited a relatively low level of fluorescence. With increasing r, the intensity rises to a plateau, which represents the unbleached background area. During irradiation (t > 0), the intensity in the center-most region gradually escalates to the level of the background intensity where it remains. This outcome is attributed to the nearly complete recovery of the bleached region. Note that significant bleaching of the whole film occurred during photoinduced recovery and this effect was accounted for. An analysis method by Axelrod was applied to determine DAN from the fluorescence recovery curves.[25] The amount of recovered fluorescence, F(t) was plotted against time t and fit using: ∞
( − K )n
n =0
1 + n (1 + τ2Dt )
F (t ) = A∑
n!
(1)
Figure 2. Reaction of multifunctional thiols with acrylates to form a network containing fluorescent arms with FMOC (fluorenylmethyloxycarbonyl) groups that become mobile upon irradiation with light. Irradiation leads to radical generation followed by addition-fragmentation in which the fragmentation decouples the FMOC groups from the network. The decoupled FMOC groups are then able to diffuse until undergoing addition into an allyl sulfide which recouples them to the network.
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COMMUNICATION Figure 3. Fluorescence intensity (IF) plotted against the distance from the center of the bleached spot following photobleaching of a thiol-acrylate Michael addition network containing fluorescent arms during irradiation. The data have been corrected to remove the additional photobleaching that occurred during irradiation (see Supporting Information, Section S3). The inset shows a plot of average fluorescence intensity in the bleached area vs. time for the experiment and the model.
where A is an amplitude parameter, K is a parameter indicating the amount of bleaching, and τD, the characteristic diffusion time, defined by:
τD =
w2 DAN
(2)
where w is the radius of the bleached region, and DAN is the diffusion coefficient. Least-squares fitting of Equation 1 to the averaged peak intensity of the bleached region resulted in a diffusion coefficient of DAN = 2.6 × 10−8 cm2 s−1. A few notable features are apparent when examining the recovery curves in Figure 3. First, the fluorescence observed within the photobleached area remains nearly constant throughout the recovery process. This behavior indicates that diffusion of unbleached fluorophores within the bleached region occurs significantly faster than diffusion outside the bleached region. We suspect that the photoinitiator is nearly depleted within the bleached region, and non-radical, mobile species (e.g., the allyl mono-sulfide) are free to diffuse without reaction. A second interesting observation is the presence of a ring of suppressed fluorescence intensity that appears at the rim of the bleached region. One possible explanation for the appearance of the dark ring follows: during bleaching, a fraction of mobile species containing deactivated fluorophores diffuses outside of the photobleached region and reacts with the network through AFCT events. For example, reactions between thiyl radicals containing deactivated fluorenyl groups with thiyl radicals attached to the network could form disulfide bonds connecting deactivated fluorophores to the network. The resulting disulfides may further react with radical species, forming oxidatively stable bonds between the network and the deactivated groups.[26] Reactions like these would explain how a fraction of deactivated fluorophore permanently accumulates
Adv. Mater. 2014, DOI: 10.1002/adma.201402097
at the rim, causing the periphery to appear darker than the rest of the bleached area. It may be possible to utilize this effect to create periodic distributions of functional groups by simple irradiation of networks using a mask or interference patterns. Photoinduced extraction was performed on specimens swollen with solvent. Films were first swollen with tetrahydrofuran resulting in an equilibrium solvent content of ca. 54 wt%. We employed a customized setup (Figure 4a), allowing for real-time monitoring of solution fluorescence during UVinduced fluorophore release. As noted in the FRAP experiment, UV irradiation at 365 nm not only releases the fluorophore via photomediated AFCT, but it also excites fluorescence enabling the solution concentration of released fluorophore to be monitored (Figure 4b). In order to correlate the observed solution fluorescence intensity with the solution fluorophore concentration, individual calibration curves were constructed for each irradiation intensity examined (Figure S4, Supporting Information). With an irradiation intensity of 60 mW cm−2, the solution fluorophore concentration gradually increased from zero and appeared to approach an asymptotic value. Raising the intensity to 77 mW cm−2 resulted in faster fluorophore release. During this run, the irradiation intensity was intermittently shut off. Without irradiation, no fluorescence emission proceeded, and upon recommencement of irradiation, continuity in the emission response was observed (Figure 4c), suggesting no diffusion took place when the light was off. Through the course of the experiments, 59% and 84% of fluorophore were extracted from the films during irradiation under UV intensity of 60 and 77 mW cm−2, respectively. A simplified mass transport model was applied to estimate the diffusion coefficient of fluorophore arms through a solvent swollen network, DAS. The model assumes: i) the initial concentration of fluorophore is uniform within the film; ii) Fickian diffusion is one dimensional, perpendicular to the film surface;
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Figure 4. Photoinduced extraction of fluorophores from a solvent-swollen polymer film. a) Illustration of the experimental setup, 365 nm light was directed into the cuvette from the top and caused the release of fluorophore from the film into solution; solution fluorescence emission was detected, orthogonally, from the side of the cuvette. b) Photograph of THF solution containing extracted fluorophore (top vial) and pure THF solvent (bottom vial) under UV irradiation. c) Plot of calculated solution fluorophore concentration (black dots) versus time during photoinduced extraction. The solid lines correspond to least-squares fits to Equation 3, and the dashed lines correspond to the times when the excitation light was turned off.
