Dynamic Covalent Hydrogels for Triggered Cell Capture and Release

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Dynamic covalent hydrogels for triggered cell capture and release Fatemeh Karimi, Joe Collins, Daniel E. Heath, and Luke A. Connal Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00360 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Bioconjugate Chemistry

Dynamic covalent hydrogels for triggered cell capture and release Fatemeh Karimi a,b*, Joe Collins a*, Daniel E Heatha, and Luke A Connala a

School of Chemical and Biomedical Engineering, Particulate Fluids Processing Centre, University of Melbourne, Parkville, Melbourne, Victoria 3010, Australia b

Polymer Science Group, Department of Chemical and Biomolecular Engineering, Particulate Fluids Processing Centre, University of Melbourne, Parkville, Melbourne, Victoria 3010, Australia *These authors contributed equally to this work Corresponding author: Dr Luke Connal Email: [email protected] Phone: +61 3 9035 3578 Fax: +61 3 8344 4153 Abstract A dual-responsive, cell capture and release surface was prepared through the incorporation of phenylboronic acid (PBA) groups into an oxime-based polyethylene glycol (PEG) hydrogel. Owing to its PEG-like properties, the unfunctionalized hydrogel was non-fouling. The use of highly efficient oxime chemistry allows for the incorporation of commercially available 3,5-diformylphenyl boronic acid into the hydrogel matrix. Thus, the surface properties of the hydrogel were modified to enable reversible cell capture and release. Boronic ester formation between PBA groups and cell surface carbohydrates enabled efficient cell capture at pH 6.8. An increase to pH 7.8 resulted in cell detachment. This capture/release procedure was performed on MCF-7 human breast cancer cells, NIH-3T3 fibroblast cells, and primary Human Vein Endothelial Cells (HUVECs) and could be cycled with negligible loss in activity. The facile preparation of PBA-functionalized surfaces presented here has applications in biomedical fields such as cell diagnostics and cell culture.

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Introduction Dynamic materials capable of triggered structural and chemical changes hold wide potential in biomedical fields. Dynamic covalent chemistry is one route for the preparation of smart polymeric materials, for example, using Diels-Alder,1,2 imine,3,4 oxime,[5–8] polypeptides,9,10 or thiol-Michael addition reactions.11 In particular, boronic (BA) and phenylboronic acid (PBA) functional materials have found utility due to their ability to reversibly bind with sugars.[11–14] This has been exploited to demonstrate their potential in drug delivery, sensing, enzymatic inhibition and cell sorting.[13–16] Furthermore, the low toxicity of these functional materials further enhances their potential in biological applications.[13–18] BAs and PBAs are known to reversibly interact with carbohydrates on cell membranes. This has enabled the realization of BA/PBA surfaces as intelligent capture and release materials for a variety of cell lines including yeast cells,18,19 murine hybridoma cells,17,20 human leukemia cells,17 and MCF-7 breast cancer cells.21 Cell binding is achieved through boronic ester formation with cell surface carbohydrates and the cell is released via a trans-esterification through the addition of competing saccharides (e.g. glucose). The benefit of this process is that it avoids the use of traditional enzymatic treatments (proteases such as trypsin) for cell release which can result in cell damage. Seminal work by Ivanov et al. in the area reported that a polymer brush morphology gives superior cell adhesion and viability owing to the freedom of movement of the end-grafted chains.20 However, the synthesis of grafted surfaces is not generalizable, is intolerant to oxygen, and is expensive and time consuming. Therefore, there is an opportunity for the development of a more robust and generalizable chemistry to realize these smart, cell capture and release properties. The oxime click reaction is fast becoming a valuable tool for the synthesis of functional materials. The attractive properties; high yielding, rapid synthesis, functional group tolerance, bioorthogonality and reversibility, have expanded the applications of oxime chemistry from its traditional use in bioconjugation22 to applications in self-healing materials,5,7 surface patterning,23,24 drug delivery,25


