Amplified photodegradation of hydrogels and methods of producing the same

Abstract

This invention is in the field of synthesis and amplified photodegradation of hydrogel network and methods of producing and using the same.

Claims

1. An allyl sulfide crosslinked hydrogel polymer network swollen in an aqueous media, wherein at least one live cell is encapsulated in the network, prepared by a process comprising the steps of: a) providing, i) a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, ii) a plurality of allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, and iii) at least one live cell, and b) mixing said tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, said allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, and at least one live cell under conditions such that a hydrogel network is produced.

2. The hydrogel network of claim 1, wherein said network comprises an allyl sulfide crosslinked strain-promoted azide-alkyne cycloaddition hydrogel network.

3. The hydrogel network of claim 2, wherein said hydrogel network produced by a reaction between a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules and a plurality of allyl sulfide poly(ethylene glycol) azide molecules.

4. The hydrogel network of claim 2, wherein said allyl sulfide poly(ethylene glycol) comprises allyl sulfide bis-(PEG.sub.3-azide).

5. The hydrogel network of claim 2, wherein said hydrogel network comprises a wound dressing.

6. The composition of claim 1, wherein said cells are human cells.

7. An allyl sulfide crosslinked strain-promoted azide-alkyne cycloaddition hydrogel polymer network swollen in an aqueous media, wherein at least one live cell encapsulated in the network and wherein said hydrogel network is in contact with a photoinitiator, prepared by a process comprising the steps of: a) providing, i) a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, ii) a plurality of allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, iii) at least one live cell, and iv) a photoinitiator, b) mixing said tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, said allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, photoinitiator, and at least one live cell under conditions such that a hydrogel network is produced.

8. The hydrogel network of claim 7, wherein said photoinitiator is in solution and at least part of said network is submerged in said solution.

9. The hydrogel network of claim 7, wherein said photoinitiator further comprises a free monothiol.

10. The hydrogel network of claim 9, wherein said free monothiol comprises mPEG-SH.

11. The hydrogel network of claim 7, wherein said hydrogel network comprises a network reaction between a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules and a plurality of allyl sulfide poly(ethylene glycol) azide molecules.

12. The hydrogel network of claim 7, wherein said allyl sulfide poly(ethylene glycol) comprises allyl sulfide bis-(PEG.sub.3-azide).

13. The hydrogel network of claim 7, wherein said photoinitiator comprises lithium phenyl-2,4,6-tri-methylbenzoylphosphinate.

14. A method of producing an allyl sulfide crosslinked strain-promoted azide-alkyne cycloaddition (SPAAC) hydrogel network, comprising: a) providing, i) a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules, ii) a plurality of allyl sulfide poly(ethylene glycol) (PEG-N.sub.3) molecules, and b) mixing said tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules and said allyl sulfide poly(ethylene glycol) molecules under conditions such that a hydrogel network is produced.

15. The method of claim 14, wherein said allyl sulfide poly(ethylene glycol) comprises allyl sulfide bis-(PEG.sub.3-azide).

16. A method comprising the steps of: a) providing, i) a plurality of tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, ii) a plurality of allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, iii) at least one live cell, and iv) a photoinitiator, b) mixing said tetrafunctional poly(ethylene glycol) dibenzocyclooctyne molecules in aqueous solution, said allyl sulfide poly(ethylene glycol) azide molecules in aqueous solution, at least one live cell, and photoinitiator under conditions such that a hydrogel network is produced.

17. The method of claim 16, wherein mixing said photoinitiator comprises immersing said hydrogel network in an aqueous solution of photoinitiator.

Description

DESCRIPTION OF THE FIGURES

(1) The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

(2) FIG. 1 shows the amplification of photodegradation by chain transfer. Addition-fragmentation chain transfer (AFCT) crosslinks exposed to a photogenerated monothiyl radical (red) transition from a crosslinked state to a non-crosslinked state and also regenerate a monothiyl radical capable of additional crosslink fragmentation reactions.

(3) FIGS. 2A&B show the network structure of AFCT-based photodegradable hydrogels. FIG. 2A shows chemical structures of cyclooctyne-terminated PEG macromer and azide crosslinker containing the allyl sulfide functionality. FIG. 2B shows that upon mixing the two species, a hydrogel network is rapidly formed incorporating the allyl sulfide reactive groups. Gelation is monitored by shear rheology in situ, and the storage modulus (solid line) and loss modulus (dotted line) are tracked. The final elastic modulus is reached within 10 min.

(4) FIG. 3 A-C shows Light-triggered radical network degradation. a) Photodegradation mechanism: in the presence of LAP and mPEG-SH, crosslinking molecules are fragmented by photoinitiator radicals or non-crosslinking monothiyl species when exposed to light. b) In the absence of free mPEG-SH, incomplete photodegradation is observed. The material is exposed to 365 nm light at 10 mW cm.sup.−2 at 30 s, and the light is shuttered for 1 min at 120 s (light exposure is indicated by purple shading). Rheological traces were performed at a frequency of 1 (black) and 10 rad s.sup.−1 (gray), monitoring the storage (solid line) and loss (dotted line) moduli, with both normalized to the initial storage modulus. Evidence for rapid network reorganization is seen in the frequency dependence of the measurements during light exposure, while the curves converge when the light is shuttered (120-180 s) and as the photoinitiator is depleted (less than 1% LAP remains after 300 s). c) Incorporation of mPEG-SH allows controlled photodegradation of the gel and tuning of the storage modulus. mPEG-SH was swollen into the network at 0 m (.diamond-solid.), 5×10.sup.−3 m (), 15×10.sup.−3 m (.box-tangle-solidup.), 25×10.sup.−3 m (⋅), and 50×10.sup.−3 m (.circle-solid.). Reverse gelation occurs when mPEG-SH concentrations of 25×10.sup.−3 and 50×10.sup.−3 m are used.