iii) the surface concentration of fluorophore within the film, at both surfaces is negligible. This model is able to predict the concentration of fluorophore in solution as a function of time (see Supporting Information, Section S5 for details): Cs (t ) =
t 8 AC f0 L ∞ 1 ⎡ − n 2π 2DAS ⎤ 1− e ∑ ⎥ n = 1,3,5... 2 2 2 ⎢ π Vs n = 1 n ⎣ L ⎦
(3)
where A is the area of the film, Cf0 is the initial fluorophore concentration within the film, L is the thickness of the film, VS is the volume of the solution and DAS is the diffusion coefficient of mobile fluorophore travelling in the swollen crosslinked film. The fit curves showed good agreement with experimental data, as shown in Figure 3. Diffusion coefficients obtained from fits to the model under irradiation intensities of 60 mW cm−2 and 77 mW cm−2 are 1.4 × 10−7 cm2 s−1 and 3.8 × 10−7 cm2 s−1, respectively. These values are almost an order of magnitude higher than that determined from FRAP studies discussed earlier (DAN = 2.6 × 10−8 cm2 s−1). This difference is attributed to the fact that mobile solvent molecules are present and open up the mesh size of the swollen crosslinked network, imparting greater mobility and decreasing the allyl sulfide concentration. The decrease in allyl sulfide concentration will increase the typical lifetime of the diffusing species in between its decoupling through fragmentation and its recoupling by addition to another allyl sulfide. Since the fluorescent groups employed here could be replaced with other functional groups, this type of photoinduced extraction could offer outstanding, spatiotemporally controlled release. The method is potentially useful for drug delivery systems because it could avoid unintended drug release. Localized transport of functional groups across an interface between two thiol-acrylate Michael addition films was demonstrated using photoirradiation through a mask. As illustrated in
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Figure 5, mobile fluorophores transport from the top, “donor” layer, across an interface, and into the bottom, “receiver” layer — but only in the regions of film that are exposed to light. In our experiment, the donor layer comprised of a thiol-acrylate Michael addition network containing fluorophores attached via a photoresponsive linkage, and the receiver layer was a similarly formed network containing allyl sulfide moieties, but no pendent fluorophores. Both networks were loaded with DMPA which forms free radicals upon appropriate light exposure. The two films were compressed against each other between two quartz slides, and the composite stack with the fluorescent film on the top was irradiated through a mask. The photogenerated radicals initiate the AFCT reaction cascade, allowing diffusion of fluorescent groups from the donor to receiver layer in spatially confined regions. The presence of the allyl sulfide groups in the receiver layer permits the film to covalently bond to the mobile fluorophores, creating contrast between the exposed and masked regions of the receiver film as shown in Figure 5. This experiment suggests that spatio-temporally controlled transport through AFT-containing networks could be used to form complex 2D or 3D patterns. In summary, we have prepared novel thiol-acrylate Michael addition networks with functional groups that become mobile only during irradiation with UV light. Functional groups are connected to the network by allyl sulfide bonds, and photogenerated free radicals initiate a cascade of AFCT reaction events, enabling transport of functional groups. Three modes of light-induced mass transport of functional arms were shown: i) photoinduced diffusion of arms through the bulk network; ii) photoinduced extraction, i.e., release of arms from a solvent-swollen film into solution; and iii) stimulated transport of arms across a material interface. Light-mediated diffusion could improve drug delivery because drug could be precisely released upon demand; this approach could possibly avoid the
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COMMUNICATION Figure 5. Localized photoinduced transport of a fluorescent species across a material interface. a) Cartoon showing the process: a fluorophore-containing donor layer in good contact with a receiver layer was placed under a mask and exposed to 365 nm light. Fluorophore transport is only permitted in the UV-exposed regions, resulting in pattern replication of the mask. b) Formulation of the receiver film; the molar ratio of the three employed components is 1.75:3:1, resulting in similar crosslink density as the donor film. c) Photograph of mask pattern. d) Fluorescence image, under 365 nm light, of pattern transfer achieved on the receiver film (“Meliora” means “ever better”). The turquoise color indicates the presence of the FMOC fluorophore.
undesirable initial “burst” effect of drug release observed in commonly encountered delivery systems.[27] The concept could also have far reaching implications relating to additive manufacturing and the fabrication of advanced optical materials. Finally, the use of the thiol-acrylate reaction with pure small molecule starting materials provides a framework to study fundamentals of molecular transport through polymer networks with well-defined architectures.