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bioadhesives26,27 and more.28,29 Previous work in our group has realized the synthesis of advanced oxime-based polymeric materials containing the PBA functionality.5,30 While BA/PBA functional monomers and polymers have been challenging to synthesize and purify in the past31 the use of the highly efficient oxime click reaction allows for the facile preparation of PBA functional materials from 3,5-diformylphenyl boronic acid. Herein we report a new cell capture and release system based on an oxime-PEG hydrogel functionalized with PBA. Simple oxime condensation polymerization allows for the facile preparation of the functional hydrogels which were found to efficiently capture and release MCF-7 human breast cancer cells, NIH-3T3 fibroblast cells, and primary Human Vein Endothelial Cells (HUVECs). Without PBA incorporation the oxime-PEG hydrogel was found to be non-fouling. The instillation of PBA modified the hydrogel properties to be capable of cell capture and release over many cycles. The PBA-PEG hydrogel was shown to be dual-responsive, sensitive to the addition of glucose (70 mM) and an increase of pH from 6.8 to 7.8. Importantly, the PBA-PEG hydrogel was responsive to pH alone, while in the presence of glucose (70 mM), making it applicable for use with standard cell culture media. This versatile platform for cell capture and release could enable potential applications in cell culture and cell diagnostics. Results and Discussion The dynamic nature of boronic acids (BA) and boronic esters (BE) is governed by the pH and the structural changes which occur above and below the pKa of the BA/BE. It is well known that only anionic, tetrahedral (sp3) BE conjugates are hydrolytically stable. Below the pKa of the BE neutral trigonal planar (sp2) species are produced which suffer from low hydrolytic stability (Scheme 1).13 Therefore, almost all previously reported cases of dynamic BA/BE systems work above the pKa of the BE, under alkaline conditions, to ensure the formation of the stable anionic conjugate. This reversible BE formation has been exploited for the development of BA/PBA-functional surfaces with cell capture and release properties.[16–23] In these systems the pH is altered between the pKa’s of the



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cell surface carbohydrates and the competing sugar (glucose). At a specific pH, above the pKa of the cell surface carbohydrate-PBA complex, stable BE formation occurs enabling efficient cell capture. Altering the pH destabilises the cell surface carbohydrate BE bond releasing the captured cells while forming stable glucose-BE bonds with the competing glucose molecules. We aim to exploit this selective BE formation to enable efficient cell capture and release under relevant cell culture conditions.

Scheme 1 (A) Equilibrium reactions between boronic acids (BA) and boronic esters (BE) from neutral or anionic species upon reaction with a diol. Uncharged boronic esters (red box) are hydrolytically unstable while anionic boronic esters (green box) are hydrolytically stable. (B) General synthesis of PBA functionalized hydrogels GBA0, GBA10, and GBA20 through the reaction between A3 and B2 with 0, 10, or 20 mol% A2, respectively. Synthesis of the oxime-based PBA functionalized scaffolds was achieved via a simple step growth polymerization mechanism using difunctional and trifunctional benzaldehyde monomers (A2 and A3 respectively) together with a difunctional hydroxylamine monomer (B2) (Scheme 1B). Polyethylene glycol was used as the base material due to its well-known biocompatibility and low fouling 4 ACS Paragon Plus Environment

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properties.34 In a simple carbo-diimide coupling reaction 4-formyl benzoic acid can be conjugated with glycerol ethoxylate to yield a PEG-based tribenzaldehyde crosslinker (A3). The Mitsunobu reaction between PEG-1k and N-hydroxyphthalimide followed by hydrazine deprotection afforded the PEG-1k bis-hydroxylamine monomer (B2) (See SI for experimental details and 1H-NMR analysis). To incorporate the PBA functionality 10 or 20 mol% 3,5-diformylphenyl boronic acid (A2) was added into the reaction mixture (GBA10 and GBA20, respectively). Hydrogel synthesis was achieved using a 2:3 mol ratio of aldehyde to hydroxylamine with gelation occurring within 5 minutes. In this way, PBAfunctionalized, PEG-based hydrogels could be prepared and examined for cell capture/release properties (Figure 1).

Figure 1. Reversible cell capture and release system based on dynamic boronate ester formation. Cell capture is achieved through the binding between cell surface carbohydrates and polymer bound PBA at pH 6.8 with 0 mM glucose (left). Cell release is achieved through the addition of 70 mM glucose and an increase to pH 7.8 (right). This cycle can be repeated to achieve multiple rounds of cell capture and release.