(5) FIG. 4A-C shows photodegradation kinetics can be adjusted by changes in either the photoinitiator concentration or light intensity. Light intensity (l) as a function of depth (d) within a hydrogel containing an absorbing molecule with molar absorptivity ε and concentration c were calculated as in Equation 4. FIG. 4A shows the Effect of photoinitiator concentration. Hydrogels were swollen with 25×10.sup.−3 in mPEG-SH and 1×10.sup.−3 m (.diamond-solid.), 2×10.sup.−3 m (.circle-solid.), 4×10.sup.−3 m (.square-solid.) or 8×10.sup.−3 m (.box-tangle-solidup.) LAP. b) Effect of light intensity. Hydrogels swollen with 4×10.sup.−3 m LAP and 25×10.sup.−3 m mPEG-SH and exposed to 2 (), 10 () or 40 (Δ) mW cm.sup.−2 365 nm light. FIG. 4C shows the apparent rate constants for photodegradation are plotted as a function of light intensity, yielding a straight line with slope k.sub.app/I0×10.sup.4 of 235 cm.sup.3 mW.sup.−1 s.sup.−1.

(6) FIG. 5A-C shows allyl sulfide-based photodegradable hydrogels can be used for large scale erosion and cytocompatible cell release. FIG. 5A shows A 1 cm thick hydrogel containing 4×10.sup.−3 m LAP and 25×10.sup.−3 m mPEG-SH was irradiated with 10 mW cm-2 365 nm light, and completed eroded over the course of ≈1 min. In this optically thick gel, 87% of the incident light is attenuated through the sample (i.e., only 13% of the light reaches the bottom of the sample). Macroscopic images of the gel are shown at 0, 20, 44, and 74 s (left to right, top to bottom). Scale bar: 5 mm. FIG. 5B shows cells that were both encapsulated for 24 h and subsequently released remained highly viable. Top panel: hMSCs 24 h after encapsulation are 90% viable. Bottom panel: released cells remain viable and spread on glass coverslips over 24 h. Cells were stained with calcein AM (green, live) and ethidium homodimer (red, dead). Scale bar: 100 μm (top panel) and 300 μm (bottom panel). FIG. 5C A 150 μm thick cell-laden hydrogel was selectively exposed to light (365 nm at 10 mW cm.sup.−2) for 1 min through a photomask to induce spatially controlled photodegradation and release of a subset of the encapsulated cells. Cells remaining in hydrogel monoliths stained with calcein AM (green) and ethidium homodimer (red). Scale bar: 100 μm.

(7) FIGS. 6A&B show modeling diffusion of mPEG-SH within a hydrogel. FIG. 6A in the left panel shows the concentration profile of mPEG-SH within a 260 μm thick hydrogel over a 5 min swell time of a mPEG-SH bath placed on the top of the hydrogel. The right panel shows the concentration profile throughout the gel has nearly reached the concentration of the bulk solution after 5 min. FIG. 6B shows the concentration profile of mPEG-SH within a 3 mm thick hydrogel over a 1 h (left panel) and 2 h (middle panel) swell time when immersed in a mPEG-SH bath. The right panel shows that the mPEG-SH concentration in the middle of the 3 mm thick hydrogel is about 40% that of the bulk at 1 h. At a bulk mPEG-SH concentration of 50 mM the minimum concentration in the hydrogel at 1 hr is about 20 mM mPEG-SH, a concentration that was shown to be sufficient for photodegradation.

(8) FIG. 7 shows encapsulated hMSC cell viability. Representative projections of 3D confocal images acquired at day 1 (left, 90% viable) and day 4 (right, 70% viable). Cells are stained with calcein AM (live—green) and ethidium homodimer (red—dead). Scale bar 100 μm.

(9) FIG. 8 shows the photodegradation and release of a cell on confocal microscope. A user defined area (red box) containing an adhered hMSC was repeatedly scanned with a 405 nm laser on a confocal laser scanning microscope, causing release of the adhered cell by 100 scans and complete gel degradation by 670 scans. Upper right=number of scans with 405 nm light. Scale bar=50 μm.

(10) FIG. 9 show an expanded reaction scheme including addition to the olefin by initiator fragments.

(11) FIG. 10 shows the diminishing returns of increasing the concentration of free monothiol. The equilibrium amount of tethered monothiol approaches the initial thioether concentration (16.5 mM) at infinite [mPEG-SH].sub.0.

(12) FIG. 11 shows theoretical light attenuation at 365 nm in a hydrogel containing 4 mM LAP (ε=218 M.sup.−1 cm.sup.−1) compared to one containing 10 mM nitrobenzyl ester linkages (assuming ε=3000 M.sup.−1 cm.sup.−1).

DETAILED DESCRIPTION OF THE INVENTION

(13) This invention is in the field of synthesis and amplified photodegradation of hydrogels and methods of producing and using the same. Hydrogels synthesized from crosslinking reactions between water soluble macromolecular precursors are widely used in a number of biomaterials applications, for example as drug or cell delivery vehicles and as cell culture scaffolds. Many of the bio-click reactions used to form hydrogels allows direct encapsulation of biologics and cells, while maintaining their activity and viability, respectively. Furthermore, many techniques exploit the ability to create multifunctional hydrogel systems with spatiotemporally controlled material properties, biological functionalities, and printed cell structures. Along with this complexity, researchers have been further interested in methods to better characterize these complex systems, control their properties on demand, and temporally tune properties such as degradation or viscoelasticity.

