Full Paper
Microfluidic Synthesis of Pharmacologically Responsive Supramolecular Biohybrid Microgelsa sire e Ho €vermann, Torsten Rossow, Raphael J. Gu € beli, Sebastian Seiffert,* De Wilfried Weber* Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfonefunctionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150 mm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications. 1. Introduction €vermann, Dr. R. J. G€ D. Ho ubeli, Prof. W. Weber Faculty of Biology, Centre for Biological Signalling Studies (BIOSS), €nzlestrasse 18 79104, Freiburg, University of Freiburg, Scha Germany E-mail: [email protected] Dr. R. J. G€ ubeli, Prof. W. Weber Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstrasse 19a 79104, Freiburg, Germany T. Rossow, Prof. S. Seiffert €t Berlin, Institute of Chemistry and Biochemistry, Freie Universita Takustr. 3 14195, Berlin, Germany E-mail: [email protected] Prof. S. Seiffert F-ISFM Soft Matter and Functional Materials Helmholtz-Zentrum Berlin, Hahn-Meitner-Platz 1 14109, Berlin, Germany E-mail: [email protected] a
1730
€vermann and T. Rossow both contributed equally to this work. D. Ho
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Biohybrid hydrogels that adapt their mechanical or biological properties in response to chemical or physical stimuli hold high promises in biomedical applications such as drug delivery or tissue engineering.[1–7] For these applications, it is of key importance that the stimulus is physiologically compatible, thereby excluding most materials that respond to physical or chemical stimuli not compatible with physiological limits.[6,8,9] In a recent effort, the development of drug-responsive biohybrid hydrogels was shown to overcome these limitations, as the stimulus responsiveness was based on drug–target interactions that have been optimized to operate under physiological conditions.[10] For example, a hydrogel was described that dissolved in response to fluorescein, a small molecule dye that is routinely used in ophthalmology as contrast agent. The hydrogel is based on two 8-arm poly(ethylene glycol) species with end groups either functionalized with fluorescein or with a humanized single chain antibody
wileyonlinelibrary.com
DOI: 10.1002/mabi.201400342
Microfluidic Synthesis of Pharmacologically Responsive . . . www.mbs-journal.de
fragment (scFv) specifically binding fluorescein. Binding of the scFv to fluorescein crosslinks the polymers to form a gel, the stability of which could subsequently be modulated by the addition of fluorescein. Fluorescein-responsive modulation of the hydrogel stability was further shown to be functional in mice, as hydrogels subcutaneously implanted were demonstrated to dissolve upon oral administration of fluorescein; this was used to release a previously embedded biopharmaceutical such as an anti human papilloma virus vaccine.[1] However, in this vaccination approach, the hydrogel-based drug depot had to be administered via a small incision, preventing further applicability. This study aims at the fabrication of micrometer-sized biohybrid materials using droplet-based microfluidics, which has been shown to be a promising production technique for smart drug delivery materials.[11–13] In this approach, emulsion droplets are formed and used as templates for the microgel synthesis, thereby controlling the size, shape, and monodispersity of the subsequent microparticles. We follow this approach and demonstrate the synthesis of micrometer-scale fluorescein-responsive biohybrid supramolecular hydrogel beads; we also demonstrate that these beads retain their fluorescein-responsive characteristics. The concept developed in this study likely represents a generally applicable approach for the formulation of biohybrid hydrogels in an injection-ready form for biomedical applications.
2. Experimental Section 2.1. Microfluidic Device for Microgel Synthesis Microfluidic devices were produced by soft lithography by pouring poly(dimethylsiloxane) (PDMS) along with crosslinker (Sylgard 184 elastomer kit, Dow Corning, base: crosslinker ¼ 10:1) onto a silicon wafer patterned with SU-8 photoresist.[14] After solidifying the material for 1 h at 65 8C, devices were fabricated by oxygen plasma bonding of the PDMS replicas onto glass slides. To render the devices hydrophobic, and hence, suitable for water-in-oil emulsification, they were treated with Aquapel1 (PPG, Pittsburgh, PA), which is a commercial windshield treatment. For this purpose, Aquapel1 was injected into the channels, allowed to sit for 3 min, and then removed by air-drying. We fabricated two different types of microfluidic devices. The microfluidic channels had a rectangular cross-section with a uniform height of (1) 180 mm or (2) 50 mm. The channel width was (1) 100 mm or (2) 80 mm at the first crossjunction and (1) 150 mm or (2) 50 mm at the second cross-junction. A wide basin channel was patterned downstream of the second crossjunction to allow for observation of the resultant droplets.