Experimental Section Materials: The asymmetric 2-((1-((2-(2-(9H-fluoren-9-yl)acetoxy)ethyl)thio)vinyl)thio) ethyl acrylate (3) was obtained through a threestep synthesis. Briefly, first, 3-chloro-2-chloromethyl-1-propene was added dropwise into a solution of sodium methoxide containing 2-mercaptoethanol and allowed to reflux overnight. Then, the purified bifunctional product was reacted with acryloyl chloride at a molar ratio of 1:1.1 under a typical base-mediated acylation mechanism. The monofunctionalized product was column-purified and reacted with fluorenyl methyloxy carbonyl chloride to receive the desired asymmetric structure. NMR spectra were acquired on a Bruker AVANCE-III 400 NMR Spectrometer system operating at 400.13 MHz for 1H observation. Solvent chemical shifts were referenced using Mestrenova chemistry software. See Supporting Information, Section S1 for the complete synthetic detail as well as the NMR analysis of the product. 2-methylenepropane-1,3-(thioethyl acrylate) was synthesized according to a method described in the literature.[28] All the other compounds were obtained from Sigma–Aldrich.
Adv. Mater. 2014, DOI: 10.1002/adma.201402097
Network Fabrication: Networks were formulated with 2 wt% DMPA in a stoichiometric (1:1 based on thiol:acrylate functional groups) 1.75:3:1 mixture of compounds 1, 2 and 3 (compounds 1, 4 and 5 for the receiver film). Soon after formulation (within 10 min), a catalytic amount of triethylamine (0.01 g) was added to approximately 1.0 g of the mixture, while vortexing. The mixture was immediately sandwiched between two glass slides separated by a 300 µm-thick polyester spacer. The samples were allowed to cure overnight and IR spectroscopy (8000S, Shimadzu) confirmed complete consumption of the thiol and acrylate functional groups. DSC (Q2000, TA instrument) was employed to measure the glass-transition temperature. Characterization: FRAP was performed on a fluorescence microscope (Zeiss Axiovert 200M). A 48 µm-diameter circular area on the sample film was bleached by a 35 mW cm−2 UV laser at 356 nm. The bleached sample was then irradiated with a (UVGL-25, Mineralight) UV lamp and observed under bright field microscopy over a 20 min recovery period. For photoinduced solvent extraction, a 1 cm × 1 cm quartz cuvette was loaded with 3 mL of THF solvent (fluorescence grade). A fluorescent film weighing 15 mg was swollen in the solvent for 20 min before the test. A mercury lamp (EXFO Acticure 4000, Lumen Dynamics) was used to deliver 365 nm light for fluorescence excitation of the solution phase. The incident intensity was measured with a radiometer (6253, International Light Technologies, Peabody, MA) within the 250–400 nm range. A fiber optic UV–vis spectrometer (USB4000FL-UV–VIS Miniature Fiber Optic Spectrometer, Ocean Optics, Dunedin, FL, USA) was employed to monitor fluorescence emission around 500 nm. Photoinduced diffusion across an interface was studied by stacking a donor fluorescent film (3 cm × 2 cm) and a receiver film (3 cm × 2 cm) between two glass slides, and bubbles between films were avoided to guarantee complete contact. A mask was placed on the top of the donor film. A mercury lamp was used to deliver 365 nm light for 10 min to induce diffusion in exposed areas, from the donor film to the receiver film.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The authors acknowledge support from funding provided by the National Science Foundation under Grant CBET-1264298 and a Pump Primer Award from the University of Rochester. The authors thank Tejas Khire for assistance with FRAP measurements and Shujie Chen for image analysis. Received: May 10, 2014 Revised: June 25, 2014 Published online:
[1] Y. Wang, M. Shim, N. Levinson, H.-W. Sung, Y. Xia, Adv. Funct. Mater. 2014, 24, 4206–4220. [2] A. M. Kloxin, A. M. Kasko, C. N. Salinas, K. S. Anseth, Science 2009, 324, 59–63. [3] Y. R. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez, W. H. Li, J. Am. Chem. Soc. 2004, 126, 4653–4663. [4] D. J. Broer, G. N. Mol, J. A. M. M. van Haaren, J. Lub, Adv. Mater. 1999, 11, 573–578. [5] D. Katsis, D. U. Kim, H. P. Chen, L. J. Rothberg, S. H. Chen, Chem. Mater. 2001, 13, 643–647. [6] K. A. Arpin, A. Mihi, H. T. Johnson, A. J. Baca, J. A. Rogers, J. A. Lewis, P. V. Braun, Adv. Mater. 2010, 22, 1084–1101. [7] K. Vats, D. S. W. Benoit, Tissue Eng., Part B 2013, 19, 455–469. [8] C. Wendeln, B. J. Ravoo, Langmuir 2012, 28, 5527–5538. [9] G. F. Meijs, T. C. Morton, E. Rizzardo, S. H. Thang, Macromolecules 1991, 24, 3689–3695.
