Short Soluble Coumarin Crosslinkers for Light-Controlled Release of

Sep 8, 2015 - We have designed and synthesized a short Bhc-bearing crosslinker to create light-degradable hydrogels .... Chase S Linsley , Benjamin M ...
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Short Soluble Coumarin Crosslinkers for Light-Controlled Release of Cells and Proteins from Hydrogels Caroline de Gracia Lux,†,⊥ Jacques Lux,†,⊥ Guillaume Collet,† Sha He,§ Minnie Chan,‡ Jason Olejniczak,‡ Alexandra Foucault-Collet,† and Adah Almutairi*,†,§,∥ †

Skaggs School of Pharmacy and Pharmaceutical Sciences, §Department of NanoEngineering, ‡Department of Chemistry and Biochemistry, and ∥Center for Excellence in Nanomedicine and Engineering, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0600, United States S Supporting Information *

ABSTRACT: Materials that degrade or dissociate in response to low power light promise to enable on-demand, precisely localized delivery of drugs or bioactive molecules in living systems. Such applications remain elusive because few materials respond to wavelengths that appreciably penetrate tissues. The photocage bromohydroxycoumarin (Bhc) is efficiently cleaved upon low-power ultraviolet (UV) and near-infrared (NIR) irradiation through one- or two-photon excitation, respectively. We have designed and synthesized a short Bhc-bearing crosslinker to create light-degradable hydrogels and nanogels. Our crosslinker breaks by intramolecular cyclization in a manner inspired by the naturally occurring ornithine lactamization, in response to UV and NIR light, enabling rapid degradation of polyacrylamide gels and release of small hydrophilic payloads such as an ∼10 nm model protein and murine mesenchymal stem cells, with no background leakage.

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and degradation rate. Research in the field can be divided into two main categories: phototriggered ligand presentation and payload release from photodegradable scaffolds. Shoichet et al. reported examples of chemical hydrogel patterning by photocontrolled immobilization of biomolecules.41,42 This strategy allows light-programmed cellular growth and migration in 3D agarose matrices. The Anseth group also pioneered this field with the first photodegradable hydrogels bearing nitrobenzyl ether linkers in PEG-based gels, allowing light-mediated control over cell culture environments.34 The vast majority of reported light-triggerable biomedical matrices for controlled delivery systems, biomolecular patterning, or programmable scaffolds13,39,43−46 respond to UV light, which does not penetrate tissues deeply due to high scattering and absorption by hemoglobin and melanin.47 The next generation of light-responsive hydrogels should employ chemistries responsive to NIR light (650−900 nm), which can penetrate tissues deeply with the high spatial and temporal resolution of multiphoton excitation while causing minimal damage. Extensive work has been done on hydrogels bearing ortho-nitrobenzyl (ONB) protecting groups,34,39,48,49 while only a few coumarin-based hydrogels have been reported.37,43,50−53 They are better candidates for two-photon triggered degradation because of coumarins’ superior two-photon action cross

INTRODUCTION Light-responsive materials promise remote control over the availability of molecules in living systems.1−6 As optogenetics has transformed neuroscience,7 broadening the range of species releasable using light should bring this precise form of light control to a wider range of biological research. In therapeutic delivery, potentially the most important field for the application of such materials, triggered release from reservoirs would stabilize therapeutics and reduce toxicity and unwanted side effects by lowering dosage and avoiding nonspecific activity. Despite remarkable advances in photoresponsive systems, few light-responsive materials have yet been designed that would allow control over the availability of bioactive hydrophilic molecules (e.g., proteins, siRNA, and pharmacological agents).8−15 Very recently, the first example of light-triggered in vivo presentation of bioligands was reported. Garciá et al. used light-regulated activation of RGD peptides on hydrogels to promote cell adhesion and vascularization while avoiding inflammation.16 Responsive hydrogels have sparked interest due to their unique stability in aqueous media and compatibility with delivery of large cargo such as proteins and cells.17−20 They show promise in a wide range of biomaterial applications such as tissue engineering (scaffolds, matrices for cell-culturing, wound healing), diagnostics, and drug delivery.21−25 Among the pool of available stimuli (temperature, 26 pH,27−29 enzymes,30,31 redox,29,32 electric signal,33 or light13,34−40), light uniquely offers spatiotemporal control over gel structure © XXXX American Chemical Society

Received: July 15, 2015 Revised: September 4, 2015

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DOI: 10.1021/acs.biomac.5b00950 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules sections52 (∼2 GM and 1 GM at 740 nm for 6-bromo-7hydroxycoumarin-4-ylmethyl acetate and Bhc-Glu, respectively,54 vs less than 0.1 GM for ONB).54,55 In addition, coumarin degradation yields alcohols, which are more biocompatible and less reactive than the aldehydes or ketones generated by ONB.54 Here, we report the synthesis, characterization, and study of UV- and NIR-degradable polyacrylamide (PAA)-based bulk hydrogels and hydrogel nanoparticles (nanogels) incorporating short ornithine-based crosslinkers containing the Bhc photocage (Scheme 1b,c). We incorporated ornithine, a non-

Our crosslinker design features a Bhc photocage through a carbamate linkage for maximal efficiency and hydrolytic stability. Halogenation of the coumarin lowers the pKa of the phenol, promoting formation of the more strongly absorbing and water-soluble phenolate under physiological pH.54,63 The presence of this heavy atom is also believed to promote intersystem crossing to the reactive triplet state. This greater efficiency means that systems incorporating the photocage can be triggered using low-power lasers that do not cause tissue damage. Finally, carbamate-linked coumarins have been reported to be more stable than ester linkages against hydrolysis.54 We designed a short crosslinker, rather than light-responsive PEG-based crosslinkers, for maximal stability and to inhibit burst release and payload leakage. In addition, our lightdegradable crosslinker is versatile and can be incorporated into other materials besides PAA, such as biodegradable polymeric scaffolds (hyaluronic acid, alginic acid, chitosan, or polylactic acid). NIR-triggered release of proteins from nanogels has not yet been shown; control over protein availability within such structures requires a tight mesh and thus a short crosslinker. To our knowledge, this is the first report of short water-soluble NIR-degradable crosslinker used in bulk or nanogels for efficient encapsulation and on-demand release of hydrophilic payloads such as proteins (∼10 nm) and cells with minimal background leakage.

