Article pubs.acs.org/Macromolecules
Photodegradable Hydrogels Made via RAFT Francesca Ercole,† Helmut Thissen,‡ Kelly Tsang,† Richard A. Evans,*,‡ and John S. Forsythe*,† †
Department of Materials Engineering, Monash University, Clayton, Victoria, Australia CSIRO Materials Science and Engineering, Clayton, Victoria, Australia
‡
S Supporting Information *
ABSTRACT: The photodegradation kinetics of reversible addition−fragmentation chain transfer (RAFT) synthesized hydrogels were investigated and compared to conventional free radical gels, using rheological monitoring. The gel polymerizations were conducted on N-acryloylmorpholine (NAM) between parallel plates of a rheometer, in aqueous media and at ambient temperature using the redox couple ammonium persulfate (APS) and N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED). The gels synthesized by RAFT are believed to have a more homogeneous network structure which is thought to be a contributing factor to the faster degradation kinetics displayed by the RAFT gels compared to conventional nonRAFT gels. Difunctional macro-RAFT agents were used to prepare the gels which insert degradable points in backbone chains, as well as the cross-links, to give significantly faster degradation rates. RAFT also provided a platform to carry out a systematic study of the photodegradable gels and demonstrated a limitation of the system, which is the unavoidable attenuation of light intensity by the light active components of the gel. This means that the concentration of photodegradable cross-linker, a parameter which is varied in order to modulate the final storage modulus G′ of the gel, will also have a consequence on the degradation kinetics.
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INTRODUCTION Hydrogels1 find use in various biomedical fields ranging from regenerative medicine,2 drug delivery systems,3 biosensing,4 and tissue engineering5−7 and can be formed from synthetic8,9 and naturally derived10 hydrophilic building blocks. By their nature, they are highly water absorbing, cross-linked, and often rubbery polymeric materials which are commonly used as scaffold materials for tissue constructs, to deliver drugs and biofactors to tissues and cell cultures, and serve as adhesives, barriers, or wound dressings for tissues. Overall, the network structure and properties of a gel, which are fundamentally important for a given application, depend on the building blocks, chemistry, and technique used for their formation.11 The three-dimensional structure of the gel is formed by linking together multifunctional and reactive precursors and can be induced using pH, temperature, and noncovalent interactions or through covalent bonds. A wide variety of chemistries have been utilized for covalent cross-linking of hydrogel materials, such as free radical polymerization (FRP), Michael addition,12 thiol−ene coupling,13 and other click chemistries.14 Regardless of the chemistry that is used to make the gel, a potential drawback of the covalent approach is that the hydrogel’s properties are largely fixed upon formation. An approach which largely mitigates this issue is to incorporate stimuli responsive units within the hydrogel.15−18 This allows the gel to undergo distinct changes in its behavior and properties in response to an externally applied stimulus. Light is a particularly interesting option as a stimulus since it can be activated remotely and can be controlled both spatially and © 2012 American Chemical Society
temporally. Further, its intensity and wavelength can easily be tuned, and by using photomasks or lasers, complex features can be fabricated into the material. Additional increases in the resolution of features can be achieved using confocal microscopy which allows patterns to be generated in a threedimensional manner.19 Frequently, such photoresponsive materials contain photochromic moieties which undergo reversible intramolecular rearrangements upon light exposure to cause a reversible change in properties.18 Another approach to elicit a response using light is through a photocleavage reaction which brings about an irreversible change in properties and behavior in a material. Several aromatic light-sensitive protecting groups have been exploited in this manner, the best known example being an o-nitrobenzyl group.20−25 A novel class of poly(ethylene glycol) (PEG)-based FRP hydrogels whose properties can be precisely controlled by the light irradiation of such moieties was developed by the Anseth group.26 In this system, copolymerization of a PEG-based photocleavable cross-linker with PEG methacrylate resulted in a photodegradable hydrogel platform that can be applied as a 3D cell culture medium to influence chondrogenic differentiation of encapsulated stem cells. Here the decomposition of its network architecture can be triggered remotely (either partially or fully) using light to allow precise spatial and temporal control over degradation of the hydrogel, along with simultaneous release of biomolecules. Received: June 26, 2012 Revised: September 28, 2012 Published: October 9, 2012 8387
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and also the reduced likelihood of residues being released on decomposition with a molecular weight above the renal threshold.54 In this work the same principles and motivations were applied to develop photodegradable hydrogels using RAFT: to simultaneously optimize photodegradation kinetics, control the size of degradation products, and produce more homogeneous networks with cleavable groups distributed more evenly throughout. The primary approach used to achieve this was to include in the hydrogel formulation a difunctional RAFT agent in which the two RAFT groups are located on opposite sides of a photocleavable, nitrobenzyl moiety. In this case, chain growth occurs biradially from a central photocleavable group so that when the gel is formed, it can be made to contain backbone cleavage points as well as cleavable cross-links. This means that when the gel is later degraded, shorter and more defined polymer segments can be released from the gel to allow more efficient degradation compared to a conventional FRP gel. This investigation was carried out by comparing the photodegradation kinetics of RAFT controlled gels, of various compositions, with those synthesized using conventional FRP, as monitored using photorheology.