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[10] G. F. Meijs, E. Rizzardo, S. H. Thang, Macromolecules 1988, 21, 3122–3124. [11] T. F. Scott, A. D. Schneider, W. D. Cook, C. N. Bowman, Science 2005, 308, 1615–1617. [12] R. Nicolay, J. Kamada, A. Van Wassen, K. Matyjaszewski, Macromolecules 2010, 43, 4355–4361. [13] W. D. Cook, T. L. Schiller, F. Chen, C. Moorhoff, S. H. Thang, C. N. Bowman, T. F. Scott, Macromolecules 2012, 45, 9734–9741. [14] C. J. Kloxin, T. F. Scott, C. N. Bowman, Macromolecules 2009, 42, 2551–2556. [15] H. Y. Park, C. J. Kloxin, A. S. Abuelyaman, J. D. Oxman, C. N. Bowman, Macromolecules 2012, 45, 5640–5646. [16] Y. Amamoto, H. Otsuka, A. Takahara, K. Matyjaszewski, ACS Macro Lett. 2012, 1, 478–481. [17] T. F. Scott, R. B. Draughon, C. N. Bowman, Adv. Mater. 2006, 18, 2128–2132. [18] Y. Amamoto, H. Otsuka, A. Takahara, K. Matyjaszewski, Adv. Mater. 2012, 24, 3975–3980. [19] Y. Amamoto, J. Kamada, H. Otsuka, A. Takahara, K. Matyjaszewski, Angew. Chem. Int. Ed. 2011, 50, 1660–1663. [20] C. J. Kloxin, T. F. Scott, H. Y. Park, C. N. Bowman, Adv. Mater. 2011, 23, 1977–1981. [21] N. R. Gandavarapu, M. A. Azagarsamy, K. S. Anseth, Adv. Mater. 2014, 26, 2521–2526. [22] D. Nair, M. Podgorski, S. Chantani, T. Gong, W. Xi, C. Fenoli, C. N. Bowman, Chem. Mater. 2014, 26, 724–744. [23] J. H. Li, K. D. Sullivan, E. B. Brown, M. Anthamatten, Soft Matter 2010, 6, 235–238. [24] A. Bertz, J. E. Ehlers, S. Wohl-Bruhn, H. Bunjes, K. H. Gericke, H. Menzel, Macromol. Biosci. 2013, 13, 215–226. [25] D. Axelrod, D. E. Koppel, J. Schlessinger, E. Elson, W. W. Webb, Biophys. J. 1976, 16, 1055–1069. [26] I. V. Koval, Russian Chem. Rev. 1994, 63, 735–750. [27] X. Huang, C. S. Brazel, J. Controlled Release 2001, 73, 121–136. [28] C. R. Fenoli, C. N. Bowman, Polym. Chem. 2014, 5, 62–68.
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Adv. Mater. 2014, DOI: 10.1002/adma.201402097
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Photoinduced Diffusion Through Polymer Networks Yuan Meng, Christopher R. Fenoli, Alan Aguirre-Soto, Christopher N. Bowman, and Mitchell Anthamatten* Improving the spatiotemporal control of molecular transport within polymer materials is increasingly desired for applications in areas such as drug delivery,[1,2] biological imaging,[3] and the fabrication of advanced optical components.[4–6] In addition to controlling molecular release, stimulated diffusion through networks could be used to deploy active molecules to surfaces for biorecognition[7] or to create chemical patterns for additive manufacturing.[8] Here, a new strategy to precisely control mass transport through bulk polymer media is demonstrated, whereby chemical species are bonded to a polymer network, but become mobile upon irradiation with light, affording spatio-temporal resolution. Photosensitive compounds are incorporated within the network and lead to active intermediates (e.g., radicals) upon light exposure. These radicals then react with selected moieties (e.g., those capable of additionfragmentation) throughout the network to enable a fraction of network bonds to rapidly undergo bond exchange, leading to concentration gradient-driven diffusion of the released functional groups. Modulated exposure can be activated by adjusting its wavelength, intensity and location, and the light source can be turned off, causing a rapid cessation of the molecular response and bond exchange. In the following, we show initial evidence that photomediated addition-fragmentation chemistry is able to precisely control mass transport. We demonstrate: i) diffusion of covalently bound functional species that become mobile upon irradiation with light; ii) photoinduced extraction of small molecule species from a solvent-swollen network; and iii) localized photoinduced transport of species across a material interface. The allyl sulfide moiety was selected as the addition-fragmentation moiety to incorporate throughout the network because it is synthetically accessible in a variety of monomer structures and it has a demonstrated ability to undergo radicalmediated addition-fragmentation with excellent exchange efficiency and stability.[9,10] Chemical networks containing allyl sulfide or trithiocarbonate functional groups in the backbone undergo efficient bond reshuffling in the presence of free radicals via addition-fragmentation chain transfer (AFCT).[11–13] This ability to reorganize network structures can bring about photoplasticity of polymer networks[14,15] and solvent-assisted Y. Meng, Prof. M. Anthamatten Department of Chemical Engineering University of Rochester 206 Gavett Hall, Rochester, New York 14627, USA E-mail: [email protected] C. R. Fenoli, A. Aguirre-Soto, Prof. C. Bowman Department of Chemical and Biological Engineering University of Colorado UCB 596, Boulder, Colorado 80303, USA
DOI: 10.1002/adma.201402097