Scheme 1a

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EXPERIMENTAL SECTION

General Procedures and Instrumentation. All chemicals were obtained from commercial sources and were used without further purification. All reactions were carried out under argon in oven-dried glassware unless otherwise noted. Flash column chromatography purification was performed using a Teledyne Isco Combiflash Companion with RediSep Rf prepacked silica. Mass determination of synthesized compounds and byproducts obtained from crosslinker degradation was performed with a HPLC-MS Agilent 160 Infinity (binary pump, UV−vis 1260 DAD, 6120 Quadrupole LC/MS ESI source) with a RP-18 column. 1H NMR spectra were acquired using a Bruker 600 MHz NMR spectrometer and 13C NMR spectra were acquired using a Bruker 150 MHz NMR spectrometer. UV−vis spectra were collected using a Shimadzu UV-3600 UV−vis spectrophotometer. Irradiation with 365 nm UV light was performed either using a Luzchem LZC-ORG photoreactor equipped with an 8UV-A lamp (316−400 nm range, resolved peaks: 313, 351 (broad), 365, 406 nm, 8 W maximum intensity, measured power: 1 mW·cm−2) or an OmniCure S2000 Curing System (30 cm from light source; power setting: 15%; measured power density: 1 mW·cm−2). NIR excitation was performed using a Ti:sapphire laser (Mai Tai HP, Spectra Physics). GFP expressing murine mesenchymal stem cells (GFPMSCs) and mMSC-cell culture media were provided by Cyagen Biosciences. Nanogel size, PDI, and count rate measurements were determined using a Malvern Zetasizer Nano ZS dynamic lightscattering instrument and direct visualization of the nanogels was imaged by transmission electron microscopy (TEM; FEI Spirit, operating at 120 kV). Bhc-PNP was prepared according to the previously reported procedure.64 Synthesis of 3. Fmoc-Orn(Boc)-OH (1.28 g, 2.8 mmol) was stirred in a mixture of TFA (1.8 mL) and DCM (9 mL) for 1 h. Solvents were removed under reduced pressure. TFA was removed by redissolution of the residue in DCM and evaporation, repeated three times. The residual white solid foam was redissolved in DCM (45 mL) with DIEA (10.8 mL, 62 mmol, 20 equiv). A solution of Bhc-PNP64 (1.35 g, 2.8 mmol, 1 equiv) in DCM (50 mL) was added dropwise to this solution, and the reaction mixture was stirred overnight. The organic mixture was extensively washed with brine and HCl (2.5 mM, pH 3−4) and

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(a) Unlike the 20 standard amino acids, ornithine is not carried by tRNA during protein synthesis because it undergoes lactamization in activated esters under biologically relevant conditions. (b) Chemical structures of the bromo-hydroxycoumarin-based crosslinkers 1 and 2. R = triethylene glycol (TEG) to increase water solubility. (c) Lightinduced degradation of PAA gels encapsulating hydrophilic payload via UV- and NIR-triggered cleavage of cross-links (upon Bhc photocleavage) through intramolecular cyclization. Payload (gray circle) is encapsulated by copolymerizing acrylamide with light-responsive bis(acrylamide) crosslinker 2. R = TEG. Detailed mechanism of Bhc photocleavage is described in Scheme S1.

proteinogenic amino acid not supported by tRNA because of its rapid fragmentation by lactamization (Scheme 1a).56,57 The unrestricted access of water throughout the hydrogel side steps the inefficient uncaging of Bhc in hydrophobic polymeric nanoparticles58 and allows high photosolvolysis quantum yield. This approach would allow low-intensity and, therefore, more cell-compatible exposure to UV or NIR light to initiate lighttriggered degradation and release, all while stably retaining cargo in the absence of irradiation (Scheme 1c). Several recent examples of live cells and bioactive protein release using biocompatible UV irradiation have been reported.39,46,59−62 However, our system uses the lowest UV power density ever reported for such platforms (365 nm, 1 vs 10 mW/cm2 generally reported).34,39,43,46,52,61,62 B

DOI: 10.1021/acs.biomac.5b00950 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

Synthesis of 7. Over a solution of tetraethylene glycol monomethyl ether (3.74 g, 17.9 mmol) in acetone (530 mL) was added dropwise 15 mL of Jones reagent ([CrO3] = 2.4 M) at 0 °C. The reaction was stirred at room temperature for 2 h followed by the addition of isopropyl alcohol (8 mL). Afterward, saturated sodium chloride (320 mL) was added to the mixture and stirred for 1 h before the removal of acetone under reduced pressure. Then, the aqueous mixture was extracted with DCM (4 × 200 mL). Organic layers were combined, then dried over MgSO4 and finally removed under reduced pressure. Yield: 3.69 g (93%). 1H NMR (600 MHz, CDCl3) δ 4.14 (s, 2H), 3.75−3.74 (m, 2H), 3.69−3.68 (m, 4H), 3.65−3.62 (m, 4H), 3.58− 3.56 (m, 2H), 3.39 (s, 3H). Synthesis of 8. 3-Amino-1,2-propane diol (3.09 g, 34 mmol) in DMF (100 mL) was added dropwise at 0 °C to a stirred solution of tert-butyl(chloro)diphenylsilane (9.8 mL, 37.4 mmol) and imidazole (2.78 g, 40.8 mmol) in DMF (200 mL). The reaction mixture was stirred overnight at RT. DMF was removed under reduced pressure and the residue was dissolved in DCM. The organic layer was washed with brine, dried over MgSO4 and finally removed under reduced pressure. The product was purified by flash chromatography on silica gel (DCM/methanol, 100:0 to 90:10). Yield: 4.14 g (37%). 1H NMR (600 MHz, CDCl3) δ 7.63 (d, J = 7.5 Hz, 4H), 7.39 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 4H), 3.86−3.82 (m, 1H), 3.63−3.61 (m, 2H), 2.95−2.85 (m, 2H), 1.04 (s, 9H). MS (ESI) m/z: [M + H]+ calcd for C19H28NO2Si, 330.2; found, 330.1. Synthesis of 9. After dissolution of 8 (3.52 g, 10.6 mmol) in EtOAc (75 mL), a solution of K2CO3 (14.79 g, 107 mmol) in water (75 mL) was added, and the resulting mixture was stirred vigorously at 0 °C. Acryloyl chloride (1.73 mL, 21.2 mmol) was then added dropwise to the reaction at 0 °C. After a few minutes, the reaction mixture was allowed to reach RT and was stirred until completion of the reaction, monitored by LCMS. After 2 h, the organic and aqueous layers were separated. The aqueous phase was extracted twice with EtOAc. Organic layers were combined, washed once with water, then dried over MgSO4, and finally removed under reduced pressure. Yield: 3.78 g (92%). 1H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.0 Hz, 4H), 7.45 (t, J = 7.5, 7.0 Hz, 2H), 7.40 (t, J = 7.5, 7.0 Hz, 4H), 6.25 (d, J = 17.5 Hz, 1H), 6.01 (dd, J = 17.5, 9.6 Hz, 1H), 5.79 (bs, 1H), 5.64 (d, J = 9.6 Hz, 1H), 3.86 (bs, OH), 3.70−3.67 (m, 1H), 3.65−3.60 (m, 2H), 3.30−3.26 (m, 1H), 3.10−3.09 (m, 1H), 1.08 (s, 9H). ES-MS: M + Na = 406.2 g·mol−1 (calcd = 406.2 g·mol−1). MS (ESI) m/z: [M + Na]+ calcd for C22H29NNaO3Si, 406.2; found, 406.2. Synthesis of 10. A mixture of 7 (3.64 g, 9.5 mmol), 9 (1.06 g, 4.7 mmol), and DMAP (0.116 g, 0.95 mmol) was dissolved in DCM (160 mL). A solution of DCC (1.1 g, 5.3 mmol) in DCM (20 mL) was added dropwise at 0 °C, and the reaction mixture was stirred overnight at RT. DCU salts were filtered off, and the solvent was removed under reduced pressure. The product was purified by flash chromatography on silica gel (Hex/EtOAc, 100:0 to 0:100). Yield: 2.11 g (76%). 1H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.0 Hz, 4H), 7.43 (d, J = 7.5 Hz, 2H), 7.39 (d, J = 7.5, 7.0 Hz, 4H), 6.29 (bs, 1H), 6.25 (d, J = 17.1 Hz, 1H), 6.03 (dd, J = 17.1, 10.1 Hz, 1H), 5.61 (d, J = 10.1 Hz, 1H), 5.14−5.11 (m, 1H), 4.16−4.04 (m, 2H), 3.82−3.78 (m, 2H), 3.68− 3.61 (m, 10H), 3.55−3.51 (m, 2H), 3.35 (s, 3H), 1.05 (s, 9H). MS (ESI) m/z: [M + H]+ calcd for C31H46NO8Si, 588.3; found, 588.2. Synthesis of 11. To a solution of 10 (716 mg, 1.2 mmol) in THF (9 mL) was added a solution of TBAF hydrate (637 mg, 2.4 mmol) in THF (10 mL) at 0 °C. After a few minutes, the reaction mixture was allowed to reach RT and was stirred until completion of the reaction, monitored by LCMS. THF was removed under reduced pressure, and the product was purified by flash chromatography on silica gel (DCM/ methanol, 100:0 to 95:5). Yield: 303 mg (71%). 1H NMR (600 MHz, CDCl3) δ 6.50 (bs, NH), 6.32 (d, J = 16.7 Hz, 1H), 6.15 (dd, J = 16.7, 11.0 Hz, 1H), 5.68 (d, J = 11.0 Hz, 1H), 4.24−4.15 (m, 4H), 4.04− 4.00 (m, 1H), 3.73−3.63 (m, 12H), 3.56−3.55 (m, 2H), 3.37 (s, 3H). MS (ESI) m/z: [M + H]+ calcd for C15H28NO8, 588.3; found, 588.2. Synthesis of 12. To a stirred solution of 3 (410.6 mg, 0.47 mmol), DMAP (115.4 mg, 0.20 mmol), and 11 (165.0 mg, 0.47 mmol) in DCM (6 mL) was added dropwise DCC (194.9 mg, 0.94 mmol) dissolved in DCM (4 mL) at 0 °C. The reaction mixture was stirred