The aforementioned photodegradable system used conventional FRP to make its network structure which is a widespread and practical approach to make gels. In this context, hydrophilic vinyl monomers are simply copolymerized in the presence of multifunctional vinyl comonomers, which act as cross-linkers.27 While conversions are high and gelation occurs easily and quickly, a drawback of using conventional FRP is that it offers little control over network microstructure.28 During the FRP it takes only seconds for an individual chain to fully grow from initiation to termination. Primary chains with a high molecular weight are formed quickly which contain numerous pendant double bonds. Because of their high dilution and slow diffusion, rapid intramolecular reactions occur between propagating radicals and vicinal pendant double bonds, generating densely cross-linked domains (microgels) which then become connected into a macrogel at later stages of the polymerization.28 Both the chain length between cross-links and the distribution of cross-links within a gel can be highly variable, leading to structural heterogeneity in the final network.29 This lack of control during network formation can result in not only poorly defined materials but also makes it difficult to correlate network structure to the final properties of the gel. Recently, controlled radical polymerization (CRP) techniques, such as reversible addition−fragmentation chain transfer (RAFT)30 and atom transfer radical polymerization (ATRP),31 have been applied to prepare hydrogels and other network structures. 32−37 One advantage these systems provide, compared to conventional FRP, is their ability to form a polymer network with a more homogeneous structure34,38−41 This is due to a number of conducive factors occurring in CRP: a lower concentration of active propagating chains, minimized termination events, and sufficient time available for polymer chains to relax and diffuse. This facilitates intermolecular reactions to occur between radical chain ends and pendant vinyl groups present on other primary chains, forming an insoluble gel with more evenly distributed branching points throughout. Along with concurrent growth and a uniform distribution of chains, all these factors ensure a more ordered network structure. In the high performance applications such as controlled drug release and biomedical materials, where the structural homogeneity of the network is important to gel characteristics, the use of CRP can offer distinct benefits over FRP. These concepts have been applied to the synthesis of gels showing improved control over the release of encapsulated agents,42 increased swelling ratios,39 faster swelling and shrinking kinetics,34,40 and improved mechanical properties.32,33 Apart from facilitating the preparation of a network structure with potentially improved properties, CRP can also be used to prepare the specific building blocks for the network, such as the macroinitiators,40,43,44 covalent cross-linkers,45−47 and gellators for physically cross-linked hydrogels.48−50 Moreover, chain-end functionalities are preserved in gels prepared by CRP so further modifications and chain extensions can also be performed postcuring by using a grafting approach.34,36 Synthetic, degradable network polymers such as hydrogels have come to represent a field of growing importance in medicine and biotechnology and several research groups have already used CRP techniques to synthesize such structures.41,43,51−53 Aside from providing more homogeneous structures, this is an attractive approach to make degradable materials owing to the predeterminable molecular weight and narrow molecular weight distribution of decomposed fragments
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RESULTS AND DISCUSSION The photocleavage of an o-nitrobenzyl ester to form the corresponding carboxylic acid and aromatic nitroso compound is shown in Scheme 1. Traditionally, the o-nitrobenzyl Scheme 1. Photodegradation of o-Nitrobenzyl Ester Derivative
functionality has had widespread application as a photolabile linker and protective group for a number of biologically relevant functional groups, including carboxylic acids, alcohols, amines, thiols, and phosphates. Chemical reagents such as acids or bases are not required for the deprotection, making the process very tolerant toward otherwise sensitive functionalities and offering flexibility in the parallel or individual synthesis of peptides, polysaccharides, nucleosides, and small molecules.20,24 It has been reported that substitution of the parent onitrobenzyl group, either on the ring or in the α-position, can affect the yield and kinetics of the photocleavage reaction.54 For example, the introduction of an α-methyl group onto the benzylic carbon enhances cleavage kinetics.24 The amount as well as position of alkoxy groups on the benzene ring alters the absorbance spectrum and therefore also the photocleavage rate since this process is dependent on the chromophore’s ability to capture a photon at a specific wavelength.55 The approach used in this study was to use a relatively slowto-cleave o-nitrobenzyl derivative which lacks both the α-methyl group on the benzylic carbon and the alkoxy functionality para 8388
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Scheme 2. Synthesis of Photodegradable Building Blocks and RAFT Agents