2.2. Production of Hydrogel Building Blocks The building blocks for the hydrogel, 8-arm PEG-fluorescein, 8-arm PEG-vinylsulfone and the fluorescein-binding scFv(FITC-E2)-cys were obtained and characterized as described previously.[1] Prior to
www.MaterialsViews.com
the microgel synthesis, scFv(FITC-E2)-cys was concentrated to 20 g L1 by ultrafiltration (10 kDa MWCO, Corning, Lowell, MA cat. no. 431483) in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The following steps were all performed under nitrogen atmosphere with degassed buffers. scFv(FITC-E2)cys was supplemented with a 20-fold molar excess of TCEP (Tris(2carboxyethyl)phosphine hydrochloride, Sigma–Aldrich, cat. no. C4706, added as 30 g L1 stock solution in 500 mM sodium carbonate buffer pH 9.0) and incubated at room temperature for 1 h. Subsequently, the buffer was exchanged to hydrogel reaction buffer (2.68 mMKCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 2 mM ethylene diaminetetra-acetic acid, pH 8.0) via a desalting column (Thermo Fisher Scientific, cat. no. 43233) and the protein was concentrated by ultrafiltration.
2.3. Microgel Synthesis For the synthesis of microgels, scFv(FITC-E2)-cys (54.5 mg ml1, 1.89 mM) was mixed with 8-arm PEG-fluorescein (7.05 g L1, 1.89 mM fluorescein groups) in hydrogel reaction buffer supplemented with 180 mM triethanolamine pH 8, filled into a syringe (1 mL, Becton Dickinson, cat. no. 309628) and connected to port 3 of the microfluidic chip (Figure 2A). Port 2 of the chip was connected to a syringe containing 8-arm PEG-vinylsulfone (65 g L1 in hydrogel reaction buffer, 13 mM vinylsulfone groups). Engerix-B (HbsAg, GlaxoSmithKline (GSK), Brentford, UK, cat. no. 7504643) was concentrated by centrifugation (13 000 x g, 1 min), resuspended in Optiprep (Sigma–Aldrich, cat. no. D1556-250ML) to 80 mg L1 and inserted via port 1 of the microfluidic device. Port 4 was connected to paraffin oil (97 wt.-% (high viscosity, Carl Roth, Germany), containing a modified polyether polysiloxane surfactant (ABIL EM 90, Evonik Industries, Germany, 2 wt.-%), and a polyglycerol polyricinoleate surfactant (PGPR 90, Danisco, Denmark, 1 wt.-%)) to separate the microgel droplets. Microgel synthesis was performed at the following flow rates:scFv(FITC-E2)-cys/PEGfluorescein mix, 110 ml h1; 8-arm PEG-vinylsulfone,40 ml h1; Engerix-B, 50 ml h1; oil, 600 ml h1. This resulted in a final amount of 30 mg scFv(FITC-E2)-cys (1.04 nmol), 3.8 mg PEG-fluorescein (1.04 nmol fluorescein groups) and 13 g PEG-VS (2.6 nmol vinylsulfone groups) per 1 ml microgel volume. The microgel suspension was stored in siliconized (Sigmacote, Sigma–Aldrich, cat. no. SL-2) glass vials and incubated for 24 h at 25 8C to achieve droplet gelation. The resulting microgels were washed four times with wash buffer (2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 1.65 mM Triton X100, pH 7.4, 2 mL buffer per 17 mL microgelvolume) and centrifuged (4 300 g, 5 min) to separate the oil and water phase.
2.4. Microgel Characterization Microgel compatibility to syringe injection was evaluated by aspirating a microgel suspension (3.4 vol.-% in phosphate-buffered saline, 2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, pH 7.4) in a 1 mL syringe (B. Braun, Melsungen, Germany, Omnifix1-F, cat. no. 9161406 V) and ejected through a 22 gauge (inner diameter 0.4 mm) needle (Becton Dickinson, cat. no. 300900). Subsequently, the microgels were visually analyzed to check for their integrity. Long-term stability of the microgel suspensions and
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1731
€vermann, T. Rossow, R. J. G€ D. Ho ubeli, S. Seiffert, W. Weber www.mbs-journal.de
maintenance of their injectability was analyzed by incubation of a fraction in wash buffer (2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 1.65 mM Triton X-100, pH 7.4, 2 mL buffer per 17 mL microgel volume) at 4 8C for 9 months before passing it through a 22 gauge (inner diameter 0.4 mm) needle. Stimulus responsiveness of the microgels was analyzed by incubation in phosphate-buffered saline (3.4 vol.-%, total volume, 500 mL) optionally supplemented with 1.65 mM Triton X-100(0.1 vol.-%) or 8.7 103 mM b-mercaptoethanol in the presence or absence of 1mM fluorescein. After 3 h, the released microgel building blocks were quantified in the supernatant using the Bradford assay (BIORad, Hercules, CA, USA, cat. no. 500–0006) with bovine serum albumin (BSA, Fluka, Rotkreuz, Switzerland, cat. no. 05479) as standard.