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reformation of network structures;[16] it has facilitated further advances involving photoinduced actuation,[17] self-healing,[18,19] mechano-photopatterning,[20] and localized functionalization of hydrogel networks.[21] In this work, allyl sulfide functional groups are used to connect pendant arms to a covalent network. Upon photomediated radical generation, the additionfragmentation process, shown in Figure 1, generates mobile free radical species that diffuse and subsequently react elsewhere in the network. Rapid termination of diffusible radicals is likely responsible for ceasing the cascade of reaction events when radical generation is stopped.[11] Thus, continuous irradiation and adequate photoinitiator supply are both necessary to drive diffusion. This mechanism then motivates and justifies the idea of photoinduced diffusion, which, in principle, can be spatio-temporally controlled. We prepared a thiol-acrylate Michael addition network with pendant arms that can release and diffuse upon UV irradiation. The base-catalyzed thiol-Michael “click” reaction between thiol and acrylate functional groups is employed to create welldefined polymer networks via a rapid, non-radical mechanism, ensuring preservation of both the allyl sulfide and photoinitiator moieties.[22] As shown in Figure 2, the polymer film was formed from a stoichiometrically balanced (i.e., a 1:1 overall ratio of thiol:acrylate groups) formulation of pentaerythritol tetra(3-mercaptopropionate), (PETMP), (1); tetraethylene glycol diacrylate (TEGDA) (2); and an asymmetric allyl sulfide containing an acrylate group and fluorophore (3). The mixture was loaded with 2 wt% of a photoinitiator that absorbs across the UV spectrum (2,2-dimethoxy-2-phenylacetophenone (DMPA)). Here, tetra-functional 1 and difunctional 2 build up the framework of the covalent network, while 3 provides the pendant arms attached to the photolabile moiety. Fourier transform infrared (FTIR) spectroscopy indicated that the resulting film was nearly fully cured, and DSC revealed a low glass-transition temperature of −27 °C, offering molecular matrix mobility at ambient temperature (see Figure S2, Supporting Information). In the fully cured network, fluorescent moieties having excitation and emission wavelengths at 365 and 500 nm, respectively, are covalently linked to the network through dynamic bonds. UV irradiation of the network plays two important roles: it enables fluorophore release and diffusion through additionfragmentation, and it leads to fluorescence emission. During irradiation, we presume that the formation of reactive termination products such as disulfides can be neglected. Termination products can only be formed at a maximum level approaching the initiator concentration, which is much less than the reactive allyl sulfide concentration used in all the gels evaluated here. Photoinduced diffusion of fluorescent arms through the bulk network was demonstrated using fluorescence recovery after photobleaching (FRAP). FRAP is most often used to study diffusion through biological media, but FRAP has also
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Figure 1. Scheme showing photomediated AFCT reaction cascade within a polymer network containing arms bearing pendant group R attached via an allyl sulfide linkage. The wavy arrow indicates that the R-S• is unattached to the network, mobile, and capable of diffusing through the network prior to adding to another allyl sulfide in a different location.
recently proven useful to characterize diffusion through covalent and transient polymer networks.[23,24] This technique involves applying intense irradiation to a small volume of material, causing photobleaching of fluorophores. The subsequent diffusion of unbleached fluorophores into the bleached region — fluorescence recovery — is then monitored to study diffusive transport. A FRAP experiment was performed on a 300 µm-thick thiol-acrylate Michael addition film using a customized setup, and the results are shown in Figure 3. A circular region of the film with a diameter of 48 µm was photobleached using a UV laser (35 mW cm−2) at 356 nm for 40 s. Fluorescence recovery was subsequently monitored for 20 minutes using bright-field microscopy while irradiating the whole film with a much lower intensity (0.6 mW cm−2) of longer wavelength light at 365 nm. In a conventional FRAP experiment, fluorescence recovery occurs right after photobleaching (typically over millisecond times) because dye molecules are unconstrained. However, during our experiment, no fluorescence recovery was observed, even after long periods of time (>30 min), in the absence of UV irradiation. Without UV light, active fluorophores outside the bleached region remain covalently bound to the network and are unable to diffuse. Only with UV light, fluorescence arms are mobile, and fluorescence recovery is nearly complete after about five minutes.
