Letter www.acsami.org
Photolabile Hydrogels Responsive to Broad Spectrum Visible Light for Selective Cell Release Vinh X. Truong,*,† Fanyi Li,†,‡ and John S. Forsythe† †
Department of Materials Science & Engineering, Monash Institute of Medical Engineering, Monash University, Clayton, 3800 Victoria, Australia ‡ CSIRO Manufacturing, Bayview Avenue, Clayton, 3168 Victoria, Australia S Supporting Information *
ABSTRACT: We introduce an efficient method for the preparation of photolabile polymer linkers to be used in the fabrication of bioorthogonal and photodegradable hydrogels. The versatility of this synthesis strategy allows for incorporation of a series of chromophores responsive to addressable wavelengths of UV and broad spectrum visible light. Consequently, selective release of different cell types from composite hydrogels by user-defined timing can be achieved by irradiating the materials with different wavelengths of light.
KEYWORDS: photodegradation, visible light, bioorthogonal click, cell release, hydrogel
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design of a different synthesis route. For certain visible-lightresponsive chromophores such as perylene20−22 and 7(diethylamino)coumarin (DEAC),23−25 potential synthesis strategies to install them into a polymer linker can be challenging. This deficiency in synthetic method to easily install a photolabile unit together with a cross-link-able functionality significantly limits the development of novel biomaterial hydrogels responsive to defined wavelengths of light in the visible region, which is more desirable for biomedical applications. The three-component Passerini reaction, which involves a carboxylic acid group, an aldehyde group, and an isocyanate to form an α-acyloxyamide, is a viable solution for the mentioned challenges because the reaction can incorporate both a chromophore and a click-able group in a one-pot procedure.26 In addition, this reaction is highly efficient and can be carried out under mild reaction conditions with high compatibility to various functional groups.27,28 The Passerini reaction therefore has been successfully applied in the synthesis of photoresponsive polymers29,30 and photocaged compounds.28,31 In this work, we apply the Passerini reaction to prepare a library of photolabile polyethylene glycol (PEG) linkers containing a clickable azide group and a chromophore responsive to various wavelengths in the UV−visible region. We subsequently fabricate photodegradable hydrogels via strain-promoted
olymeric hydrogels have been researched extensively for applications in drug delivery, tissue engineering, and regenerative medicine due to their high water content, mechanical stability, and synthetic flexibility.1 Encapsulation of bioactive components within hydrogels has been used to allow controlled release until a stimulus triggers a change in the hydrogel network structure. Among the various triggers possible for hydrogel degradation, light stimulation is highly attractive because it can be achieved remotely with excellent spatial and temporal precision.2−4 Thus, there have been numerous reports on photoresponsive hydrogels designed for biological applications, including manipulation of the microenvironment to direct cell migration and cellular function,2−9 or on-demand release of therapeutics10−15 and living cells.13,16−18 Current photodegradable hydrogel platforms mostly utilize the photocleavage of ortho-nitrobenzyl (o-NB)2−7,9−12,14,16−18 or coumarin derivatives13,19 that absorb light in the 320−410 nm wavelength, restricting the degradation triggered by UV light. The preparation of these photodegradable hydrogels requires the synthesis of chromophores containing two reactive handles - one for selective conjugation to a polymer chain-end and the other for attachment of a polymerize-able functional group, such as acrylate2−4,6−8,10,13,14,17,18 or azide,9,12,16,19 for subsequent polymer cross-linking. The syntheses of such photolabile compounds follow multistep procedures with generally low total yield (5−20%).2,10,17 A second handle attached to the chromophore may also change the light absorption properties and reduce the degradation efficiency.17 Furthermore, switching to a new chromophore necessitates the © XXXX American Chemical Society
Received: August 3, 2017 Accepted: September 11, 2017 Published: September 11, 2017 A
DOI: 10.1021/acsami.7b11517 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 1. (a) Synthesis strategy for incorporation of a chromophore and a linker to the polymer end-group. (b) Chemical structure of the nitrobenzyl with different substituents (P1-P6), DEAC (P7), and perylene (P8) as chromophores for photocleave of the acyloxyamide and the azide, cyclooctyne for SPAAC. (c) Schematic presentation of polymer cross-linking and photodegradation of the hydrogel structure.
Figure 2. Degradation kinetics of the investigated hydrogels under irradiation (with intensity of 10 mW cm−1) at wavelengths of (a) 365, (b) 420, (c) 455, (d) 470, and (e) 530 nm, and (f) the resultant rate constants.