3. Results and Discussion The present approach for the synthesis of pharmacologically responsive supramolecular microgels is based on two 8-arm PEG molecules end-functionalized with either fluorescein isothiocyanate (FITC) (PEG amine, M w ¼ 20 kDa) or with a humanized single chain antibody fragment (scFv(FITC-E2)-cys) (PEG-VS, M w ¼ 40 kDa) specific for fluorescein. Binding of the scFv to fluorescein crosslinks the PEG polymers and entails hydrogelation, whereas subsequent addition of free fluorescein competitively disrupts the crosslinking resulting in dissolution of the hydrogel (Figure 1A).[1] For the synthesis of the hydrogels, the 8-arm PEG-fluorescein is first incubated with the scFv(FITC-E2) containing one C-terminal cysteine. Subsequent addition of 8-arm PEG-vinylsulfone covalently crosslinks the scFv(FITC-E2) moieties via Michael-type addition (Figure 1B).[1] To scale the previously optimized synthesis protocol to the fabrication of micrometer-sized hydrogel particles with controlled and narrowly dispersed sizes, poly(dimethylsiloxane) (PDMS) microchannels with two sequential crossjunctions fabricated by soft lithography were used in a droplet-based microfluidic approach.[11,14] The hydrogel synthesis protocol was scaled to the microfluidic format by first mixing 8-arm PEG-fluorescein with scFv(FITC-E2)-cys (fluorescein: scFv ¼ 1:1, mol:mol, 1.04 mmol L1 with a final concentration of scFv and fluorescein groups of 30 and 3.8 g L1, respectively) and connecting this mix via port 3 to the microfluidic chip.[1] Port 2 of the chip was connected to 8-arm PEG-vinylsulfone (2.6 mmol vinylsulfone per L final concentration equal to 13 g L1) and the reactants were supplied at a flow rate of 110 and 40 ml h1 respectively, while commercially available hepatitis B vaccine (EngerixB, GlaxoSmith-Kline16), composed of recombinantly expressed hepatitis B surface antigen (HBsAg, concentrated in Optiprep to 80 mg L1), was inserted via port 1 of the microfluidic device at a flow rate of 50 ml h1, resulting in a final concentration of 20 mg L1 hydrogel. After injection,
1732
Figure 1. Design and synthesis of biohybrid hydrogels based on complementary linkage of 8-arm PEG precursor polymers. (A) Crosslinking of two 8-arm PEG derivatives, one functionalized with fluorescein and one functionalized with a fluoresceinspecific humanized single-chain variable antibody fragment (scFv(FITC-E2)-cys). Binding of the scFv to fluorescein results in gelation, whereas addition of free fluorescein competitively weakens the crosslinks. (B) Synthesis of the biohybrid hydrogel. 8-arm PEG-fluorescein is allowed to bind to scFv(FITC-E2) that is further functionalized with a C-terminal cysteine (scFv(FITC-E2)cys). Subsequent addition of 8-arm PEG-vinylsulfone crosslinks the scFv by Michael-type addition to result in gelation.
these three fluids met at the first cross-junction, forming a laminar coflowing stream in the microchannel (Figure 2A). In the second junction, periodic break-up of this stream was induced by flow-focusing with immiscible paraffin oil as a fourth fluid at a flow rate of 600 ml h1 via port 4 (Figure 2B). The droplets were allowed to undergo gelation for 24 h at 25 8C prior to separating the resulting microgel particles from the oil phase by addition of phosphatebuffered saline supplemented with 0.1 vol.-% Triton X-100 and subsequent centrifugation. The beads remained in the aqueous phase and showed a mean diameter of 150 mm (Figure 2C). In an additional experiment, we prepared smaller microgels with a mean diameter of 30 mm and evaluated whether these microgels were compatible with standard syringe-based injection. To achieve this aim, a
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
Microfluidic Synthesis of Pharmacologically Responsive . . . www.mbs-journal.de
Figure 2. Microfluidic microgel synthesis. (A) Schematic of the microfluidic system. scFv(FITC-E2)-cys-bound 8-arm PEG-fluorescein is added via port 3,while 8-arm PEG-vinylsulfone is supplied via port 2. Port 1 is connected to the vaccine HbsAg, concentrated in Optiprep. Oil supplied via port 4 is used to separate the hydrogel reaction mixture into pre-microgeldroplets. (B) Image of the microfluidic chip. Port 3, scFv(FITC-E2)-cys-loaded 8-arm PEG-fluorescein, 30 ml h1; port 2, 8-arm PEG-vinylsulfone, 110 ml h1; port 1, Engerix B suspension, 50 ml h1; ports 4, oil, 600 ml h1 per port. (C) Micrograph of the microgels obtained from the experiment in Figure 2B. Capturing of the vaccine is visible and indicated by an arrow. Scalebar, 100 mm.