(14) With respect to temporally controlling hydrogel properties, many regenerative medicine applications that embed cells in hydrogels require degradation of the network structure to allow formation of focal adhesions, proliferation, migration, deposition of matrix components, and even to avoid fibrotic encapsulation of the implanted biomaterials. Hydrogels are routinely degraded by hydrolytic, enzymatic, or photolytic mechanisms, and each mechanism provides specific advantages. More recently, hydrogels with photocleavable crosslinks have been developed that allow spatiotemporal control of the degradation process. Spatiotemporal control of degradation has found numerous applications in guiding cellular proliferation, migration, and differentiation. One limitation to man; of the photodegradable groups used in biomaterial applications to date, however, is that the reactions rely on “one-photon one-event” processes. A photolabile molecule incorporated directly into the polymer backbone absorbs a photon and undergoes a cleavage reaction. This subsequently requires either long exposure times or high quantum yields.

(15) With this in mind, hydrogels are described herein that can be degraded via a radical addition-fragmentation chain transfer (AFCT) process. Allyl sulfides have been used as efficient AFCT functionalities to introduce plasticity into crosslinked networks and recently to reversibly photopattern biomolecules within a hydrogel. The allyl sulfide moiety can be incorporated into the network backbone either through a radical process or via an orthogonal reaction such as strain-promoted azide-alkyne cycloaddition (SPAAC). Subsequent exposure to light in the presence of a photoinitiator and a monofunctional thiol causes the crosslinked system to revert to soluble branched macromers. Photogenerated thiyl radicals rapidly propagate through thiol-ene addition reactions and chain transfer events. Allyl sulfide moieties in a crosslinking state participate in a reversible thiol-ene addition with non-crosslinking thiyl radicals, converting the allyl sulfides into a non-crosslinked state. Subsequent thiyl-thiol chain transfer events allow one absorbed photon to cleave multiple crosslinks, whereas current biocompatible photodegradation strategies are limited to a maximum of one crosslink cleavage by one photon. Furthermore, the low absorptivity of the photoactive compounds in this system allow for deep penetration of light through the samples. While the primary focus is upon allyl sulfides, this system could be adapted for a wide range of existing AFCT agents.

(16) The allyl sulfide degradable hydrogel system of the present invention is cytocompatible and may be used for many cellular applications. Cells encapsulated in the present invention degradable hydrogel system can be released from the material at a user-defined time point for further analysis of intracellular protein and mRNA production as well as fluorescently activated cell sorting (FACS). This system would also be beneficial in a wound healing application, where the hydrogel could initially serve as a protective barrier and then could be rapidly removed on-demand with a low light dose. Additionally, this material can be used for the photolithographic production of three dimensional cell laden structures and can also be used as a templating material to produce void-forming hydrogels.

(17) Hydrogels synthesized from crosslinking reactions between water soluble macromolecular precursors are widely used in a number of biomaterials applications, for example, as drug or cell delivery vehicles and as cell culture scaffolds [3, 4]. Many of the bioclick reactions used to form hydrogels allow direct encapsulation of biologics and cells, while maintaining their activity and viability, respectively [5-9]. Furthermore, many techniques exploit the ability to create multifunctional hydrogel systems with spatiotemporally controlled material properties [10, 11], biological functionalities [9, 12, 13], and printed cell structures [14, 15]. Along with this complexity, researchers have been further interested in methods to better characterize these complex systems, control their properties on demand, and temporally tune properties such as degradation or viscoelasticity.

(18) With respect to temporally controlling hydrogel properties, many regenerative medicine applications that embed cells in hydrogels require degradation of the network structure to allow formation of focal adhesions, proliferation, migration, deposition of matrix components, and even to avoid fibrotic encapsulation of the implanted biomaterials. Hydrogels are routinely degraded by hydrolytic [16], enzymatic [17], or photolytic mechanisms [10, 18], and each mechanism provides specific advantages. With their high water content, hydrogels with hydrolytically cleavable crosslinks typically degrade through a uniform, bulk process; whereas hydrogels that are proteolytically cleavable often degrade through a local mechanism that depends on cell-secreted enzymes. More recently, hydrogels with photocleavable crosslinks have been developed that allow spatiotemporal control of the degradation process. In one example, Kloxin et al. demonstrated on demand control of network crosslinking density and elastic modulus, and used materials with photocleavable crosslinks to study the effects of mechanical properties on the reversibility of the fibroblast-to-myofibroblast transition in heart valve cells [19]. Since these early studies, spatiotemporal control of degradation has found numerous applications in guiding cellular proliferation, migration, and differentiation [18, 20-25]. One limitation to many of the photodegradable groups used in biomaterial applications to date, however, is that the degradation relies on “one-photon one-event” reactions. A photolabile molecule, such as a nitrobenzyl or coumarin group, incorporated directly into the polymer backbone absorbs a photon and undergoes a cleavage reaction. This subsequently requires either long exposure times or high quantum yields.