The observed fluorescence recovery during UV-irradiation was analyzed to determine the diffusion coefficient of arms moving through the thiol-Michael network, DAN. Images were acquired every 6 s during recovery, and analysis was conducted to determine how the average grayscale pixel intensity varies radially with time. The results are shown in Figure 3. Immediately following photobleaching (t = 0), the center-most region (0 < r < 48 µm) was uniformly bleached and exhibited a relatively low level of fluorescence. With increasing r, the intensity rises to a plateau, which represents the unbleached background area. During irradiation (t > 0), the intensity in the center-most region gradually escalates to the level of the background intensity where it remains. This outcome is attributed to the nearly complete recovery of the bleached region. Note that significant bleaching of the whole film occurred during photoinduced recovery and this effect was accounted for. An analysis method by Axelrod was applied to determine DAN from the fluorescence recovery curves.[25] The amount of recovered fluorescence, F(t) was plotted against time t and fit using: ∞
( − K )n
n =0
1 + n (1 + τ2Dt )
F (t ) = A∑
n!
(1)
Figure 2. Reaction of multifunctional thiols with acrylates to form a network containing fluorescent arms with FMOC (fluorenylmethyloxycarbonyl) groups that become mobile upon irradiation with light. Irradiation leads to radical generation followed by addition-fragmentation in which the fragmentation decouples the FMOC groups from the network. The decoupled FMOC groups are then able to diffuse until undergoing addition into an allyl sulfide which recouples them to the network.
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COMMUNICATION Figure 3. Fluorescence intensity (IF) plotted against the distance from the center of the bleached spot following photobleaching of a thiol-acrylate Michael addition network containing fluorescent arms during irradiation. The data have been corrected to remove the additional photobleaching that occurred during irradiation (see Supporting Information, Section S3). The inset shows a plot of average fluorescence intensity in the bleached area vs. time for the experiment and the model.
where A is an amplitude parameter, K is a parameter indicating the amount of bleaching, and τD, the characteristic diffusion time, defined by:
τD =
w2 DAN
(2)
where w is the radius of the bleached region, and DAN is the diffusion coefficient. Least-squares fitting of Equation 1 to the averaged peak intensity of the bleached region resulted in a diffusion coefficient of DAN = 2.6 × 10−8 cm2 s−1. A few notable features are apparent when examining the recovery curves in Figure 3. First, the fluorescence observed within the photobleached area remains nearly constant throughout the recovery process. This behavior indicates that diffusion of unbleached fluorophores within the bleached region occurs significantly faster than diffusion outside the bleached region. We suspect that the photoinitiator is nearly depleted within the bleached region, and non-radical, mobile species (e.g., the allyl mono-sulfide) are free to diffuse without reaction. A second interesting observation is the presence of a ring of suppressed fluorescence intensity that appears at the rim of the bleached region. One possible explanation for the appearance of the dark ring follows: during bleaching, a fraction of mobile species containing deactivated fluorophores diffuses outside of the photobleached region and reacts with the network through AFCT events. For example, reactions between thiyl radicals containing deactivated fluorenyl groups with thiyl radicals attached to the network could form disulfide bonds connecting deactivated fluorophores to the network. The resulting disulfides may further react with radical species, forming oxidatively stable bonds between the network and the deactivated groups.[26] Reactions like these would explain how a fraction of deactivated fluorophore permanently accumulates
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at the rim, causing the periphery to appear darker than the rest of the bleached area. It may be possible to utilize this effect to create periodic distributions of functional groups by simple irradiation of networks using a mask or interference patterns. Photoinduced extraction was performed on specimens swollen with solvent. Films were first swollen with tetrahydrofuran resulting in an equilibrium solvent content of ca. 54 wt%. We employed a customized setup (Figure 4a), allowing for real-time monitoring of solution fluorescence during UVinduced fluorophore release. As noted in the FRAP experiment, UV irradiation at 365 nm not only releases the fluorophore via photomediated AFCT, but it also excites fluorescence enabling the solution concentration of released fluorophore to be monitored (Figure 4b). In order to correlate the observed solution fluorescence intensity with the solution fluorophore concentration, individual calibration curves were constructed for each irradiation intensity examined (Figure S4, Supporting Information). With an irradiation intensity of 60 mW cm−2, the solution fluorophore concentration gradually increased from zero and appeared to approach an asymptotic value. Raising the intensity to 77 mW cm−2 resulted in faster fluorophore release. During this run, the irradiation intensity was intermittently shut off. Without irradiation, no fluorescence emission proceeded, and upon recommencement of irradiation, continuity in the emission response was observed (Figure 4c), suggesting no diffusion took place when the light was off. Through the course of the experiments, 59% and 84% of fluorophore were extracted from the films during irradiation under UV intensity of 60 and 77 mW cm−2, respectively. A simplified mass transport model was applied to estimate the diffusion coefficient of fluorophore arms through a solvent swollen network, DAS. The model assumes: i) the initial concentration of fluorophore is uniform within the film; ii) Fickian diffusion is one dimensional, perpendicular to the film surface;