microgel suspension (3.4 vol.-% microbeads in phosphatebuffered saline supplemented with 0.1 vol.-% of Triton X100 buffer) was incubated at 4 8C for 9 months, aspirated in a syringe, and then passed through a 22 gauge needle (inner diameter: 0.4 mm). Visual analysis revealed that the passage through the needle did not have any visible influence on the microgels (Figure 3A and B). To evaluate whether the microgels retained the pharmacological stimulus responsiveness, we incubated them in phosphate-buffered saline in the presence or absence of 1 mM fluorescein for 24 h at 25 8C prior to quantifying the release of the hydrogel building blocks via Bradford assay. In the absence of fluorescein, no release of hydrogel building blocks was observable, whereas in fluorescein-containing sample the gel building blocks were released to the supernatant, thereby demonstrating the stimulus responsiveness of the microgels (Figure 3C). To evaluate whether
www.MaterialsViews.com
Figure 3. Characterization of the microgels. (A, B) Compatibility of microgels to needle-based injection. Microgels (3.4 vol.-% suspension) were passed through a 22 gauge (inner diameter: 0.4 mm) needle and visually analyzed by transmitted light and fluorescence microscopy. Scalebar: 100 mm. (A) Before passage through the needle; (B) after passage. (C) Stimulus responsiveness of the microgels. Microgels (synthesized as described in Figure 2) were incubated in phosphate-buffered saline(17 ml microgels per 0.5 ml buffer) in the presence or absence of 1 mM fluorescein, 0.1 vol.-% Triton X-100 or 8.7 103 mM b-mercaptoethanol. After 5 h, the concentration of the released microgel building blocks was quantified in the supernatant. Data represent the mean and the standard deviation of four replica.
hydrogel stability was potentially in part due to disulfide crosslinks between the antibody fragments (which may result from a possible side reaction during gelation) or due to non-specific protein adsorption, the stimulus-responsive dissolution experiment was performed in the presence of 8.7 103 mM b-mercaptoethanol or 0.1 vol.-% Triton X100. The identical dissolution characteristics in the presence or absence of these agents, however, suggest that hydrogel stability is not conferred by these effects (Figure 3C).
4. Conclusion In this work, we demonstrate the microfluidic synthesis of pharmacologically responsive biohybrid hydrogel particles with sizes and functionalities relevant for biomedical applications. This protocol opens the opportunity for formulating biohybrid hydrogels in an injection-ready form for drug delivery. This new way of injectioncompatible formulation combined with previous studies where such hydrogels have successfully been applied for single-injection vaccination represents a step forward
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1733
€vermann, T. Rossow, R. J. G€ D. Ho ubeli, S. Seiffert, W. Weber www.mbs-journal.de
towards the biomedical application of biohybrid materials as externally controlled drug depots.[1,2]
Acknowledgements: This work was supported by the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement n8 259043-CompBioMat, the INTERREG IV Upper Rhine project no. A20, the Excellence Initiative of the German Federal and State Governments (EXC-294 and GSC-4), and the Berlin Joint Lab for Supramolecular Polymer Systems. During conduction of this work, T. Rossow was an Elsa Neumann fellow of the state of Berlin and S. Seiffert was a Liebig fellow of the Fund of the Chemical Industry (FCI).
Received: July 25, 2014; Revised: August 8, 2014; Published online: September 3, 2014; DOI: 10.1002/mabi.201400342 Keywords: drug depot; hydrogel; poly(ethylene glycol); stimulusresponsive; single-injection vaccination
1734
€ beli, D. Ho €vermann, H. Seitz, B. Rebmann, R. G. [1] R. J. Gu Schoenmakers, M. Ehrbar, G. Charpin-El Hamri, M. Daoud-El € ller, W. Weber, Adv. Funct. Mater. Baba, M. Werner, M. Mu 2013, 23, 5355. € beli, M. Ehrbar, M. Fussenegger, C. Friedrich, W. Weber, [2] R. J. Gu Macromol. Rapid Commun. 2012, 33, 1235. [3] M. Kwak, A. Herrmann, Chem. Soc. Rev. 2011, 40, 5745. [4] M. P. Lutolf, J. A. Hubbell, Nat. Biotechnol. 2005, 23, 47. [5] K. C. Koehler, K. S. Anseth, C. N. Bowman, Biomacromolecules 2013, 14, 538. [6] C. Wang, R. J. Stewart, J. Kopecek, Nature 1999, 397, 417. [7] J. Kopecek, J. Y. Yang, Angew. Chem. Int. Edit 2012, 51, 7396. [8] K. Jakobus, S. Wend, W. Weber, Chem. Soc. Rev. 2012, 41, 1000. [9] W. Weber, M. Fussenegger, Nat. Rev. Genet. 2012, 13, 21. [10] M. Ehrbar, R. Schoenmakers, E. H. Christen, M. Fussenegger, W. Weber, Nat. Mater. 2008, 7, 800. [11] S. Y. Teh, R. Lin, L. H. Hung, A. P. Lee, Lab on a Chip 2008, 8, 198. [12] C. X. Zhao, Adv. Drug Deliv. Rev. 2013, 65, 1420. [13] D. Jagadeesan, I. Nasimova, I. Gourevich, S. Starodubtsev, E. Kumacheva, Macromol. Biosci. 2011, 11, 889. [14] J. C. McDonald, D. C. Duffy, J. R. Anderson, D. T. Chiu, H. Wu, O. J. Schueller, G. M. Whitesides, Electrophoresis 2000, 21, 27.