dried over MgSO4. After filtration, addition of diethyl ether to the organic layer induced the precipitation of the product (white solid, 864 mg). The filtrate was evaporated and redissolved in DCM. Similar treatment gave an additional 365 mg, for an overall yield of 1.23 g (65%), Alternatively, the product was recovered from the DCM phase without ether precipitation (85% yield, with residual DIEA). 1H NMR (600 MHz, DMSO-d6) δ (ppm) 7.97 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 5.6 Hz, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.28 (s, 1H), 6.29 (s, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 4.27 (m, 2H), 4.22 (m, 1H), 3.94 (m, 1H), 3.42 (s, 3H), 3.04 (q, J = 6.5 Hz, 2H), 1.74 (m, 1H), 1.60 (m, 1H), 1.51 (m, 2H). MS (ESI) m/z: [M − H]− calcd for C33H30BrN2O10, 693.1; found, 693.0. Synthesis of 4. To a stirred solution of 3 (680 mg, 0.98 mmol), DMAP (24 mg, 0.20 mmol) and 2-hydroxyethylacrylamide (0.4 mL, 0.45 g, 3.9 mmol) in DCM (9 mL) was added dropwise a solution of DCC (202 mg, 0.98 mmol) in DCM (4 mL) at 0 °C. The reaction mixture was stirred overnight at RT. The crude mixture was washed three times with water and once with brine. The organic layer was removed under reduced pressure and the gray solid residue (960 mg) was used in the next step without further purification. MS (ESI) m/z: [M + H]+ calcd for C38H39BrN3O11, 792.2; found, 792.1. Synthesis of 5. Crude 4 (960 mg) was dissolved in DMF (10 mL) with piperidine (94.1 mg, 1.1 mmol), stirred for 1 h 30 min and monitored by LCMS. Upon completion, the reaction mixture was filtered (1 μm filter) and the solvent was removed under reduced pressure. DCM was added, and the precipitate formed (remaining DCU salts) was filtered out (0.45 μm filter). The product was purified by flash chromatography on silica gel (DCM/methanol). Yield: 331 mg (60%, over two steps). 1H NMR (600 MHz, DMSO) δ (ppm) 8.22 (t, J = 5.2 Hz, 1H), 7.97 (s, 1H), 7.55 (t, J = 5.7 Hz, 1H), 7.28 (s, 1H), 6.28 (s, 1H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H), 6.08 (dd, J = 17.1, 1.8 Hz, 1H), 5.58 (dd, J = 10.1, 1.8 Hz, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 4.08 (m, 2H), 3.42 (s, 3H), 3.38 (m, 2H), 3.30 (m, 1H), 3.02 (q, J = 6.1 Hz, 2H), 1.59 (m, 1H), 1.52−1.39 (m, 3H). MS (ESI) m/z: [M + H]+ calcd for C23H29BrN3O9, 570.1; found, 570.2. Synthesis of 6. After dissolution of 5 (57 mg, 0.1 mmol) in EtOAc (1.5 mL), a solution of K2CO3 (138.1 mg, 1 mmol) in water (1.5 mL) was added, and the resulting mixture was stirred vigorously at 0 °C. Acryloyl chloride (16.2 μL, 0.2 mmol) was then added dropwise to the reaction at 0 °C. After a few minutes, the reaction mixture was allowed to reach RT and was stirred until completion of the reaction, monitored by LC-MS. After 30 min, the organic solvent was removed under reduced pressure and DCM was added to the crude. The organic layer was washed extensively with water and evaporated under reduced pressure to give a white solid (63 mg, 100%). 1H NMR (600 MHz, DMSO-d6) δ (ppm) 8.48 (d, J = 7.5 Hz, 1H), 8.18 (t, J = 5.7 Hz, 1H), 7.97 (s, 1H), 7.56 (t, J = 5.7 Hz, 1H), 7.28 (s, 1H), 6.31 (dd, J = 17.1, 10.1 Hz, 1H), 6.28 (s, 1H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H), 6.10 (td, J = 16.7, 2.2, 1.8 Hz, 2H), 5.64 (dd, J = 10.1, 1.8 Hz, 1H), 5.59 (dd, J = 10.1, 2.2 Hz, 1H), 5.43 (s, 2H), 5.28 (s, 2H), 4.33 (m, 1H), 4.15−4.06 (m, 2H), 3.42 (s, 3H), 3.38 (q, J = 11.6, 5.7 Hz, 2H), 3.03 (q, J = 12.8, 6.6 Hz, 2H), 1.80−1.72 (m, 1H), 1.68−1.58 (m, 1H), 1.51−1.44 (m, 2H). Synthesis of 1. Compound 6 (63.4 mg, 0.1 mmol) was stirred in DCM (2 mL) and TFA (0.2 mL) for 1 h. TFA was removed under high vacuum. TFA was removed by redissolution of the residue in DCM and evaporation, repeated three times, yielding 59 mg of whitish solid foam (100%). 1H NMR (600 MHz, DMSO-d6) δ (ppm) 8.48 (d, J = 7.4 Hz, 1H), 8.18 (t, J = 5.4 Hz, 1H), 7.87 (s, 1H), 7.55 (t, J = 5.4 Hz, 1H), 6.91 (s, 1H), 6.31 (dd, J = 17.1, 10.1 Hz, 1H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H), 6.18 (bs, 1H), 6.10 (td, J = 17.1, 1.8 Hz, 2H), 5.64 (dd, J = 10.1, 1.8 Hz, 1H), 5.59 (dd, J = 10.1, 1.8 Hz, 1H), 5.25 (s, 2H), 4.33 (m, 1H), 4.15−4.06 (m, 2H), 3.37 (m, 2H), 3.03 (q, J = 12.7, 6.6 Hz, 2H), 1.78−1.72 (m, 1H), 1.66−1.60 (m, 1H), 1.50−1.43 (m, 2H). 13C NMR (150 MHz, DMSO) δ (ppm) 25.76, 28.24, 37.55, 51.82, 60.77, 62.92, 103.15, 106.14, 108.48, 110.48, 125.27, 125.90, 128.33, 130.98, 131.42, 151.06, 153.73, 155.24, 157.38, 159.63, 164.68, 164.83, 171.79. HRMS (ESI) m/z: [M + Na] + calcd for C24H26BrN3NaO9, 602.0745; found, 602.0744. C