(19) With this in mind, hydrogels were synthesized that can be degraded via a radical addition-fragmentation chain transfer (AFCT) process, where a single photon initiates multiple events and amplifies the degradation process. Allyl sulfides have been used as efficient AFCT functionalities to introduce plasticity into crosslinked networks [26-28] and recently to reversibly photopattern biomolecules within a hydrogel [12]. To incorporate this moiety into biomaterial systems, a symmetric allyl sulfide crosslinker flanked with azide functionalities was synthesized for formation of a hydrogel network through a strain-promoted azide-alkyne cycloaddition (SPAAC). Subsequent exposure to light in the presence of a photoinitiator and a monofunctional thiol causes the crosslinked system to revert to soluble branched macromolecules. Upon exposure, photogenerated thiyl radicals rapidly propagate through thiol-ene addition reactions and chain transfer events (FIG. 1) [26, 27, 29]. Allyl sulfide moieties in a crosslinking state participate in a reversible thiol-ene addition with noncrosslinking thiyl radicals, converting the allyl sulfides into a non-crosslinked state and generating a thiyl radical bound to the network (“network thiyl”). Alternating cycles of thiol-ene addition and chain transfer from a liberated network thiyl to a free thiol replace crosslinking allyl sulfides with non-crosslinking counterparts. Thiyl-thiol chain transfer events allow one absorbed photon to cleave multiple crosslinks, whereas current biocompatible photodegradation strategies typically rely on mechanisms where there is a maximum of one crosslink cleavage by one photon, with typical quantum yields being much lower [30]. Moreover, a lower concentration of photoinitiator is required for radical mediated photodegradation of allyl sulfide-containing hydrogels, compared to traditional photodegradable hydrogels that have one or more photoactive constituents per crosslink. Crosslinked hydrogel networks were formed through a SPAAC reaction between a tetrafunctional poly(ethylene glycol) dibenzocyclooctyne (PEG-DBCO) and an allyl sulfide bis-(PEG3-azide) (FIG. 2A). This bio-orthogonal “click” reaction proceeds rapidly at physiological conditions, and has been used in numerous studies as a cytocompatible crosslinking strategy [18] [22][31] [32]. Specifically, a 7.5 wt % solution of PEG-DBCO (15×10.sup.−3 m DBCO) and allyl sulfide crosslinker (16.5×10.sup.−3 m azide) was polymerized in situ on a rheometer. Excess azide was chosen to ensure complete conversion of the DBCO functionalities and to avoid side reactions during the subsequent thiol-ene reactions, as strained alkynes are known to react with thiols in a Michael-type addition and also participate in thiolyne radical additions [33-39]. The gel point was estimated by the crossover of G′ and G″, which occurred in <30 s, while a final modulus of 3500} 660 Pa was achieved in ≈10 min for this formulation (FIG. 2B).

(20) For subsequent photodegradation, the hydrogel was equilibrium swollen, and then placed in a solution containing varying concentrations of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) [40, 41] (1×10.sup.−3 to 8×10.sup.−3 m) and a methoxy-PEG-thiol (mPEG-SH, Mn≈500 Da, from 0 to 50×10.sup.−3 m). Exposure to light generates photoinitiator radical species, which can add directly to the olefin of the allyl sulfide or undergo chain transfer to a free thiol, which in turn reacts with the allyl sulfide (FIG. 3A). After 20 min of swelling, the gels were irradiated with 365 nm light (2-40 mW cm.sup.−2), and the degradation was tracked via changes in the shear storage and loss moduli with exposure time.

(21) When the exchange solution contained only 4×10.sup.−3 in LAP and no free thiol, significant photodegradation occurs, but not to an extent that results in reverse gelation (FIG. 3B). In this condition, there are initially no free thiols present in the hydrogel system; the only sulfur atoms are in the thioethers of the crosslinker. Thus, photodegradation is likely due to the direct addition of the photoinitiator radical fragments to the olefin of the allyl sulfide crosslinker. According to FIG. 9, this reaction generates a new olefin and also a pendant thiyl radical. In the presence of thiyl radicals, the network is expected to reorganize rapidly as the system propagates through a number of thiol-ene additions, each consuming one thiyl radical and generating another [26, 29, 42]. Evidence for this network reorganization is shown in FIG. 3B. The large increase in the loss modulus upon exposure to light indicates a significant shift the viscoelastic properties of the network from an almost purely elastic material to one that is more fluid in nature. This behavior is typical of networks that are crosslinked by dynamic linkages, such as hydrazone bonds [43, 44], host-guest interactions [14, 45], electrostatics [46-48], and others (see reviews by Kloxin and Bowman [49], Rosales and Anseth [50], and Wang and Heilshorn [51]), which display frequency dependent mechanical properties. This is further emphasized by a near crossover of G′ and G″, indicating that the material is approaching the reverse gel point (i.e., behaving as soluble, highly branched polymer) at the given strain rate (1 rad s.sup.−1).