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
Microfluidic Synthesis of Pharmacologically Responsive Supramolecular Biohybrid Microgelsa sire e Ho €vermann, Torsten Rossow, Raphael J. Gu € beli, Sebastian Seiffert,* De Wilfried Weber* Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfonefunctionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150 mm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications. 1. Introduction €vermann, Dr. R. J. G€ D. Ho ubeli, Prof. W. Weber Faculty of Biology, Centre for Biological Signalling Studies (BIOSS), €nzlestrasse 18 79104, Freiburg, University of Freiburg, Scha Germany E-mail: [email protected] Dr. R. J. G€ ubeli, Prof. W. Weber Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Albertstrasse 19a 79104, Freiburg, Germany T. Rossow, Prof. S. Seiffert €t Berlin, Institute of Chemistry and Biochemistry, Freie Universita Takustr. 3 14195, Berlin, Germany E-mail: [email protected] Prof. S. Seiffert F-ISFM Soft Matter and Functional Materials Helmholtz-Zentrum Berlin, Hahn-Meitner-Platz 1 14109, Berlin, Germany E-mail: [email protected] a
1730
€vermann and T. Rossow both contributed equally to this work. D. Ho
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Biohybrid hydrogels that adapt their mechanical or biological properties in response to chemical or physical stimuli hold high promises in biomedical applications such as drug delivery or tissue engineering.[1–7] For these applications, it is of key importance that the stimulus is physiologically compatible, thereby excluding most materials that respond to physical or chemical stimuli not compatible with physiological limits.[6,8,9] In a recent effort, the development of drug-responsive biohybrid hydrogels was shown to overcome these limitations, as the stimulus responsiveness was based on drug–target interactions that have been optimized to operate under physiological conditions.[10] For example, a hydrogel was described that dissolved in response to fluorescein, a small molecule dye that is routinely used in ophthalmology as contrast agent. The hydrogel is based on two 8-arm poly(ethylene glycol) species with end groups either functionalized with fluorescein or with a humanized single chain antibody
wileyonlinelibrary.com
DOI: 10.1002/mabi.201400342
Microfluidic Synthesis of Pharmacologically Responsive . . . www.mbs-journal.de
fragment (scFv) specifically binding fluorescein. Binding of the scFv to fluorescein crosslinks the polymers to form a gel, the stability of which could subsequently be modulated by the addition of fluorescein. Fluorescein-responsive modulation of the hydrogel stability was further shown to be functional in mice, as hydrogels subcutaneously implanted were demonstrated to dissolve upon oral administration of fluorescein; this was used to release a previously embedded biopharmaceutical such as an anti human papilloma virus vaccine.[1] However, in this vaccination approach, the hydrogel-based drug depot had to be administered via a small incision, preventing further applicability. This study aims at the fabrication of micrometer-sized biohybrid materials using droplet-based microfluidics, which has been shown to be a promising production technique for smart drug delivery materials.[11–13] In this approach, emulsion droplets are formed and used as templates for the microgel synthesis, thereby controlling the size, shape, and monodispersity of the subsequent microparticles. We follow this approach and demonstrate the synthesis of micrometer-scale fluorescein-responsive biohybrid supramolecular hydrogel beads; we also demonstrate that these beads retain their fluorescein-responsive characteristics. The concept developed in this study likely represents a generally applicable approach for the formulation of biohybrid hydrogels in an injection-ready form for biomedical applications.
2. Experimental Section 2.1. Microfluidic Device for Microgel Synthesis Microfluidic devices were produced by soft lithography by pouring poly(dimethylsiloxane) (PDMS) along with crosslinker (Sylgard 184 elastomer kit, Dow Corning, base: crosslinker ¼ 10:1) onto a silicon wafer patterned with SU-8 photoresist.[14] After solidifying the material for 1 h at 65 8C, devices were fabricated by oxygen plasma bonding of the PDMS replicas onto glass slides. To render the devices hydrophobic, and hence, suitable for water-in-oil emulsification, they were treated with Aquapel1 (PPG, Pittsburgh, PA), which is a commercial windshield treatment. For this purpose, Aquapel1 was injected into the channels, allowed to sit for 3 min, and then removed by air-drying. We fabricated two different types of microfluidic devices. The microfluidic channels had a rectangular cross-section with a uniform height of (1) 180 mm or (2) 50 mm. The channel width was (1) 100 mm or (2) 80 mm at the first crossjunction and (1) 150 mm or (2) 50 mm at the second cross-junction. A wide basin channel was patterned downstream of the second crossjunction to allow for observation of the resultant droplets.