DOI: 10.1021/acs.biomac.5b00950 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules overnight at RT. DCU salts were filtered and the solvent were removed under reduced pressure. The product was purified by flash chromatography on silica gel (DCM/MeOH, 100:0 to 95:5). Yield: 284 mg (59%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.72 (d, J = 7.5 Hz, 2H), 7.65 (bs, 1H), 7.57 (d, J = 7.5 Hz, 2H), 7.55 (bs, 1H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.5 Hz, 2H), 7.11 (s, 1H), 6.89−6.77 (m, 1H), 6.35 (s, 1H), 6.26 (t, J = 15.8 Hz, 1H), 6.13 (m, 1H), 5.88 (m, 1H), 5.59 (m, 1H), 5.19 (m, 4H), 4.38 (m, 4H), 4.26−4.11 (m, 5H), 3.59 (m, 12H), 3.49 (bs, 5H), 3.32 (s, 3H), 3.24 (m, 2H), 1.88 (m, 1H), 1.88 (m, 1H), 1.60 (m, 2H). Synthesis of 13. Compound 12 (370.7 mg, 0.36 mmol) was dissolved in DMF (5.1 mL) with piperidine (30.7 mg, 0.36 mmol), stirred for 3 h until completion of the reaction (LCMS monitoring). DMF was removed under reduced pressure and the product was purified by flash chromatography on silica gel (DCM/methanol, 100:0 to 80:20). Yield: 261 mg (90%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.68 (s, 1H), 7.13 (s, 1H), 6.86−6.66 (m, 1H), 6.35 (bs, 1H), 6.28 (d, J = 16.7 Hz, 1H), 6.15 (m, 1H), 6.03 (m, 1H), 5.63 (m, 1H), 5.26 (m, 2H), 5.21 (m, 2H), 4.45−4.10 (m, 4H), 3.73−3.61 (m, 12H), 3.54−3.45 (m, 4H), 3.49 (s, 3H), 3.35 (bs, 3H), 3.23 (m, 2H), 1.78 (m, 1H), 1.63 (m, 3H). MS (ESI) m/z: [M + H]+ calcd for C33H47BrN3O15, 804.2; found, 804.0. Synthesis of 14. After dissolution of 13 (260 mg, 0.32 mmol) in EtOAc (10 mL), a solution of K2CO3 (446.5 mg, 3.23 mmol) in water (10 mL) was added, and the resulting mixture was stirred vigorously at 0 °C. Acryloyl chloride (54.3 μL, 0.67 mmol) was then added dropwise to the reaction at 0 °C. After a few minutes, the reaction mixture was allowed to reach RT and was stirred until completion of the reaction, monitored by LCMS. Portion-wise additions of acryloyl chloride (108 μL) were needed to reach complete removal of the MOM groups. After 4 h, the organic and aqueous layers were separated. The aqueous phase was extracted twice with EtOAc. Organic layers were combined, washed twice with water and brine, then dried over MgSO4 and finally removed under reduced pressure. Yield: 268.6 mg (97%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.66 (s, 1H), 7.12 (s, 1H), 7.08−6.95 (m, 1H), 6.34−6.00 (m, 7H), 5.65 (m, 1H), 5.59 (m, 1H), 5.27 (m, 2H), 5.21 (m, 2H), 4.65−4.20 (m, 5H), 3.73−3.59 (m, 12H), 3.51 (m, 2H), 3.48 (s, 3H), 3.36 (m, 3H), 3.33 (bs, 3H), 3.24 (m, 2H), 1.89 (m, 1H), 1.79 (m, 1H), 1.61 (m, 2H). MS (ESI) m/z: [M + H]+ calcd for C36H49BrN3O16, 858.2; found, 858.0. Synthesis of 2. After dissolution of 14 (260 mg, 0.30 mmol) in DCM (6.8 mL), TFA (1.3 mL) was added, and the resulting mixture was stirred 1 h at room temperature. Additional TFA (0.3 mL) was added to reach the completion of the reaction. After 2 h of reaction, the solvents were removed under reduced pressure to give the target molecule as a pale yellow solid. Yield: 239.2 mg (97%). 1H NMR (600 MHz, CDCl3) δ (ppm) 7.62 (s, 1H), 7.23−7.08 (m, 1H), 6.98 (s, 1H), 6.34−6.21 (m, 5H), 6.00−5.86 (m, 1H), 5.75 (d, 1H, J = 9.6 Hz), 5.69 (dd, 1H, J = 14.5 Hz, 10.5 Hz), 5.30−5.16 (m, 3H), 4.44 (m, 3H), 4.26−4.14 (m, 3H), 3.78−3.52 (m, 14H), 3.37 (bs, 3H), 3.28 (m, 2H), 1.94 (m, 1H), 1.81 (m, 1H), 1.65 (m, 2H). HRMS (ESI) m/z: [M + Na]+ calcd for C34H44BrN3NaO15, 836.1848; found, 836.1847. Crosslinker 1 Photolysis and Degradation. 1H NMR Study. A 1 mg/mL solution of compound 1 in deuterated PBS 1× (pH 7.4)/ DMSO-d6 (95:5) was placed into a NMR tube and irradiated using a Luzchem LZC-ORG photoreactor equipped with 8UV-A lamp, for 15 min (1 mW·cm−2). The crosslinker solution was incubated at 37 °C and 1H NMR (600 MHz) spectra were taken periodically to estimate the degree of crosslinker degradation as the percent of the cleaved ester bonds. HPLC-MS Study. A 0.1 mg/mL solution of compound 1 in PBS 1× (pH 7.4)/DMSO (95:5) was placed into a quartz semimicrocuvette and irradiated using a Luzchem LZC-ORG photoreactor equipped with 8UV-A lamp, for 15 min (1 mW·cm−2). The solution was incubated at 37 °C and aliquots (10 μL) were removed periodically and injected into HPLC-MS to determine the presence of the intermediates by integrating the peaks of the single ions, m/z = 580 (1, [M + H]+), m/z = 284 (uncaged crosslinker, [M + H]+), m/z = 169 (lactam byproduct obtained by the intramolecular cyclization, [M +