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Figure 4. Photoinduced extraction of fluorophores from a solvent-swollen polymer film. a) Illustration of the experimental setup, 365 nm light was directed into the cuvette from the top and caused the release of fluorophore from the film into solution; solution fluorescence emission was detected, orthogonally, from the side of the cuvette. b) Photograph of THF solution containing extracted fluorophore (top vial) and pure THF solvent (bottom vial) under UV irradiation. c) Plot of calculated solution fluorophore concentration (black dots) versus time during photoinduced extraction. The solid lines correspond to least-squares fits to Equation 3, and the dashed lines correspond to the times when the excitation light was turned off.
iii) the surface concentration of fluorophore within the film, at both surfaces is negligible. This model is able to predict the concentration of fluorophore in solution as a function of time (see Supporting Information, Section S5 for details): Cs (t ) =
t 8 AC f0 L ∞ 1 ⎡ − n 2π 2DAS ⎤ 1− e ∑ ⎥ n = 1,3,5... 2 2 2 ⎢ π Vs n = 1 n ⎣ L ⎦
(3)
where A is the area of the film, Cf0 is the initial fluorophore concentration within the film, L is the thickness of the film, VS is the volume of the solution and DAS is the diffusion coefficient of mobile fluorophore travelling in the swollen crosslinked film. The fit curves showed good agreement with experimental data, as shown in Figure 3. Diffusion coefficients obtained from fits to the model under irradiation intensities of 60 mW cm−2 and 77 mW cm−2 are 1.4 × 10−7 cm2 s−1 and 3.8 × 10−7 cm2 s−1, respectively. These values are almost an order of magnitude higher than that determined from FRAP studies discussed earlier (DAN = 2.6 × 10−8 cm2 s−1). This difference is attributed to the fact that mobile solvent molecules are present and open up the mesh size of the swollen crosslinked network, imparting greater mobility and decreasing the allyl sulfide concentration. The decrease in allyl sulfide concentration will increase the typical lifetime of the diffusing species in between its decoupling through fragmentation and its recoupling by addition to another allyl sulfide. Since the fluorescent groups employed here could be replaced with other functional groups, this type of photoinduced extraction could offer outstanding, spatiotemporally controlled release. The method is potentially useful for drug delivery systems because it could avoid unintended drug release. Localized transport of functional groups across an interface between two thiol-acrylate Michael addition films was demonstrated using photoirradiation through a mask. As illustrated in
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Figure 5, mobile fluorophores transport from the top, “donor” layer, across an interface, and into the bottom, “receiver” layer — but only in the regions of film that are exposed to light. In our experiment, the donor layer comprised of a thiol-acrylate Michael addition network containing fluorophores attached via a photoresponsive linkage, and the receiver layer was a similarly formed network containing allyl sulfide moieties, but no pendent fluorophores. Both networks were loaded with DMPA which forms free radicals upon appropriate light exposure. The two films were compressed against each other between two quartz slides, and the composite stack with the fluorescent film on the top was irradiated through a mask. The photogenerated radicals initiate the AFCT reaction cascade, allowing diffusion of fluorescent groups from the donor to receiver layer in spatially confined regions. The presence of the allyl sulfide groups in the receiver layer permits the film to covalently bond to the mobile fluorophores, creating contrast between the exposed and masked regions of the receiver film as shown in Figure 5. This experiment suggests that spatio-temporally controlled transport through AFT-containing networks could be used to form complex 2D or 3D patterns. In summary, we have prepared novel thiol-acrylate Michael addition networks with functional groups that become mobile only during irradiation with UV light. Functional groups are connected to the network by allyl sulfide bonds, and photogenerated free radicals initiate a cascade of AFCT reaction events, enabling transport of functional groups. Three modes of light-induced mass transport of functional arms were shown: i) photoinduced diffusion of arms through the bulk network; ii) photoinduced extraction, i.e., release of arms from a solvent-swollen film into solution; and iii) stimulated transport of arms across a material interface. Light-mediated diffusion could improve drug delivery because drug could be precisely released upon demand; this approach could possibly avoid the
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COMMUNICATION Figure 5. Localized photoinduced transport of a fluorescent species across a material interface. a) Cartoon showing the process: a fluorophore-containing donor layer in good contact with a receiver layer was placed under a mask and exposed to 365 nm light. Fluorophore transport is only permitted in the UV-exposed regions, resulting in pattern replication of the mask. b) Formulation of the receiver film; the molar ratio of the three employed components is 1.75:3:1, resulting in similar crosslink density as the donor film. c) Photograph of mask pattern. d) Fluorescence image, under 365 nm light, of pattern transfer achieved on the receiver film (“Meliora” means “ever better”). The turquoise color indicates the presence of the FMOC fluorophore.