2.2. Production of Hydrogel Building Blocks The building blocks for the hydrogel, 8-arm PEG-fluorescein, 8-arm PEG-vinylsulfone and the fluorescein-binding scFv(FITC-E2)-cys were obtained and characterized as described previously.[1] Prior to
www.MaterialsViews.com
the microgel synthesis, scFv(FITC-E2)-cys was concentrated to 20 g L1 by ultrafiltration (10 kDa MWCO, Corning, Lowell, MA cat. no. 431483) in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The following steps were all performed under nitrogen atmosphere with degassed buffers. scFv(FITC-E2)cys was supplemented with a 20-fold molar excess of TCEP (Tris(2carboxyethyl)phosphine hydrochloride, Sigma–Aldrich, cat. no. C4706, added as 30 g L1 stock solution in 500 mM sodium carbonate buffer pH 9.0) and incubated at room temperature for 1 h. Subsequently, the buffer was exchanged to hydrogel reaction buffer (2.68 mMKCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 2 mM ethylene diaminetetra-acetic acid, pH 8.0) via a desalting column (Thermo Fisher Scientific, cat. no. 43233) and the protein was concentrated by ultrafiltration.
2.3. Microgel Synthesis For the synthesis of microgels, scFv(FITC-E2)-cys (54.5 mg ml1, 1.89 mM) was mixed with 8-arm PEG-fluorescein (7.05 g L1, 1.89 mM fluorescein groups) in hydrogel reaction buffer supplemented with 180 mM triethanolamine pH 8, filled into a syringe (1 mL, Becton Dickinson, cat. no. 309628) and connected to port 3 of the microfluidic chip (Figure 2A). Port 2 of the chip was connected to a syringe containing 8-arm PEG-vinylsulfone (65 g L1 in hydrogel reaction buffer, 13 mM vinylsulfone groups). Engerix-B (HbsAg, GlaxoSmithKline (GSK), Brentford, UK, cat. no. 7504643) was concentrated by centrifugation (13 000 x g, 1 min), resuspended in Optiprep (Sigma–Aldrich, cat. no. D1556-250ML) to 80 mg L1 and inserted via port 1 of the microfluidic device. Port 4 was connected to paraffin oil (97 wt.-% (high viscosity, Carl Roth, Germany), containing a modified polyether polysiloxane surfactant (ABIL EM 90, Evonik Industries, Germany, 2 wt.-%), and a polyglycerol polyricinoleate surfactant (PGPR 90, Danisco, Denmark, 1 wt.-%)) to separate the microgel droplets. Microgel synthesis was performed at the following flow rates:scFv(FITC-E2)-cys/PEGfluorescein mix, 110 ml h1; 8-arm PEG-vinylsulfone,40 ml h1; Engerix-B, 50 ml h1; oil, 600 ml h1. This resulted in a final amount of 30 mg scFv(FITC-E2)-cys (1.04 nmol), 3.8 mg PEG-fluorescein (1.04 nmol fluorescein groups) and 13 g PEG-VS (2.6 nmol vinylsulfone groups) per 1 ml microgel volume. The microgel suspension was stored in siliconized (Sigmacote, Sigma–Aldrich, cat. no. SL-2) glass vials and incubated for 24 h at 25 8C to achieve droplet gelation. The resulting microgels were washed four times with wash buffer (2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 1.65 mM Triton X100, pH 7.4, 2 mL buffer per 17 mL microgelvolume) and centrifuged (4 300 g, 5 min) to separate the oil and water phase.
2.4. Microgel Characterization Microgel compatibility to syringe injection was evaluated by aspirating a microgel suspension (3.4 vol.-% in phosphate-buffered saline, 2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, pH 7.4) in a 1 mL syringe (B. Braun, Melsungen, Germany, Omnifix1-F, cat. no. 9161406 V) and ejected through a 22 gauge (inner diameter 0.4 mm) needle (Becton Dickinson, cat. no. 300900). Subsequently, the microgels were visually analyzed to check for their integrity. Long-term stability of the microgel suspensions and
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1731
€vermann, T. Rossow, R. J. G€ D. Ho ubeli, S. Seiffert, W. Weber www.mbs-journal.de
maintenance of their injectability was analyzed by incubation of a fraction in wash buffer (2.68 mM KCl, 1.47 mM KH2PO4, 8.03 mM Na2HPO4, 137 mM NaCl, 1.65 mM Triton X-100, pH 7.4, 2 mL buffer per 17 mL microgel volume) at 4 8C for 9 months before passing it through a 22 gauge (inner diameter 0.4 mm) needle. Stimulus responsiveness of the microgels was analyzed by incubation in phosphate-buffered saline (3.4 vol.-%, total volume, 500 mL) optionally supplemented with 1.65 mM Triton X-100(0.1 vol.-%) or 8.7 103 mM b-mercaptoethanol in the presence or absence of 1mM fluorescein. After 3 h, the released microgel building blocks were quantified in the supernatant using the Bradford assay (BIORad, Hercules, CA, USA, cat. no. 500–0006) with bovine serum albumin (BSA, Fluka, Rotkreuz, Switzerland, cat. no. 05479) as standard.