(22) An apparent anomaly is also seen in the 1 rad s.sup.−1 rheological trace, wherein G′ reaches a minimum after ≈20 s of exposure and then increases before reaching a plateau (FIG. 3B—black). This observation is likely due to a combination of photoinitiator consumption during light exposure and the generation of pendant network thiyls after allyl sulfide cleavage (FIG. 9). Initially, LAP is at its highest concentration (4×10.sup.−3 m), leading to the highest rate of radical generation and allyl sulfide crosslink cleavage. The product of the allyl sulfide cleavage by LAP is a pendant thiyl radical. The pendant thiyl radicals have limited mobility, but are reactive toward other allyl sulfide species and can reform a crosslink upon addition to a network allyl sulfide molecule, which results in network reorganization, but not photodegradation [26]. Consequently, the measured G′ during the radical mediated network reorganization is lower than the value that would be obtained without radical generation, because crosslinked strands dissipate their potential energy upon crosslink reorganization. Here, it was observed that the generation of network thiyls leads to a shift from a purely elastic network to a viscoelastic network with a frequency dependent storage and loss modulus. For comparison, the same experiment was performed at 10 rad s.sup.−1 and the results superimposed (FIG. 3B—gray). At the higher sampling frequency, the hydrogel is less capable of dissipating the imposed force, and the storage modulus is a closer representation of what would be measured in the absence of radicals. To demonstrate the effect of network rearrangement on the storage modulus, the light was briefly shuttered at 120 s. The hydrogel subsequently reverted back to more purely elastic behavior, which led to an increase in G′ and a decrease in G″. At this point, the traces from the 1 and 10 rad s.sup.−1 experiments converge. Re-exposure of the sample at 180 s returns the gel to its previous viscoelastic storage modulus as it continues toward the final elastic modulus and the photoinitiator is completely consumed (photoinitiator half-life=45 s, Equation 1). Clearly, these reaction conditions result in rapid but incomplete photodegradation. Examination of the relevant functional groups—LAP (4×10.sup.−3 m) and initial network thioethers (16.5×10.sup.−3 m)—can help explain this. According to the Flory-Stockmayer equation [52], the gel point for this hydrogel is estimated as ≈61% (Equation 2), meaning that 39%, or 6.5×10.sup.−3 m, of crosslinks would need to be cleaved to achieve reverse gelation. Theoretically, if every initiator fragment added directly to a crosslinking olefin, it would be possible to cause reverse gelation. However, proton abstraction from the increasing number of liberated pendant thiols may bias the system toward reactions that do not result in a net change in the crosslink density.

(23) $\begin{array}{cc}\frac{d\left[\mathrm{LAP}\right]}{\mathrm{dt}}=\frac{\mathrm{.Math.}\ln \left(10\right)\varphi I_0\lambda }{N_{\mathrm{AV}}\mathrm{hc}}& \mathrm{Equation}1\end{array}$
Equation 1 shows the Photoinitiator lifetime. For a thin film, the photoinitiator lifetime can be calculated as shown above, where molar absorptivity ε=218 M.sup.−1 cm.sup.−1, intensity of incident light I.sub.0=10 mW/cm.sup.2, wavelength λ=365 nm, quantum yield ϕ is assumed to be unity, Avogadro's number N.sub.AV, Planck's constant h, and the speed of light c. Solving, the photoinitiator follows first order decay with a rate constant of 0.015 s.sup.−1. This corresponds to a half-life of 45 s.

(24) Equation 2 shows the Flory-Stockmayer percolation threshold for a step growth polymer network:

(25) $\begin{array}{cc}{p}_c=\frac{1}{\sqrt{r\left(f_a-1\right)\left(f_b-1\right)}}& \mathrm{Equation}2\end{array}$
with functionalities of the PEG-DBCO (f.sub.a=4) and the azide crosslinker (f.sub.b=2) and a stoichiometric ratio r=0.91 gives the critical conversion for gelation as pc=0.61

(26) To increase the efficiency of the photodegradation reaction and allow complete network degradation, the AFCT reaction in the presence of a free monothiol (mPEG-SH) was studied. In contrast to network-bound thiols, addition by a monofunctional thiol changes the overall network connectivity and effectively cleaves crosslinks. These replacement reactions can be favored by increasing the concentration of free monothiol relative to the concentration of thioethers initially present in the network. mPEG-SH was introduced at concentrations ranging from 5×10.sup.−3 to 50×10.sup.−3 m, along with 4×10.sup.−3 m LAP and irradiated with 365 nm light at 10 mW cm.sup.−2. As seen in FIG. 3C, 25×10.sup.−3 and 50×10.sup.−3 m of mPEG-SH resulted in reverse gelation, while concentrations of 0×10-, 5×10.sup.−3, or 15×10.sup.−3 m did not. If one assumes similar reactivity of the alkyl thiols from mPEG-SH and the liberated network thiols, it may be expected that the various thiolated molecules in FIG. 1 would be expected to approach an equilibrium defined by Equation 3:

(27) $\begin{array}{cc}{k}_{\mathrm{eq}}=\frac{\left[\mathrm{free}\mathrm{mPEG}\text{-}\mathrm{SH}\right]\left[\mathrm{network}\mathrm{thioether}\right]}{\left[\mathrm{liberated}\mathrm{network}\mathrm{thiol}\right]\left[\mathrm{tethered}\mathrm{mPEG}\text{-}\mathrm{Sh}\right]}=\frac{\left({\left[\mathrm{mPEG}\text{-}\mathrm{SH}\right]}_0-x\right)\left(16.5\times {10}^{-3}M-x\right)}{x^2}-1& \mathrm{Equation}3\end{array}$

(28) FIG. 10 shows the expected equilibrium as a function of initial monothiol concentration. The predicted amount of initial free monothiol needed to cleave 6.5×10.sup.−3 m crosslinks is 11×10.sup.−3 m, and thus, one may expect complete degradation at 15×10.sup.−3 m. Instead, a very weak gel with G′≈1% of its initial value was found. It is possible that increasing the photoinitiator concentration would allow this equilibrium to be reached, but the competing reaction of initiator with olefin makes this analysis complex.