UV−vis analysis (Figure S20) of the resultant polymers revealed that all o-NB containing linkers (P1-P6) have the absorbance band extending up to 400 nm. Incorporation of an electron withdrawing group at the para-position with regards to the α-acyloxyamide group does not significantly affect the UV− vis absorbance and the absorbance of these linkers at 365 nm is lower than that of linker P6 (ε365 = 3912 M−1 cm−1), which contains the methoxy-substituted o-NB moiety widely used in photodegradable hydrogels.4,9,17 As expected, the incorporation of the DEAC and perylene chromophores resulted in polymers (P7 and P8) having the absorbance extending into the blue light region up to 480 nm. Notably, both P7 and P8 have very high molar absorptivity at 420 nm, with values of 52730 M−1 cm−1 and 48128 M−1 cm−1 respectively. Irradiation of the o-NB linkers with UV light (365 nm) resulted in, with the exception of P6, an increase in absorbance at the long wavelength region, whereas a decrease in the UV−vis absorbance was observed for linkers P7 and P8. Further analysis of the photolyzed products by 1H NMR shows the disappearance of both the amide and
azide−alkyne cycloaddition (SPAAC) and demonstrate selective and sequential release of different cell types encapsulated within a composite hydrogel matrix. In our synthesis strategy, 4-arm poly(ethylene glycol) endgroups were first modified to contain carboxylic groups, which then participated in the Passerini reaction with an aldehyde and azido-propane-isocyanate to form α-acyloxyamide end-groups (Figure 1a). This method enables the incorporation of a series of photoresponsive moieties, as shown in Figure 1b, in the polymer end-group. Except for 7-(diethylamino)coumarin-3carbaldehyde (for the preparation of P7), which was synthesized via a one-step procedure, all other aldehyde starting materials could be obtained from commercial sources. Notably, with slight excess of aldehyde and isocyanate in the reacting mixture, good yield (80−90%) of the polymer products with near complete conversion (95%−99%) of the carboxylic end-group to the photocleavable ester end-groups (see Experimental Section and NMR analysis of the polymer products in the Supporting Information) was obtained. B
DOI: 10.1021/acsami.7b11517 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
°C).16 This degradation rate is much slower than the rates of photocleavage under a light-trigger as shown above, which suggests that photocontrolled release of encapsulated macromolecules or cells is possible within a certain time frame before hydrolysis occurs. Controlled release of different cell types or peptides by userdefined timing creates synergetic interactions that are highly beneficial in tissue engineering and regenerative medicine.11,17 Although faster release of one cell population over another from a multihydrogel construct using o-NB linkers with different degradation kinetics was reported,17 this photorelease strategy resulted in a mixture of cell types with prolonged UV light exposure. To demonstrate the potential utility of our hydrogels in selective release of cells from a biomaterial matrix, we encapsulated 2 different cell types, human mesenchymal stem cells (hMSC) and L929 murine fibroblast cells in gel P2 and gel P8, respectively. The two hydrogels were fabricated so that they adhered and formed an interface (Figure 3). Live/
ester protons, indicating that photoinduced cleavage may happen to both the ester and the amide from the αacyloxyamide group. We next prepared hydrogels by cross-linking the photolabile linkers with 4arm PEG20k-DBCO via strain-promoted azide alkyne cycloaddition (SPAAC) and the polymer network was subsequently degraded by photolysis of the ester linkage (Figure 1c). Catalyst free bioorthogonal SPAAC was chosen so that the hydrogel system can be used for 3D cell encapsulation in biological applications.9,12,16,32−34 Rheological analysis of the gelation kinetics revealed formation of stable hydrogels with moduli of ca. 4.8 kPa within 20 min (Figures S22 and S23) of mixing the polymers in aqueous solution at a concentration of 10 wt % (5 mM). The resultant hydrogels have a swelling ratio (Q) of 2.6−2.8 and a gel fraction of 0.89−0.90 (Figure S26). Subsequently, degradation of the as-prepared gels under irradiation (intensity of 10 mW cm−2) with UV light (365 nm), blue light (420 nm, 455 nm, 470 nm) and green light (530 nm) were investigated by rheological analysis. The kinetic constants of degradation kapp were calculated from the slope of ln(G′/ G′o) vs time curve and the data are presented in Figure 2. Except for hydrogel P5, all other hydrogels display fast degradation under UV light irradiation. Hydrogel P6 has a kinetic constant of 0.0026 s−1 and this value is comparable to the kapp (0.0029 s−1) from photodegradation of a similar hydrogel network reported by De Forest and Anseth.9 Interestingly, hydrogel containing o-NB (P1) without the methoxy substituent, while having a lower molar absorptivity at 365 nm, displays a faster degradation rate with a kapp of 0.0047 s−1. Incorporation of a chloride or bromide at the para- osition (hydrogels P2 and P3) results in an increase in the rate, whereas the presence of the nitro group (hydrogel P4) decreases the rate of photodegradation compared to the kinetics of the hydrogel containing unsubstituted o-NB group. The photodegradation kinetics of hydrogels P7 and P8 at 365 nm are both much faster than that of the o-NB containing hydrogels, which may be due to the higher molar absorptivity of the corresponding DEAC and pyrelene chromophores. Furthermore, fast photodegradation of hydrogels P7 and P8 was observed under irradiation of blue light (420−470 nm), whereas very slow degradation occurred for hydrogels P1-P6 at 420 nm irradiation and no degradation could be seen for these gels under irradiation of light at wavelength above 420 nm. Within the blue light region, the perylene containing hydrogel (P8) showed a faster rate of disintegration compared to DEAC containing hydrogel (P7). In addition, the degradation of hydrogel P7 was negligible under green light illumination (530 nm) while hydrogel P8 displayed good degradation kinetics (comparable to degradation rate of gel P6 under UV light irradiation) at this wavelength. Altogether these data indicate that DEAC and perylene are excellent chromophores to be used in the preparation of photodegradable hydrogels responsive to long wavelength UV light and broad spectrum blue light; key to incorporation of these chromophores into hydrogel network is the utilization of the Passerini reaction. The chromophores used for this chemical approach must contain an aldehyde group for insertion into the polymer chainend and subsequent photolysis. The gels prepared by this method contain esters groups which are prone to hydrolysis. A previous study from our group on hydrogels containing the nitrobenzyl group similar to gel P6 showed that the gels degraded, via ester hydrolysis, after one month under physiological conditions (PBS solution pH 7.4 and at 37
Figure 3. Image of composite gel from P2 and P8 linkers cross-linked with PEG20k-DBCO together with fluorescent live dead staining image of cell laden gel at the interface, and graph showing viability of the encapsulated L929 cells and hMSC (live cell = green, dead cell = red; scale bar = 100 μm).