H]+), and m/z = 187 (byproduct from hydrolysis, [M + H]+). Area under the peak corresponding to each intermediate was measured and these integrations were then normalized. A similar experiment was done with nonirradiated samples. Preparation of Photodegradable Hydrogels. Hydrogels at 2.7 mol % cross-linking density were formed by dissolving 4.65 mg (5.7 μmol) of Bhc crosslinker 2 and acrylamide (15 mg, 211 μmol) with fluorescein-labeled BSA (0.5 mg) in 100 mM sodium phosphate buffer, pH 8.5 (100 μL). A total of 1 μL of 50% ammonium persulfate (APS) in 100 mM sodium phosphate buffer, pH 8.5, was added, followed by the addition of TMED (1 μL). The resulting mixture was gently mixed in a 1 mL syringe and left at room temperature for 1 h. After this time, hydrogels were stiff enough to be manipulated with a spatula and were used for protein release experiments. Empty hydrogels were also prepared and analyzed as blanks. Protein Release Study. The hydrogel was added to a 20 mL scintillation vial and washed in PBS 1× (pH 7.4, 10 mL) under mild swirling (14 h, rt, in the dark). Washing solution was removed and replaced by fresh PBS (5 mL). Hydrogel was incubated at 37 °C. At each time point, complete solution was removed and replaced by fresh PBS (5 mL). The irradiated samples were irradiated at each time point (15 min UV, 1.0 mW/cm2). On each collected sample, the amount (mi, μg) of FTC-BSA released was assessed by micro-BCA protein assay kit (Thermo Scientific). Percentage released at each point was calculated by mi/Σmi × 100%, with mi = ci × Vi + Σmi−1. Cell Culture. Mouse mesenchymal stem cells stably expressing GFP (Cyagen, OriCell, strain C57BL/6, mMSC-GFP, Cat. No. MUBMX-0110) were cultured in OriCell mouse mesenchymal stem cell growth medium (Cyagen, Cat. No. MUXMX-90011) supplemented according to provider with cell-qualified fetal bovine serum, glutamine, and penicillin-streptomycin. Cells were routinely cultured at 37 °C in a humidified incubator in a 95% air/5% CO2 atmosphere and passaged by detaching cells with 0.25% trypsin−0.05% EDTA (w/ v) solution (Gibco Invitrogen). Preparation of Hydrogels Encapsulating mMSCs-GFP. Hydrogels at 2.9 mol % cross-linking density were formed by dissolving 5 mg (6.1 μmol) of Bhc crosslinker 2 and acrylamide (15 mg, 211 μmol) in sterile DMEM, pH 9.1 (75 μL). pH reached back 7.4 upon saturation with CO2 for 5 min. A total of 1 μL of 50% APS in DMEM, pH 7.4, was added, followed by the addition of TMED (1 μL). The resulting mixture was combined with fibronectin (4 μL, 1 mg/mL) and GFP-MSCs solution (50000 cells, 21 μL DMEM) and gently vortexed for a few seconds. The solution was aliquoted in 12 samples (8 μL) in two Lab-Tek 8 chambers, and the hydrogels were allowed to polymerize for 45 min in the incubator (37 °C, 5% CO2). Then, 100 μL of mMSC-cell culture media (wash #1) was added, and the hydrogels were allowed to equilibrate for 40 min (37 °C, 5% CO2). Wash #1 was replaced by fresh mMSC-cell culture media (100 μL, wash 2) for further equilibration (37 °C, 5% CO2). Washes #1 and #2 allow us to remove cells that are weakly entrapped at the gel surface in order to evaluate the accurate number of light triggered cell released from the gel. GFP-mMSCs Release Study. Cell culture media was replaced by PBS 1× (pH 7.4, 100 μL). Hydrogels (N = 6) were exposed to UV light (365 nm, OmniCure S2000 Curing System, 30 cm from light source, 1 mW/cm2) for 15 min intervals. After each exposure time, additional cell culture media was added (100 μL) and the hydrogels were incubated 1.5 h (37 °C, 5% CO2). Before each irradiation, the supernatant solution was moved to a new well and cells were allowed to settle for few hours before counting the cells released by microscopy (irradiated fractions 1−6, Figure S9). Nonirradiated hydrogels (N = 6) were studied in parallel (control fractions 1−6, Figure S9). A total of six irradiations were performed on the hydrogels, and the final incubation time was 10 h. Preparation of Photodegradable Nanogels. Nanogels at 1.7 mol % cross-linking density were formed by dissolving 12 mg (13 μmol) of Bhc crosslinker 2 and acrylamide (61.3 mg, 862 μmol) with or without 10 nm iron oxide (II,III) magnetic nanoparticles (50 μL of a 5 mg/mL solution in H2O) in 10 mM Tris-HCl, pH 8 (250 μL). The resulting solution was added into 9% docusate sodium (AOT; 1 g) D