(29) The effect of varying photoinitiator concentration was also investigated while keeping free thiol concentration constant at 25×10.sup.−3 m and light intensity constant at 10 mW cm.sup.−2 (FIG. 4A). Light intensity (l) as a function of depth (d) within a hydrogel containing an absorbing molecule with molar absorptivity ε and concentration c were calculated as in Equation 4. As expected, the photoinitiator concentration had a strong effect on both the rate and extent of photodegradation. By increasing the photoinitiator concentration to 8×10.sup.−3 m, complete reverse gelation was achieved after <13 s of irradiation, corresponding to a rate constant of k.sub.app/I.sub.0×10.sup.−4 of 580 cm.sup.2 mW.sup.−1 s.sup.−1 and a photodegradation half-life of under 2 s. This result was compared to the rate of photodegradation of the widely used orthonitrobenzyl group and coumarin groups, and the AFCT mode of degradation was 70-2000 times faster than photodegradation methods based on alpha cleavage [10, 18, 21, 23, 53-55]. The effect of light intensity on the degradation rate was then studied (FIG. 4B). The rate of photodegradation was easily tuned by setting the intensity of the illuminating light to 2, 10, or 40 mW cm.sup.−3. For 4×10.sup.−3 m LAP and 25×10.sup.−3 m mPEG-SH, a plot of k.sub.app versus I0 yields a straight line with a slope of 235×10.sup.−4 cm.sup.2 mW.sup.−1 s.sup.−1 (FIG. 4C).

(30) $\begin{array}{cc}\frac{I}{I_0}=e^{-\ln \left(10\right)\mathrm{.Math.}\mathrm{cd}}& \mathrm{Equation}4\end{array}$

(31) AFCT-based photodegradable hydrogels also benefit from decreased light attenuation. There are two underlying causes to this effect. First, the molar absorptivity of one chosen photoinitiator, LAP, at 365 nm is 218 m.sup.−1 cm.sup.−1 [41]. This is in contrast to the ortho-nitrobenzyl ester and coumarin photolabile groups which have molar absorptivities on the order of 3000 to 7000 m.sup.−1 cm.sup.−1 at 365 nm [18, 23, 54], and while more transparent at longer wavelengths, the quantum yield and efficiency also decrease. The other contributing factor is that the concentration of photoactive species in this case can be lower because the radicals generated can propagate through numerous photocleavage events. In practice, 4×10.sup.−3 m LAP was found to be sufficient for complete photodegradation, in comparison to 10×10.sup.−3 and 40×10.sup.−3 m nitrobenzyl photodegradable groups commonly employed in photodegradable polymer strands. The combination of these factors allowed for photodegradation of much thicker hydrogel samples. To demonstrate the power of this effect, a 1 cm thick hydrogel with 4×10.sup.−3 m LAP was created and subsequently swelled in mPEG-SH to a final concentration of 25×10.sup.−3 m. The swollen hydrogen was then exposed to 365 nm light at 10 mW cm.sup.−2 along the 1 cm axis. For the LAP concentration employed, this 1 cm sample still allows ≈13% transmission of the incident light at the bottom of the sample (Equation 5). As observed macroscopically in FIG. 5A, this hydrogel rapidly erodes in ≈1 min. which renders this hydrogel chemistry particularly useful for certain biomaterials applications. For example, in applications where one may wish to harvest selected cells or the entire cell population (e.g., for fluorescence-activated cell sorting or other analyses), light exposure in defined regions can allow cell capture in a manner akin to laser capture microdissection. In addition, as the field of mechanotransduction transitions from 2D to 3D culture systems, there is an ever increasing need for ways to expand and passage cells in 3D environments. One major roadblock in this approach is how to harvest cells from these 3D materials for further expansion or characterization. Photodegradation is one attractive option, due to the spatial and temporal control that can be leveraged to release defined regions of cells at user-specified time points. However, rapid and spatially defined erosion is required, and current systems can be limited by relatively slow degradation kinetics and significant light attenuation. The aforementioned qualities of allyl sulfide crosslinked hydrogels give potential utility in this regard.

(32) Equation 5 shows the equilibrium approached by thiolated molecules of FIG. 9.

(33) $\begin{array}{cc}{K}_{\mathrm{eq}}=\frac{\left[\mathrm{free}\mathrm{mPEG}\text{-}\mathrm{SH}\right]\left[\mathrm{network}\mathrm{thioether}\right]}{\left[\mathrm{liberated}\mathrm{network}\mathrm{thiol}\right]\left[\mathrm{cleaved}\mathrm{xlinks}\right]}=\frac{\left({\left[\mathrm{mPEG}\text{-}\mathrm{SH}\right]}_0-x\right)\left(16.5-x\right)}{x^2}& \mathrm{Equation}5\end{array}$
where x=tethered mPEG-SH=liberated network thiol=cleaved crosslinks

(34) To demonstrate some of these advantages, primary human mesenchymal stem cells (hMSCs) were encapsulated in 3 mm thick hydrogels at a density of 5×10.sup.6 cells mL.sup.−1. For all of the cell culture experiments, an azide-functionalized RGD peptide was added to the gel formulation at 1×10.sup.−3 m to provide cell-matrix interactions. Encapsulation using the SPAAC reaction is known to proceed with high cell viability [18]; indeed, hMSC viability was quantified as 90% and 74% after 1 and 4 d of encapsulation, respectively (FIG. 7). This reduction in cell viability over time may be attributed, in part, to the inability of these encapsulated cells to remodel the PEG networks [56], but the chemistry is readily modified to include protease degradable peptide linkers.