dead staining analysis of the cell-laden gels after 1 day of encapsulation showed very high cell viability for both cell types (98.8% ± 0.5% for L929 cell and 97.6% ± 0.6% for hMSC), indicating both the chromophores and cross-linking process are cytocompatible. Irradiation of the combined gels with blue light (470 nm, 20 mW cm−2) was performed to selectively degrade gel P8, followed by UV light (365 nm, 10 mW cm−2) to degrade gel P2. We selected these wavelengths and intensities, based on the degradation rates from Figure 2, to ensure rapid degradation (within 10 min of light irradiation) of the hydrogels and minimize the time that cells are exposed to light irradiation. It was reported previously that exposure to low dose UV light at 365 nm does not disturb gene expression of hMSCs.35 The selection of these wavelengths also allows the degradation of one gel component without affecting the other, so that complete separation of one cell type from another by wavelength-selective triggered release can be achieved. After each light irradiation treatment, the cells were collected, transferred on tissue culture poly(styrene) (TCPS) plate, cultured for 3 days and the cell growth was assessed by live/ dead staining (Figure 4). We observed complete degradation of the respective gels within the multigel construct after each light treatment and consequently complete release of the encapsulated cells. Both cell types are not affected by the photoregulated degradation, demonstrated by high cell viability and C
DOI: 10.1021/acsami.7b11517 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
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swelling, and gel fraction; cell culture procedures and additional cell data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Vinh X. Truong: 0000-0001-5553-6097 John S. Forsythe: 0000-0003-2849-229X Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
The research was supported under the Australian Research Council’s Discovery Projects funding scheme (DP160101591). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS F.L. acknowledges support from the New Horizons MonashCSIRO Council Joint PhD Scholarship Program. The authors acknowledge the facilities and scientific and technical assistance of Monash Micro Imaging, Monash University, Victoria, Australia.
Figure 4. (a) Complete degradation of hydrogel P8 under blue light irradiation (470 nm) after 5 min. The released L929 cells showed high viability as well as normal cellular activity after 3 days postrelease. (b) Subsequent degradation of the hMCS laden hydrogel P2 by UV light (365 nm). The collected hMSCs also displayed high cell viability and normal cellular activity compared to cells without encapsulation and release (live cells = green, dead cells = red; scale bar = 100 μm; ns = not significant).
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normal cell proliferation compared to untreated cells on a 2D cell culture platform (Figure S27). Green light (530 nm) triggered degradation of hydrogel P8 for the release of hMSCs was also undertaken, however a longer time (20 min) of light irradiation was required to fully degrade the cell-laden hydrogel (Figure S28). The longer exposure to light resulted in a slight decrease in hMSC viability (93.1% ± 0.7%) compared to cells released under UV or blue light irradiation. In summary, we show that the Passerini reaction is a valuable tool for incorporation of photoremovable protecting groups (photocages) within hydrogel structures to produce photodegradable matrices responsive to biologically benign visible light. In turn, we demonstrate selective release of different cell types in a spatiotemporal manner from a multihydrogel platform by utilizing different wavelengths of cytocompatible light. With increasing development of photocages in organic chemistry, we expect that this methodology will extend to the fabrication of materials having mechanical properties responsive to long wavelength visible light, which is highly applicable for clinical settings. Furthermore, we anticipate that this synthesis method will be utilized in the preparation of light-responsive materials for spatiotemporal delivery of therapeutics, advanced treatment of diseases, and tissue regeneration.
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ABBREVIATIONS o-NB, o-nitrobenzyl PEG, poly(ethylene glycol) DEAC, 7-(diethylamino)coumarin NMR, nuclear magnetic resonance SPAAC, strain-promoted azide alkyne cycloaddition hMSC, human mesenchymal stem cell TCPS, tissue culture poly(styrene) REFERENCES
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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/acsami.7b11517. Experimental procedures for the synthesis of the precursors; 1H NMR spectra of the precursors; UV−vis spectra of the compounds; procedures and characterization data for hydrogels including rheological data, D
DOI: 10.1021/acsami.7b11517 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsami.7b11517 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