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Biomacromolecules solution in hexane (10 mL). After vortexing for 1 min, 16 μL of 50% APS in 10 mM Tris-HCl, pH 8, was added. The reaction mixture was vortexed for 1 min and then filtered through a 0.22 μm PVDF filter. After portionwise addition of 500 μL of 5% tetramethylethylenediamine (TMEDA) in hexane (10 μL × 50), the particle solution was left on a shaker at 60 rpm for 30 min, followed by removal of hexane by rotary evaporation at 40 °C. Nanogels were washed with acetone (10 mL × 3) and collected each time by centrifugation at 5000g for 10 min. The white precipitate was then dispersed in 1 mL of PBS 1× (pH 7.4). Dynamic Light Scattering. All DLS experiments were done in triplicate. Degradation upon UV Exposure. In a typical experience, 25 μL of the nanogel solution mentioned above was diluted with PBS 1× (pH 7.4, 100 μL) in a UV transparent disposable cuvette. Then the solution was irradiated in the Luzchem LZC-ORG photoreactor for 15 min. A nonirradiated mixture was studied in parallel. Time course of nanogels degradation was monitored as a function of incubation time at 37 °C. Degradation upon NIR Exposure. In a typical experience, 8 μL of the nanogel solution mentioned above was diluted with PBS 1× (pH 7.4, 50 μL). Then the solution was irradiated at 740 nm with a Ti:sapphire laser for 1 h in a quartz cuvette (50 μL, 3 mm, Z = 15 mm). A nonirradiated mixture was studied in parallel. Time course of nanogels degradation was monitored as a function of incubation time at 37 °C. Transmission Electron Microscopy of Nanogels. Samples before and after irradiation/incubation were diluted five times with DI water and one droplet of the diluted solution for each sample was dropcast onto a Pelco carbon-coated 400 square mesh copper grid (Ted Pella, inc.). The grids were naturally dried in air for 3−5 h before imaging.

Figure 1. 1H NMR and HPLC-MS analysis confirm that crosslinker 1 degrades exclusively via lactamization upon light-triggered deprotection (15 min UV irradiation, 1 mW/cm2) in PBS (1×, pH 7.4)/ DMSO-d6 (95:5) at 37 °C. (a) Potential products formed by photolysis of the light-cleavable crosslinker 1. (b) 1H NMR spectra of 1, following irradiation, as a function of incubation time. Integration of C and c were slightly off because the intensity of the signal decreases due to water signal suppression, required to avoid domination of the spectra by the water residual signal (at 4.79 ppm). (c) Kinetics of lighttriggered degradation of 1 (15 min UV irradiation) or by hydrolysis, as determined by 1H NMR spectroscopy. (d) HPLC-MS analysis confirms degradation of 1 via lactamization following irradiation ([M + H]+ = 116 was not detected).

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RESULTS AND DISCUSSION We combined ornithine, which spontaneously forms a sixmembered lactam upon deprotection, with a coumarin-based photocage to yield light-degradable crosslinkers 1 and 2 (Scheme 1). Absorption spectra of 1 and 2 were recorded in diluted organic and aqueous solutions to determine their molar absorption coefficients (Table 1 and Figure S3). Table 1. Absorption Spectral Data of 1 and 2 compound

solvent

λmaxa

εb

1 1 1 2 2 2

DMSO PBS (1×, pH 7.4)/DMSO (95:5) DMEM DMSO PBS (1×, pH 7.4)/DMSO (95:5) DMEM

330 372 371 330 373 371

10846 14889 10274 14696 18454 27119

was then introduced through a carbamate linkage. The first acrylamide group was then added by DCC coupling with N-(2hydroxyethyl)acrylamide. For ease of synthesis, the crude intermediate was treated with piperidine to remove the Fmoc protecting group. The second acrylamide moiety was attached by addition of acryloyl chloride using Schotten−Baumann conditions. In the last step, TFA treatment removed the methoxymethyl acetal protecting group (MOM) to give the target crosslinker structure 1. 1H and 13C NMR spectra of 1 are reported in the Supporting Information (Figures S1 and S2). 1 H NMR Spectroscopic Degradation Study. 1H NMR spectroscopy confirmed that crosslinker 1 degrades exclusively by lactamization following photocleavage in deuterated PBS (1×, pH 7.4)/DMSO-d6 (95:5). Degradation products were identified and degradation kinetics were quantified after incubation at 37 °C with or without UV irradiation (Figures 1a−c and S4 and Table 2). Upon irradiation, all resonance signals from the protons of the Bhc (7.8, 6.6, 6.1, and 5.3 ppm) disappeared, indicating complete removal of the photocage (Figure 1, 0 h); nonsoluble cleaved Bhc residues typically precipitate and are no longer detected. In addition, the shift from 3.2 to 3.1 ppm is characteristic of the methylene protons in α position of the amine (D′ → D). In the nonirradiated sample, no shift of signal D′ is observed (Figure S4), indicating

a

Maximum absorption wavelengths. bMolar absorption coefficient (M−1·cm−1). Concentrations ranged from 3.10−6 to 8.10−5 M.

Before investigating hydrogel stability and light-triggered degradation at the bulk and nanoscale, model compound 1 was exposed to UV light to confirm degradation via lactamization. Detailed characterization of the photodegradation reaction was performed using 1H NMR spectroscopy and HPLC-MS (Figure 1). Both techniques show (1) high stability of the crosslinker at 37 °C in the absence of irradiation, (2) near complete light-triggered degradation within less than 24 h of incubation at 37 °C, and (3) formation of only lactam products. Synthesis of Model Crosslinker 1. The ornithine-inspired light-degradable crosslinker 1 was obtained via a five-step synthesis (Scheme 2; global yield: 39%). Briefly, commercially available Fmoc-Orn(Boc)-OH was first treated with TFA to selectively remove the Boc protecting group. The Bhc moiety E

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light-triggered degradation, we needed a more hydrophilic crosslinker (compound 2, Scheme 3) to formulate hydrogels

Scheme 2. Synthesis of Ornithine-Inspired Light-Degradable Crosslinker 1a

Scheme 3. Synthesis of Ornithine-Inspired Light-Degradable Crosslinker 2a

a

Reagents and conditions: (a) TFA/DCM; Bhc-PNP, DIEA, DCM/ DMF, o.n., 65%; (b) N-(2-hydroxyethyl)acrylamide, DCC, DMAP, DCM/DMF, o.n.; (c) piperidine, DMF, 1.5 h, 60% over 2 steps; (d) acryloyl chloride, K2CO3, EtOAc/H2O, 30 min, 100%; (e) TFA/ DCM, 1 h, 100%. R = methoxymethyl acetal (MOM).

Table 2. Mechanism of Degradation and Half-Life (t1/2) of 1 Incubated at 37 °C, as Determined by 1H NMR

b

a Reagents and conditions: (a) CrO3/H2SO4, acetone, 2 h, 93%; (b) tert-butyl(chloro)diphenylsilane, imidazole, DMF, o.n., 37%; (c) acryloyl chloride, K2CO3, EtOAc/H2O, 2 h, 92%; (d) 7, DCC, DMAP, DCM, o.n., 76%; (e) tetrabutylammonium fluoride hydrate, THF, 3 h, 71%; (f) 3, DCC, DMAP, DCM, o.n., 59%; (g) piperidine, DMF, 3 h, 90%; (h) acryloyl chloride, K2CO3, EtOAc/H2O, 4 h, 97%; (i) TFA, DCM, 2 h, 97%.