In order to illustrate the non-fouling properties of the unfunctionalized hydrogel, which contains 0 mol% PBA (GBA0), MCF-7 cancer cells were cultured on the surface of standard tissue culture polystyrene (TCPS) and compared to those grown on the surface of GBA0 (Figure S1). In all cell culture experiments the cells were added to fully swollen hydrogels (the hydrogels were incubated in PBS buffer for 24 hrs prior to use) to ensure that there was no change in the hydrogel surface properties



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or mechanical properties upon addition of the cell culture media. The adhesion of cultured cells was analyzed using CCK-8 assay and presented as absorbance that is proportional to the metabolic activity of the adhered cells on various surfaces. The cells on the TCPS surface showed the expected high metabolic activity indicating healthy cells adhering to the cell culture surface. Owing to the PEGlike properties of GBA0 very low numbers of cells were found to adhere onto the unfunctionalized hydrogel surface confirming its non-fouling properties (Figure S1A). Fluorescence microscopy was further used to confirm the non-fouling properties of GBA0. The cells were fluorescently stained with live/dead staining. Compared to the TCPS surface (Figure S1B) very little fluorescence is observed on the GBA0 surface (Figure S1C) strongly suggesting that the surface possess non-fouling properties. In order to test the viability of the cells grown on GBA0 a re-plating assay was used.

Briefly, the suspension of cells initially cultured on the GBA0 interface was

transferred to a new TCPS well and incubated for 4 h. As expected, essentially all of the transferred cells adhered and spread on the TCPS surface, confirming the cells cultured on GBA0 retained viability, illustrating the cytocompatibility of the materials (Figure S1D). Moreover, the adhered cells strongly fluoresced green (live staining), further supporting the high viability on this substrate. These results illustrate the non-fouling and cytocompatible properties of GBA0 and provide the basis for functionalisation with PBA in order to achieve a cell capture and release system. The incorporation of 10 or 20 mol% PBA into the PEG-based hydrogel (GBA10 and GBA20, respectively) was hypothesized to modify the properties of the initial PEG-based hydrogel, GBA0, from a nonfouling surface to a surface being capable of specific and reversible cell capture and release. In order to demonstrate this process MCF-7 human breast carcinoma cells and NIH-3T3 fibroblast normal mammalian cells were seeded on GBA0, GBA10 and GBA20 and incubated at pH 6.8. As expected, the unfunctionalized GBA0 surface captured very few cells of either cell line (Figure 2). Cell adhesion was obtained through the incorporation of PBA into the hydrogel matrix. Interestingly, we observed increased adhesion of MCF-7 cancer cells, which over express sialic acid on their cell surface


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membrane, compared to the 3T3 fibroblast cells (Figure 2). The superior adhesion of cancer cells expressing high levels of sialic acid on their surfaces has been reported previously.21 MCF-7 cancer cells were captured with an efficiency of approximately 40% which dropped to about 10% when compared to the 3T3 fibroblast cells. The efficiency of MCF-7 cancer cell capture is lower than some previously reported boronic acid-functional brush polymers, 60%.21 The brush-polymer architecture is reported to be the most efficient cell capture platform.20 We believe however that the reduced cell capture number of our system is offset by the far more simple means of hydrogel synthesis and its robust properties. Further to this, the hydrogel presented here displays higher cell capture ability than other previously reported boronic acid functionalized hydrogels and silicon wafers.20,21 Interestingly, no statistical difference in the density of captured cells was observed between GBA10 and GBA20, indicating that successful cell capture and release can be achieved with low PBA incorporation. Therefore, all following experiments were undertaken on GBA10 alone.

Figure 2. Capture of MCF-7 cancer cells and NIH-3T3 fibroblast cells at pH 6.8 on GBA0, GBA10 and GBA20 surfaces. Very little cell adhesion of both MCF-7 cancer cells and NIH-3T3 fibroblast cells was observed on the unfunctionalized GBA0 surface owing to the lack of PBA groups. High cell adhesion was observed for MCF-7 cancer cells on GBA10 and GBA20 which we believe is due to the specific binding of PBA to sialic acid. Adhesion of normal NIH-3T3 fibroblast was also observed on both GBA10 and GBA20. **P

Dynamic Covalent Hydrogels for Triggered Cell Capture and Release

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