to the benzylic carbon, factors which promote photocleavage. This allowed us to focus on the effect of the gel’s structure on the photocleavage rates, as opposed to substitution patterns which may override or mask any possible effect arising from the methodology that was used to synthesize the hydrogel. Therefore, in the photocleavable linker that was applied here, R1 and R2 = H and R3 = alkoxy substitution, a substitution pattern that has been applied by other researchers for studying other photocleavable constructs.56−60 Monomers and RAFT Agents. Scheme 2 shows the building blocks (1−8) that were used for the preparation of the cross-linker and RAFT agents, which were easily accessed from a commercially available starting material, 5-hydroxy-2-nitrobenzaldehyde. A primary requirement for the hydrogel preparations was to use reagents that are water-soluble. The photodegradable cross-linker (9) was therefore synthesized using the same procedure as Anseth and co-workers26,61 in which a water-soluble linear PEG tail is covalently bound in between two acrylated o-nitrobenzyl handles, essentially making a telechelic functionalized PEG, as in Scheme 3. In an analogous fashion we synthesized a difunctional photodegradable macro-RAFT agent (10) containing a watersoluble PEG tail in the middle of two o-nitrobenzyl RAFT groups, as in Scheme 4. Overall, we found this method to be very convenient for producing water-soluble macromonomers and RAFT agents but also found that it introduced some variation in the number of EG groups in the polymers isolated. This could be the result of the multiple precipitation steps used in the work-up as well as the different reactants and coupling agents that were applied.
Scheme 3. Synthesis of Photodegradable (PD) PEG CrossLinker 9
The other approach used to make a water-soluble difunctional photodegradable RAFT agent was to first assemble the difunctional RAFT agent (7) in which two RAFT groups are located on opposite sides of one photocleavable, nitrobenzyl moiety. Two polymer arms were then grown biradially from the central photocleavable group, using RAFT, to give watersoluble, photodegradable poly(N-acryloylmorpholine), pNAM macro-RAFT agents 11 and 12, as shown in Scheme 5. As part of our studies we also synthesized some control gels which served as useful comparisons for degradation studies. 8389
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soluble redox couple, ammonium persulfate (APS), and 1,2bis(dimethylamino)ethane (TEMED), yielded photodegradable gels, with idealized structures as depicted in Figures 1 and 2. Both the cross-linker and the RAFT agent serve as sources of degradable groups with resulting hydrogels containing photodegradable units both in the primary chains and in their crosslinks. As depicted in Figure 1, PEG macro-RAFT agent 10 is chain extended on both ends with NAM units during the gel formation with two degradable points being inserted in the primary chains during the gelation process. On degradation, PEG sections are then released from the primary chains as well as from the cross-linker (9) component. Further, cleaved pNAM chains are also released from the primary chains as a result of scission points existing in the macro-RAFT agent itself. In the case of pNAM macro-RAFT agents 11 and 12, the situation is slightly different in that only one degradable point is inserted in the middle of the primary chains during gel formation. Therefore, PEG sections would only be released from the cross-linker, 9, and only pNAM chains, essentially halved, would be released from the main chains. Rheological Monitoring: Gelation and Photodegradation. The gelation and photodegradation properties of the aforementioned systems were studied using isothermal rheological measurement at 25 °C. The dynamic rheology experiments monitored the evolution and variation of the storage modulus G′, first during the polymerization and then during the photodegradation period when the cured gel samples were irradiated in situ using UV light (365 nm). The loss of the storage modulus G′ vs time data was fitted to a monoexponential decay equation:
Scheme 4. Synthesis of Photodegradable (PD) PEG MacroRAFT Agent 10
Scheme 5. Synthesis of Photodegradable (PD) Poly(Nacryloylmorpholine), pNAM, and Macro-RAFT Agents 11 and 12
G′(t ) = G′0 e−kt + G′th
where G′ is the storage modulus, G′0 the initial storage modulus, and G′th the residual storage modulus, to obtain k, a first-order rate constant. This essentially provided a practical method to determine relative cleavage kinetics, therefore allowing the overall kinetic rates for photodegradation of the different hydrogel samples to be compared. When the storage modulus G′ exhibited a pronounced plateau during the curing period which lasted for minutes and, concurrently, the loss modulus G″ displayed a much smaller value than the storage modulus G′, we assigned this G′ value as the final storage modulus for the hydrogel samples. These factors are discussed herein and are tabulated in Tables 1 for reference throughout the discussion. Variation of PD Cross-Linker 9 Concentration in RAFT and Non-RAFT Hydrogels. The first set of experiments
These gels contained either no RAFT component or were made using nonphotodegradable RAFT agent 13 or crosslinker 14. Their synthesis from PEG is shown as Scheme 6. Gel Synthesis. The RAFT polymerization of a mixture of NAM, macro-RAFT agent (10, 11, or 12), and cross-linker (9) in water, initiated under ambient conditions using the water-
Scheme 6. Synthesis of Nonphotodegradable PEG Macro-RAFT Agent 13 and Diacrylate 14
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Figure 1. Synthesis and degradation of photodegradable RAFT hydrogels.