that the Bhc group remains intact. New resonance signals (e and f) correspond to the formation of N-(2-hydroxyethyl) acrylamide, upon the cleavage of the ester linkage. The shift from 3.1 to 3.4 ppm (D → d) confirmed the formation of lactam derivatives after irradiation and incubation.65 HPLC-MS Degradation Study. After irradiation, no remaining intermediate (deprotected 1, [M + H]+ = 284) is detected in the media within 20 h (Figure 1d). This result is in agreement with the time needed to completely degrade ornithine-based dimers when exposed to UV light.65 Moreover, only lactam derivatives ([M + H]+ = 169) were formed from the irradiated crosslinker, as no primary amine intermediates resulting from ester hydrolysis ([M + H]+ = 187) were detected. Interestingly, both 1H NMR and HPLC-MS are in agreement with no hydrolysis of the ester bound but an exclusive degradation by cyclization. No degradation species were detected in nonirradiated crosslinker (Figure S5). To validate that our hydrogel degrades in response to NIR light, we irradiated our crosslinker for 1 h at 740 nm (1 W) and incubated at RT overnight. 1H NMR spectroscopy confirmed the efficiency of photocleavage (shift from 3.2 to 3.1 ppm, indicating the formation of a free amine; only around 5% of protecting group signal remains; red rectangles, Figure S6a). The extent of lactamization was calculated to be near 30%, which is close to the percentage obtained upon UV irradiation at RT (Figure S6b). Synthesis of Crosslinker 2. While crosslinker 1 was a model compound used to validate the mechanism of fast and

and to assess the feasibility of NIR/UV-triggered hydrogel and nanogel degradation. Without modifying the overall length of the crosslinker (14 carbons (2) vs 13 (1)), a triethylene glycol (TEG) arm was introduced within the ornithine-inspired structure. Briefly, commercially available tetraethylene glycol monomethyl ether was first treated with Jones reagent mixture to oxidize the terminal alcohol to form carboxylic acid 7. In parallel, silyl ether 8 was obtained from 3-amino-1,2-propanediol using tert-butyl(chloro)diphenylsilane and imidazole. The first acrylamide moiety was attached by addition of acryloyl chloride to 8. The remaining alcohol in 9 was then coupled to the TEG chain of 7 by DCC coupling to obtain TBDPSprotected TEG compound 10. Finally, similar procedures as used in the synthesis of 1 were followed to synthesize 2 from 10. The key and unique feature of the reported light-degradable hydrogels (bulk and nano) is the short length between the two acrylamide functional groups of crosslinker 2 (15.4 Å, as measured by ChemBio3D after MM2 energy minimization). Upon copolymerization with acrylamide (2−3 mol % crosslinking density), the obtained hydrogels are stable in the absence of irradiation and degrades in a rapid and controlled fashion upon irradiation. We found that these polymerization conditions yielded a suitable mesh size for the quantitative entrapment and release of proteins or other hydrophilic payloads with a hydrodynamic diameter of >7 nm.66,67 Light-Triggered BSA Release Kinetics. Fluo-BSA-loaded bulk hydrogels were prepared by copolymerizing 2 and acrylamide in sodium phosphate buffer (100 mM, pH 8.5) in

UV no UV

time

lactamizationa (%)

hydrolysisb (%)

t1/2

45 h 45 h

82.3 ± 5.7 0

0 13.8 ± 2.5

12 h 20 d

% lactamization = ∫ d/(∫ D + ∫ d) ≈ ∫ f/(∫ F + ∫ f) ≈ ∫ e/(∫ E + ∫ e). % hydrolysis = ∫ f/(∫ F + ∫ f) ≈ ∫ e/(∫ E + ∫ e). ∫ refers to the integration below each respective peak.

a

F

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(measured by infrared camera, Figure S7). No significant heating by water absorption is expected with a femtosecond laser operating at a water-transparent wavelength70 (740 nm) with low powers. As a proof of concept, we formulated acrylamide-based hydrogels encapsulating murine mesenchymal stem cells expressing green fluorescent protein (GFP-mMSCs) in bicarbonate-buffered cell culture medium (Dulbecco’s modified Eagle’s medium, DMEM). Taking advantage of the sodium bicarbonate−carbon dioxide system, 2, was solubilized in DMEM (pH 9, after competing the CO2 by bubbling argon for 1 h, Figure S8). Neutral pH was recovered prior to cell addition simply by bubbling the solution with CO2 for a few minutes. After formation and equilibration of the hydrogels in mMSC cell-culture medium, some were exposed to UV light for six periods of 15 min (365 nm, 1 mW/cm2) and incubated 1.5 h between each exposure. The medium was changed between each irradiation and the number of collected cells was counted in both irradiated and nonirradiated hydrogels (Figures 2c and S9). Quantifying nonencapsulated cells in the wash revealed that the encapsulation efficiency was above 92% (±1.8%, n = 12). In addition, mMSCs were released 15× faster when hydrogels were exposed to repeated UV irradiation. Nonirradiated hydrogels did not release appreciable numbers of cells (Figure 2c). While this is not the first report of light-triggered cell release, our design for release at low power is an important property for biologically relevant applications.39 Light-Triggered Release of Iron Oxide Nanoparticles from Nanogels. Using the same chemistry, crosslinker 2 was also employed to synthesize light-degradable nanogels. Because they can efficiently encapsulate, transport, and release hydrophilic payload in a preprogrammed manner, nanogels are ideal carriers for the delivery of small therapeutic agents or biomacromolecules, combining small size with large surface area, efficient swelling, and high loading.71−74 Empty nanogels were formulated by means of inverse emulsion redox polymerization at 1.7 mol % crosslinker density in Tris HCl (1×, pH 8.0) to verify stability and determine light-triggered degradation kinetics. No change in hydrodynamic diameter and PDI was observed in the absence of irradiation, confirming that light-responsive nanogels (200 nm) are stable for at least 1 week at 37 °C (Figures 3a,d,e and S10). Conversely, brief exposure to UV (15 min) or NIR (1 h) triggers disassembly within a few hours of incubation (Figure 3b−f). After 5 h, only nonirradiated nanogels yielded reliable DLS data; the difficulty in analyzing irradiated nanogels is a result of aggregated or sedimented particles and small fragments. At the first stage of degradation, photocage cleavage leads to nanogel swelling due to increased hydrophilicity from unmasking free amines. Further incubation of the loosened network led to an additional increase in diameter in combination with diffusion of free hydrophilic PAA chains. No swelling was observed in the absence of light, indicating stability of the polymeric network at this crosslinking density. This absence of initial swelling, likely a result of the crosslinker’s chemical structure and short length, is crucial to prevent burst release and payload leakage. Polymer mesh morphology and chemical composition are known to affect release characteristics.75,76 In these hydrogels, hydrophobic coumarin moieties covalently attached to the network reduce the initial penetration of water, and the short crosslinker limited mesh space available for diffusion preirradiation. Finally, we investigated whether nanogels release contents in response to light. PAA nanogels containing crosslinker 2 and

the presence of the protein. To confirm light-induced degradation and release, both irradiated and nonirradiated gels were incubated in PBS (1×, pH 7.4) at 37 °C, and the amount of protein released over time in the supernatant was measured by microbicinchoninic acid assay (micro-BCA assay; Figure 2b). BCA analysis of the solution used to wash the