3. Results and Discussion The present approach for the synthesis of pharmacologically responsive supramolecular microgels is based on two 8-arm PEG molecules end-functionalized with either fluorescein isothiocyanate (FITC) (PEG amine, M w ¼ 20 kDa) or with a humanized single chain antibody fragment (scFv(FITC-E2)-cys) (PEG-VS, M w ¼ 40 kDa) specific for fluorescein. Binding of the scFv to fluorescein crosslinks the PEG polymers and entails hydrogelation, whereas subsequent addition of free fluorescein competitively disrupts the crosslinking resulting in dissolution of the hydrogel (Figure 1A).[1] For the synthesis of the hydrogels, the 8-arm PEG-fluorescein is first incubated with the scFv(FITC-E2) containing one C-terminal cysteine. Subsequent addition of 8-arm PEG-vinylsulfone covalently crosslinks the scFv(FITC-E2) moieties via Michael-type addition (Figure 1B).[1] To scale the previously optimized synthesis protocol to the fabrication of micrometer-sized hydrogel particles with controlled and narrowly dispersed sizes, poly(dimethylsiloxane) (PDMS) microchannels with two sequential crossjunctions fabricated by soft lithography were used in a droplet-based microfluidic approach.[11,14] The hydrogel synthesis protocol was scaled to the microfluidic format by first mixing 8-arm PEG-fluorescein with scFv(FITC-E2)-cys (fluorescein: scFv ¼ 1:1, mol:mol, 1.04 mmol L1 with a final concentration of scFv and fluorescein groups of 30 and 3.8 g L1, respectively) and connecting this mix via port 3 to the microfluidic chip.[1] Port 2 of the chip was connected to 8-arm PEG-vinylsulfone (2.6 mmol vinylsulfone per L final concentration equal to 13 g L1) and the reactants were supplied at a flow rate of 110 and 40 ml h1 respectively, while commercially available hepatitis B vaccine (EngerixB, GlaxoSmith-Kline16), composed of recombinantly expressed hepatitis B surface antigen (HBsAg, concentrated in Optiprep to 80 mg L1), was inserted via port 1 of the microfluidic device at a flow rate of 50 ml h1, resulting in a final concentration of 20 mg L1 hydrogel. After injection,
1732
Figure 1. Design and synthesis of biohybrid hydrogels based on complementary linkage of 8-arm PEG precursor polymers. (A) Crosslinking of two 8-arm PEG derivatives, one functionalized with fluorescein and one functionalized with a fluoresceinspecific humanized single-chain variable antibody fragment (scFv(FITC-E2)-cys). Binding of the scFv to fluorescein results in gelation, whereas addition of free fluorescein competitively weakens the crosslinks. (B) Synthesis of the biohybrid hydrogel. 8-arm PEG-fluorescein is allowed to bind to scFv(FITC-E2) that is further functionalized with a C-terminal cysteine (scFv(FITC-E2)cys). Subsequent addition of 8-arm PEG-vinylsulfone crosslinks the scFv by Michael-type addition to result in gelation.
these three fluids met at the first cross-junction, forming a laminar coflowing stream in the microchannel (Figure 2A). In the second junction, periodic break-up of this stream was induced by flow-focusing with immiscible paraffin oil as a fourth fluid at a flow rate of 600 ml h1 via port 4 (Figure 2B). The droplets were allowed to undergo gelation for 24 h at 25 8C prior to separating the resulting microgel particles from the oil phase by addition of phosphatebuffered saline supplemented with 0.1 vol.-% Triton X-100 and subsequent centrifugation. The beads remained in the aqueous phase and showed a mean diameter of 150 mm (Figure 2C). In an additional experiment, we prepared smaller microgels with a mean diameter of 30 mm and evaluated whether these microgels were compatible with standard syringe-based injection. To achieve this aim, a
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
Microfluidic Synthesis of Pharmacologically Responsive . . . www.mbs-journal.de
Figure 2. Microfluidic microgel synthesis. (A) Schematic of the microfluidic system. scFv(FITC-E2)-cys-bound 8-arm PEG-fluorescein is added via port 3,while 8-arm PEG-vinylsulfone is supplied via port 2. Port 1 is connected to the vaccine HbsAg, concentrated in Optiprep. Oil supplied via port 4 is used to separate the hydrogel reaction mixture into pre-microgeldroplets. (B) Image of the microfluidic chip. Port 3, scFv(FITC-E2)-cys-loaded 8-arm PEG-fluorescein, 30 ml h1; port 2, 8-arm PEG-vinylsulfone, 110 ml h1; port 1, Engerix B suspension, 50 ml h1; ports 4, oil, 600 ml h1 per port. (C) Micrograph of the microgels obtained from the experiment in Figure 2B. Capturing of the vaccine is visible and indicated by an arrow. Scalebar, 100 mm.