(35) After 1 d of encapsulation, the cell laden hydrogels were swollen with mPEG-SH (50×10.sup.−3 m) and LAP (4×10.sup.−3 m) for 1 h, followed by exposure to 10 mW cm.sup.−2 of 365 nm light for 1 min on a gelatin coated glass coverslip (FIG. 5B). Under these exposure conditions, 2.4×10.sup.−3 m photoinitiator is consumed, which is similar to photoinitiator concentrations that have been widely found to be cytocompatible for photoencapsulation and photopatterning [5, 41, 57-59]. The encapsulated cells were released from the photodegradable hydrogel and allowed to adhere to the underlying glass coverslip under standard culture conditions. hMSCs were viable upon release, and spread on the coverslip over 24 h, which demonstrates that these reaction conditions are mild enough to be useful for 3D cell culture and capture. Spatial control over cell release was also achieved under the same conditions by selective exposure of a 150 μm thick hydrogel through a chrome photomask (FIG. 5C). In addition, the absorption spectrum of LAP was taken advantage of, which extends up to 450 nm, to demonstrate that these hydrogels are capable of photodegradation under 405 nm light for cell release using a conventional microscope setup. hMSCs were seeded onto hydrogels at 1×10.sup.4 cells cm.sup.−2 and using a confocal laser scanning microscope with a 405 nm laser (DAPI channel) at 60% power, a user defined area of the gel was degraded to release an adhered cell (FIG. 8).

(36) In conclusion, a photodegradable hydrogel system was synthesized incorporating an allyl sulfide functionality that allowed for a radical-initiated thiol-ene exchange reaction. By the introduction of monothiols, the network connectivity and mechanical properties could be controlled on-demand by exposure to light. Under conditions that proved cytocompatible (4×10.sup.−3 m LAP, 25×10.sup.−3 m mPEG-SH, 10 mW cm.sup.−2 365 nm light), reverse gelation occurred in under 30 s and samples up to 1 cm thick could be eroded in ≈1 min, representing a significant benefit over conventional photodegradable hydrogels in both respects.

(37) Importantly, both the SPAAC gel formation and photodegradation processes were designed to be compatible with biological systems, allowing new-found experiments to study cells in dynamic environments, and to readily capture cells from 3D laden systems. This new class of photodegradable hydrogels is unique in its mechanism, speed of degradation, and depths attainable, and provides access to experiments previously limited by light dose and attenuation.

EXAMPLES

(38) The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. .sup.1H-NMR spectra were performed on a 400 MHz Bruker NMR spectrometer, where the residual proton resonance of the solvent is used as an internal standard for each molecule. Chemical shifts are shown in parts per million (ppm). The multiplicities of each peak are given in abbreviations such as: s, singlet; d, doublet; t, triplet; m, multiplet; q, quartet. All chemicals used were purchased from commercial sources and used as such, unless otherwise mentioned.

Example 1

(39) Synthesis of Compound 1 [Allyl Sulfide Bis(Methyl Ester)]

(40) ##STR00001##

(41) Sodium (1.1 g, 48 mmol) was added to a flame dried flask charged with 150 mL anhydrous methanol and allowed to react for 10 minutes. After dissolution of the sodium, methyl thioglycolate (3.93 mL, 44 mmol) was added to the flask and the mixture was heated to reflux under an argon atmosphere and reacted for 20 minutes. 3-chloro-2-chloromethyl-1-propene (2.31 mL, 20 mmol) dissolved in 20 mL anhydrous methanol was then added dropwise to the reaction vessel over 45 minutes. The reaction mixture was purged with argon and stirred for 18 h at 60° C. The resulting mixture was filtered and concentrated through rotary evaporation to yield a yellow crude oil. The crude oil was taken up in 100 mL MQ water and extracted 6× with 100 mL diethyl ether (Et.sub.2O). The combined organic phases were washed with 200 mL brine, dried over sodium sulfate and concentrated by rotary evaporation to yield a pale yellow oil (2.88 g, 55%). TLC in 80% hexanes:20% ethyl acetate reveals a single spot at R.sub.f=0.27 under UV, I.sub.2, and PMA. .sub.1H NMR (400 MHz, Methanol-d.sub.4) δ 5.11 (q, J=0.6 Hz, 2H), 3.72 (s, 6H), 3.42 (t, J=0.7 Hz, 4H), 3.21 (s, 4H).

Example 2

(42) Synthesis of Compound 2 [Allyl Sulfide Bis(Acetic Acid)]

(43) ##STR00002##

(44) 100 ml of 1M LiOH (aq) was added to a solution of 1 (2.88 g, 10.9 mmol) in 100 ml THF on ice. The turbid solution was stirred for 5 h on ice, after which the solution was acidified (to pH=0) by addition of ˜100 mL 2M HCl. 50 ml brine was added and the solution was extracted with EtOAc (4×125 ml). The combined organics were dried over Na.sub.2SO.sub.4 and concentrated to yield a dark brown oil (2.57 g, 100%)

(45) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 12.58 (s, 2H), 5.04 (s, 2H), 3.32 (d, J=0.8 Hz, 4H), 3.14 (s, 4H).

Example 3

(46) Synthesis of Compound 3 [Allyl Sulfide Bis(PEG3-Azide)]

(47) ##STR00003##

(48) A flame dried RBF was charged with 2 (360 mg, 1.53 mmol), diisopropylethylamine (DIEA, Sigma) (2.7 mL, 15.3 mmol), O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, Chem-Impex Int'l) (1.16 g, 3.05 mmol), and 250 mL ethyl acetate. The resulting slurry was purged with argon and reacted for 1 hr at room temperature. 11-Azido-3,6,9-trioxaundecan-1-amine (TCI America) (1 g, 4.58 mmol) was added to the flask and the mixture was stirred overnight at room temperature under an argon atmosphere. The resulting mixture was vacuum filtered and washed with 1N HCl (1×250 mL), saturated aq. sodium bicarbonate (1×250 mL), water (1×100 mL), and brine (1×100 mL). The organic phase was dried over sodium sulfate and concentrated to yield a dark yellow oil (646 mg, 66%). TLC in 80% acetone:20% hexanes revealed a single spot at R.sub.f=0.3 with b staining.