Figure 2. (a) Accelerated hydrogel erosion and degradation upon UV irradiation. (b) UV irradiation-dependent release of Fluo-BSA from hydrogels analyzed by micro-BCA assay. Hydrogels were irradiated (365 nm, 15 min, 1 mW·cm−2) prior to incubation for each time point. Results are presented as means ± SEM; n = 3. (c) UV irradiationtriggered release of encapsulated GFP-mMSCs. Hydrogels (n = 6) were irradiated (15 min, 365 nm, 1 mW/cm2), and the released cells were counted after each incubation interval (1.5 h). Results are presented as means ± SEM; n = 6.

hydrogels indicated that 80% of proteins were entrapped in the gel. This is in agreement with the gels’ small pore size capable of efficiently retaining BSA-size payload (7 nm).68 Similar BSA release kinetics were obtained with hydrogels prepared from acid-labile acetal crosslinkers (comparable linker length, crosslinking ratio, and gelation conditions).27 Light acceleration of hydrogel degradation and BSA release were clearly observed (Figure 2a). Upon irradiation, the hydrogel released 100% of the entrapped protein within 10−20 h, and the hydrogel was no longer visible (fully degraded) in the media after 20 h incubation. Conversely, the nonirradiated hydrogel retained its integrity and more than 80% of the payload over the same amount of time. Light-Triggered Release of GFP-mMSCs from Hydrogels. Despite substantial challenges, hydrogels hold great potential as cell delivery implants for regenerative medicine.69 This work demonstrates the ability of crosslinker 2 to build cell reservoir matrices for the encapsulation and on-demand delivery of therapeutic cells. No appreciable increase in temperature was observed after 15 min of UV irradiation G

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Figure 4. Nanogels fall apart within 5 h upon UV or NIR irradiation. Representative TEM microphotographs of nanogels after 5 h incubation at 37 °C in phosphate buffer (pH 7.4, 10 mM), either (a) nonirradiated or (b) UV-irradiated (365 nm, 15 min, 1 mW·cm−2) or NIR-irradiated (740 nm) for (c) 1 h (1 W, 1.9 × 106 mW·cm−3) and (d) 2 h (0.5 W, 9.5 × 105 mW·cm−3).

Figure 3. Upon UV and NIR exposure, nanogel particles first swell then degrade. DLS profiles of (a) nonirradiated, (b) UV-, and (c) NIR-irradiated nanogels (15 min, 365 nm, 1 mW·cm−2 and 1 h, 740 nm, 1 W, 1.9 × 106 mW·cm−3, respectively). Individual DLS parameters of nonirradiated and irradiated nanogels over time: (d) hydrodynamic diameter, (e) PDI, and (f) mean count rate. Results are presented as means ± SEM; n = 3.

encapsulation and light-activated release of hydrophilic payloads as small as 7 nm in diameter. As these materials also degrade in response to NIR light, they hold promise for spatiotemporal control over protein and cell presentation and delivery in cultured and engineered tissues as well as within living tissue. The next step in this line of research is to use this crosslinker to develop light-degradable hydrogels from biodegradable materials in place of PAA, such as methacrylated hyaluronic acid (HA), alginic acid, chitosan, or polylactic acid, all known to be enzyme-degradable. Our design may find application where complete on-demand degradation is of high importance, especially to avoid surgical removal of materials following cell delivery.79

superparamagnetic iron oxide nanoparticles (SPIONs) were prepared following the same emulsion technique. SPIONs are effective contrast agents for magnetic resonance imaging (MRI) and are widely used for clinical oncology imaging and therapy.77,78 Hydrophilic SPIONs (Fe3O4, 10 nm) display high electron density, facilitating detection of nanogels by TEM. TEM micrographs of these nanogels show that nanogels are spherical, dispersible in water, and efficiently encapsulate iron oxide particles (Figure S11). No precipitation in acetone of nonencapsulated SPIONs was observed during the formulation. As expected from the crosslinker design, UV- or NIRirradiated nanogels completely fell apart and aggregated within 5 h, corroborating degradation kinetics, as determined by DLS (Figure 3). Nonirradiated nanogels were intact after 5 h (8 h incubation in Figure S12), with iron oxide particles located only inside nanogels (Figure 4a). After UV or NIR irradiation, intact particles are no longer visible; only fragments of material coexist with iron oxide aggregates (Figure 4b−d).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00950. 1 H and 13C NMR of crosslinker 1, absorbance spectra in DMEM of crosslinkers 1 and 2, 1H NMR and HPLC-MS photolysis and degradation studies, photographs and thermal images, and release experiments, DLS, TEM (PDF).

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CONCLUSION Two new ornithine-based photodegradable coumarin-bearing short crosslinkers were synthesized and used to formulate PAAbased bulk hydrogels and nanogels encapsulating protein (BSA), cells (GFP-mMSCs), and iron oxide nanoparticles. Both the crosslinker and the resulting hydrogels are stable in PBS (pH 7.4) in the absence of irradiation with minimal or no leakage of their contents, depending on the payload size. However, upon irradiation with low-power UV or NIR light, the gels fully degrade into short fragments through intramolecular cyclization. As expected from the molecular design of the crosslinker, these hydrogels are capable of efficient

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address ⊥

UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8896

Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acs.biomac.5b00950 Biomacromolecules XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS We thank the NIH (R01EY024134) for funding. NMR data was acquired at the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences NMR facility.

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ABBREVIATIONS BCA, bicinchoninic acid; Bhc, bromohydroxycoumarin; BhcGlu, N-(6-bromo-7-hydroxycoumarin-4-yl)methoxycarbonyl-Lglutamic acid; Bhc-PNP, (6-bromo-7-(methoxymethoxy)-2oxo-2H-chromen-4-yl)methyl(4-nitrophenyl) carbonate; BSA, bovine serum albumin; DCC, N,N′-dicyclohexylcarbodiimide; DCM, dichloromethane; DLS, dynamic light scattering; DIEA, N,N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s modified Eagle’s medium; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; Fluo-BSA, fluorescein-labeled bovine serum albumin; GM, Goeppert−Mayer; kcps, kilo counts per second; HA, hyaluronic acid; HPLC, high-performance liquid chromatography; mMSCs-GFP, mice mesenchymal stem cells expressing green fluorescent protein; MOM, methoxymethyl acetal; MS, mass spectrometry; NIR, near-infrared; o.n., overnight; ONB, ortho-nitrobenzyl; PBS, phosphate buffer saline; PDI, polydispersity index; PEG, polyethylene glycol; PMMA, poly(methyl methacrylate); PNP, para-nitrophenyl; TBDPS, tertbutyldiphenylsilane; TEG, triethylene glycol; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; tRNA, trans-ferribonucleic acid; UV, ultraviolet; Zavg, Z average

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