microgel suspension (3.4 vol.-% microbeads in phosphatebuffered saline supplemented with 0.1 vol.-% of Triton X100 buffer) was incubated at 4 8C for 9 months, aspirated in a syringe, and then passed through a 22 gauge needle (inner diameter: 0.4 mm). Visual analysis revealed that the passage through the needle did not have any visible influence on the microgels (Figure 3A and B). To evaluate whether the microgels retained the pharmacological stimulus responsiveness, we incubated them in phosphate-buffered saline in the presence or absence of 1 mM fluorescein for 24 h at 25 8C prior to quantifying the release of the hydrogel building blocks via Bradford assay. In the absence of fluorescein, no release of hydrogel building blocks was observable, whereas in fluorescein-containing sample the gel building blocks were released to the supernatant, thereby demonstrating the stimulus responsiveness of the microgels (Figure 3C). To evaluate whether
www.MaterialsViews.com
Figure 3. Characterization of the microgels. (A, B) Compatibility of microgels to needle-based injection. Microgels (3.4 vol.-% suspension) were passed through a 22 gauge (inner diameter: 0.4 mm) needle and visually analyzed by transmitted light and fluorescence microscopy. Scalebar: 100 mm. (A) Before passage through the needle; (B) after passage. (C) Stimulus responsiveness of the microgels. Microgels (synthesized as described in Figure 2) were incubated in phosphate-buffered saline(17 ml microgels per 0.5 ml buffer) in the presence or absence of 1 mM fluorescein, 0.1 vol.-% Triton X-100 or 8.7 103 mM b-mercaptoethanol. After 5 h, the concentration of the released microgel building blocks was quantified in the supernatant. Data represent the mean and the standard deviation of four replica.
hydrogel stability was potentially in part due to disulfide crosslinks between the antibody fragments (which may result from a possible side reaction during gelation) or due to non-specific protein adsorption, the stimulus-responsive dissolution experiment was performed in the presence of 8.7 103 mM b-mercaptoethanol or 0.1 vol.-% Triton X100. The identical dissolution characteristics in the presence or absence of these agents, however, suggest that hydrogel stability is not conferred by these effects (Figure 3C).
4. Conclusion In this work, we demonstrate the microfluidic synthesis of pharmacologically responsive biohybrid hydrogel particles with sizes and functionalities relevant for biomedical applications. This protocol opens the opportunity for formulating biohybrid hydrogels in an injection-ready form for drug delivery. This new way of injectioncompatible formulation combined with previous studies where such hydrogels have successfully been applied for single-injection vaccination represents a step forward
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1733
€vermann, T. Rossow, R. J. G€ D. Ho ubeli, S. Seiffert, W. Weber www.mbs-journal.de
towards the biomedical application of biohybrid materials as externally controlled drug depots.[1,2]
Acknowledgements: This work was supported by the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement n8 259043-CompBioMat, the INTERREG IV Upper Rhine project no. A20, the Excellence Initiative of the German Federal and State Governments (EXC-294 and GSC-4), and the Berlin Joint Lab for Supramolecular Polymer Systems. During conduction of this work, T. Rossow was an Elsa Neumann fellow of the state of Berlin and S. Seiffert was a Liebig fellow of the Fund of the Chemical Industry (FCI).
Received: July 25, 2014; Revised: August 8, 2014; Published online: September 3, 2014; DOI: 10.1002/mabi.201400342 Keywords: drug depot; hydrogel; poly(ethylene glycol); stimulusresponsive; single-injection vaccination
1734
€ beli, D. Ho €vermann, H. Seitz, B. Rebmann, R. G. [1] R. J. Gu Schoenmakers, M. Ehrbar, G. Charpin-El Hamri, M. Daoud-El € ller, W. Weber, Adv. Funct. Mater. Baba, M. Werner, M. Mu 2013, 23, 5355. € beli, M. Ehrbar, M. Fussenegger, C. Friedrich, W. Weber, [2] R. J. Gu Macromol. Rapid Commun. 2012, 33, 1235. [3] M. Kwak, A. Herrmann, Chem. Soc. Rev. 2011, 40, 5745. [4] M. P. Lutolf, J. A. Hubbell, Nat. Biotechnol. 2005, 23, 47. [5] K. C. Koehler, K. S. Anseth, C. N. Bowman, Biomacromolecules 2013, 14, 538. [6] C. Wang, R. J. Stewart, J. Kopecek, Nature 1999, 397, 417. [7] J. Kopecek, J. Y. Yang, Angew. Chem. Int. Edit 2012, 51, 7396. [8] K. Jakobus, S. Wend, W. Weber, Chem. Soc. Rev. 2012, 41, 1000. [9] W. Weber, M. Fussenegger, Nat. Rev. Genet. 2012, 13, 21. [10] M. Ehrbar, R. Schoenmakers, E. H. Christen, M. Fussenegger, W. Weber, Nat. Mater. 2008, 7, 800. [11] S. Y. Teh, R. Lin, L. H. Hung, A. P. Lee, Lab on a Chip 2008, 8, 198. [12] C. X. Zhao, Adv. Drug Deliv. Rev. 2013, 65, 1420. [13] D. Jagadeesan, I. Nasimova, I. Gourevich, S. Starodubtsev, E. Kumacheva, Macromol. Biosci. 2011, 11, 889. [14] J. C. McDonald, D. C. Duffy, J. R. Anderson, D. T. Chiu, H. Wu, O. J. Schueller, G. M. Whitesides, Electrophoresis 2000, 21, 27.
Macromol. Biosci. 2014, 14, 1730–1734 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