(49) .sub.1H NMR (400 MHz, Methanol-d.sub.4) δ 5.12 (s, 2H), 4.66 (s, 2H), 3.73-3.62 (m, 20H), 3.58 (t, J=5.4 Hz, 4H), 3.43-3.36 (m, 12H), 3.16 (s, 4H).

(50) ESI+ HRMS: calc'd for [C.sub.24H.sub.44N.sub.8O.sub.8S.sub.2+H]+: 637.2797, found 637.2805.

Example 4

(51) Synthesis of PEG-DBCO

(52) ##STR00004##

(53) A solution of HATU (125 mg, 0.33 mmol). DIEA (78 mg, 0.6 mmol), and dibenzocyclooctyne acid (Click Chemistry Tools) (100 mg, 0.3 mmol) in DMF was stirred for 10 minutes, followed by addition of 4 arm PEG amine (MW=20 kDa, Jenkem Technology USA) (750 mg, 0.037 mmol). The solution was Argon purged and allowed to react for 24 h at room temperature. The product was precipitated in cold Et.sub.2O (200 ml), and washed with cold Et.sub.2O (2×200 ml). The precipitate was dissolved in deionized water, dialyzed (MW cutoff 8 kDa) for 48 h, and lyophilized to yield 762 mg (95% yield). PEG functionalization was confirmed with .sup.1H NMR to be ca. 95% by comparing characteristic peaks from DBCO to the PEG backbone peaks.

Example 5

(54) Synthesis of RGD-Azide

(55) ##STR00005##

(56) The peptide GRGDS was synthesized via standard Fmoc solid phase peptide synthesis (Protein Technologies Tribute peptide synthesizer) and HATU activation using Rink amide resin. 4-azidobutanoic acid (prepared as previously described) [18] was coupled to the free N-terminus on resin via HATU coupling. The peptide was subsequently cleaved from the resin by treatment with 88:5:5:2 (trifluoroacetic acid:phenol:water:triisoproylsilane) for 2 hours and precipitated in cold Et.sub.2O. Crude peptide was purified as needed by reverse phase HPLC (Waters Delta Prep 4000). MALDI-TOF: calc'd [M+H]+ 601.28, found 601.98 g/mol.

Example 6

(57) Characterization of SPAAC Hydrogel Formation

(58) Oscillatory rheology was performed on a TA Instruments DHR-3 rheometer with an 8 mm parallel plate geometry and a quartz lower plate to allow UV illumination. Allyl sulfide crosslinked SPAAC hydrogels were prepared by mixing stock solutions of 20 wt % PEG-DBCO in phosphate buffered saline (PBS) and 30 mM allyl sulfide bis(azide) 2 in 1:1 DMSO:distilled H.sub.2O to a final concentration of 7.5 wt % PEG-DBCO and 8.25 mM 2 in PBS. The precursor solution was vortexed for 5 s and placed on the rheometer with the gap immediately lowered to 260 μm. Hydrogel gelation kinetics were characterized and evaluated in situ by measuring the evolution of the storage and loss moduli (G′ and G″). Measurements were taken with an oscillatory shear strain of 1% and a frequency of 1 rad/s (within the linear viscoelastic range).

Example 7

(59) Characterization of Hydrogel Photodegradation

(60) This rheometer was fitted with an adaptor to allow for light exposure from a mercury arc lamp (Omnicure) fitted with a 365 nm bandpass filter in order to monitor gel degradation during exposure to UV light. Allyl sulfide crosslinked hydrogels were synthesized as described in the previous section and were allowed to gel completely (as determined by a negligible change in G′ with respect to time; about 10 minutes). After this point, the tool was carefully lifted and the gel immersed in a bath to bring the final concentration to the desired level of photoinitiator and monothiol. After 20 min, a swell time which was determined to be sufficient to attain a near uniform concentration profile throughout the sample (see next section), the gel was exposed to 365 nm light at an intensity of 2, 10, or 40 mW cm.sup.−2. Measurements were taken with an oscillatory shear strain of 1% and a frequency of 1 rad/s. Due to the rapid reorganization of the network, it was necessary to shutter the light after consecutive 1 s exposures to obtain accurate G′ readings. The measurement was allowed to stabilize in the dark between each 1 s exposure, and the storage modulus was recorded.

Example 8

(61) Calculation of Swelling Time

(62) A diffusion model using Ficks Laws was set up in Mathematica to determine the minimum amount of time required to achieve a nearly uniform concentration profile of the photodegradation components throughout the hydrogel samples in the experiments. Do was assumed equal to that of PEG.sub.600 (2.6×10.sup.−6 cm.sup.2 s.sup.−1) [60] in all simulations. For the rheological experiments modeled in FIG. 6A, a no-flux boundary condition was used at the bottom of the hydrogel and the bulk mPEG-SH concentration was used as the boundary condition at the top of the hydrogel; it was assumed that there was no mPEG-SH within the hydrogel initially. For the cell-laden hydrogel photodegradation experiments modeled in FIG. 6b, it was assumed that there was no mPEG-SH in the gel initially and the concentration at the top and bottom of the hydrogel were that of the bulk solution for all time.

(63) Thus, specific compositions and methods of amplified photodegradation of hydrogels and methods of producing the same have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

(64) Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

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