undesirable initial “burst” effect of drug release observed in commonly encountered delivery systems.[27] The concept could also have far reaching implications relating to additive manufacturing and the fabrication of advanced optical materials. Finally, the use of the thiol-acrylate reaction with pure small molecule starting materials provides a framework to study fundamentals of molecular transport through polymer networks with well-defined architectures.
Experimental Section Materials: The asymmetric 2-((1-((2-(2-(9H-fluoren-9-yl)acetoxy)ethyl)thio)vinyl)thio) ethyl acrylate (3) was obtained through a threestep synthesis. Briefly, first, 3-chloro-2-chloromethyl-1-propene was added dropwise into a solution of sodium methoxide containing 2-mercaptoethanol and allowed to reflux overnight. Then, the purified bifunctional product was reacted with acryloyl chloride at a molar ratio of 1:1.1 under a typical base-mediated acylation mechanism. The monofunctionalized product was column-purified and reacted with fluorenyl methyloxy carbonyl chloride to receive the desired asymmetric structure. NMR spectra were acquired on a Bruker AVANCE-III 400 NMR Spectrometer system operating at 400.13 MHz for 1H observation. Solvent chemical shifts were referenced using Mestrenova chemistry software. See Supporting Information, Section S1 for the complete synthetic detail as well as the NMR analysis of the product. 2-methylenepropane-1,3-(thioethyl acrylate) was synthesized according to a method described in the literature.[28] All the other compounds were obtained from Sigma–Aldrich.
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Network Fabrication: Networks were formulated with 2 wt% DMPA in a stoichiometric (1:1 based on thiol:acrylate functional groups) 1.75:3:1 mixture of compounds 1, 2 and 3 (compounds 1, 4 and 5 for the receiver film). Soon after formulation (within 10 min), a catalytic amount of triethylamine (0.01 g) was added to approximately 1.0 g of the mixture, while vortexing. The mixture was immediately sandwiched between two glass slides separated by a 300 µm-thick polyester spacer. The samples were allowed to cure overnight and IR spectroscopy (8000S, Shimadzu) confirmed complete consumption of the thiol and acrylate functional groups. DSC (Q2000, TA instrument) was employed to measure the glass-transition temperature. Characterization: FRAP was performed on a fluorescence microscope (Zeiss Axiovert 200M). A 48 µm-diameter circular area on the sample film was bleached by a 35 mW cm−2 UV laser at 356 nm. The bleached sample was then irradiated with a (UVGL-25, Mineralight) UV lamp and observed under bright field microscopy over a 20 min recovery period. For photoinduced solvent extraction, a 1 cm × 1 cm quartz cuvette was loaded with 3 mL of THF solvent (fluorescence grade). A fluorescent film weighing 15 mg was swollen in the solvent for 20 min before the test. A mercury lamp (EXFO Acticure 4000, Lumen Dynamics) was used to deliver 365 nm light for fluorescence excitation of the solution phase. The incident intensity was measured with a radiometer (6253, International Light Technologies, Peabody, MA) within the 250–400 nm range. A fiber optic UV–vis spectrometer (USB4000FL-UV–VIS Miniature Fiber Optic Spectrometer, Ocean Optics, Dunedin, FL, USA) was employed to monitor fluorescence emission around 500 nm. Photoinduced diffusion across an interface was studied by stacking a donor fluorescent film (3 cm × 2 cm) and a receiver film (3 cm × 2 cm) between two glass slides, and bubbles between films were avoided to guarantee complete contact. A mask was placed on the top of the donor film. A mercury lamp was used to deliver 365 nm light for 10 min to induce diffusion in exposed areas, from the donor film to the receiver film.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The authors acknowledge support from funding provided by the National Science Foundation under Grant CBET-1264298 and a Pump Primer Award from the University of Rochester. The authors thank Tejas Khire for assistance with FRAP measurements and Shujie Chen for image analysis. Received: May 10, 2014 Revised: June 25, 2014 Published online:
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