the non-RAFT FR hydrogel G1, but not as efficiently as G5, which was made out of macro-RAFT 10, since their primary chains would only have one cleavage point, vs two in the latter. And in fact, their degradation kinetics showed this to be the case. Interestingly, the use of a shorter pNAM macro-RAFT agent 12 (with MW 3700 g/mol), to give G8 vs G7, made using 11 (with MW 6000 g/mol) did not give a corresponding increase in degradation rates. This is because after chain extension with NAM and cross-linker 9 to give the final hydrogel, the original macro-RAFT agent only accounts for a minor portion of its final MW and network structure. Therefore, by using a shorter macro-RAFT agent 12, this would mean that degraded chains would not be significantly shorter than in the case of macro-RAFT agent 11, and therefore this would make little difference to the degradation kinetics of their resulting gels. We therefore decided to continue our investigations by focusing on hydrogels made with macroRAFT agent 10. Variation of Macro-RAFT Agent Concentration. Another aspect that we tested was whether using a higher proportion of RAFT agent with respect to both monomers
centered on RAFT hydrogels prepared with photodegradable macro-RAFT agent 10. The ratio of NAM:10 was kept constant (66.6:1), and the amount of PD cross-linker 9 was varied (Table 1, G3−G6). Non-RAFT free radical (FR) hydrogels, G1 and G2, were run as controls using only NAM and PD crosslinker 9, with ratios shown in rows 1 and 2 of Table 1. The first thing to note from comparing these samples was that the RAFT hydrogels gave storage moduli G′ which could be tuned efficiently by variation of the cross-linker concentration, and further to that, their photodegradation kinetics were found to be consistently faster than the non-RAFT controls. The k value of G1 can be compared with G5 and that of G2 with G6, since the pairs were made using the same ratio of [NAM]:[9]. In both instances the RAFT gels showed more than double the speed of photodegradation compared to the non-RAFT controls. Figure 3 further depicts this with overlaid photodegradation plots of RAFT and non-RAFT hydrogels. Variation of Macro-RAFT Agent Type. We also tested RAFT hydrogels G7 and G8, synthesized with photodegradable pNAM macro-RAFT agents 11 and 12, respectively. We hypothesized that these hydrogels would degrade faster than 8391
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Figure 2. Synthesis and degradation of photodegradable RAFT hydrogels.
linker 2.2 to 3.3 only resulted in hyperbranched polymers which were too slow to proceed into forming gels for practical applications. In any case, the degradation kinetics had plateaued at this ratio. We also ran G5 using a lower quantity of redox initiators (G5* with 2 × 10−6 mol vs 1 × 10−5 mol of each redox couple). This slowed down the curing kinetics, as expected, but did not result in gels that degraded any faster. RAFT Hydrogels Made Using Nonphotodegradable Components. Our investigation also involved testing several other control gels such as G11 which was made using nonphotodegradable RAFT agent 13. Interestingly, this gel was found to have a slightly lower storage modulus compared to the corresponding RAFT gel G5 (9 kPa vs 12 kPa). This is due to a higher number of EG repeating units being present in the PEG chain of the macro-RAFT agent 13 (EG = 100) vs RAFT agent 10 (EG = 75). A longer PEG spacer located in the middle of the original RAFT agent results in a greater distance between the first junction points that appear along the primary chains from incorporation of the cross-linker. During the network evolution, the local cross-linking density would therefore be lower, eventually leading to a softer gel characterized by a lower storage modulus value. The photodegradation for G11 was also found to be slower than for G5 (0.046 min−1 vs 0.066 min−1 for k). Given that they are both RAFT synthesized gels, this result shows that it is not only a more homogeneous structure that is important for providing faster degradation but also the insertion of in-chain degradation sites. G12 was made using a combination of photodegradable cross-linker 9 and nonphotodegradable cross-linker 14, also with a ratio of [monomer]:[macro-RAFT]:[combined cross-
(NAM and 9) would improve degradation kinetics, as in hydrogels G9 and G10. We found this to be the case, with both hydrogels showing faster degradation kinetics compared to G5, which was made using the same ratio of NAM and 9 but with a lower amount of RAFT agent. This is due to the fact that adding more RAFT agent results in a gel with a higher proportion of backbone scission points with respect to monomer units. The gels made with a higher proportion of RAFT agent gave low storage moduli (
