Dynamic Covalent Hydrogels for Triggered Cell ...

Communication pubs.acs.org/bc

Dynamic Covalent Hydrogels for Triggered Cell Capture and Release Fatemeh Karimi,†,‡,∥ Joe Collins,†,∥ Daniel E. Heath,† and Luke A. Connal*,† †

School of Chemical and Biomedical Engineering, Particulate Fluids Processing Centre and ‡Polymer Science Group, Department of Chemical and Biomolecular Engineering, Particulate Fluids Processing Centre, University of Melbourne, Parkville, Melbourne, Victoria 3010, Australia S Supporting Information *

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 nonfouling. The use of highly efficient oxime chemistry allows the incorporation of commercially available 3,5diformylphenyl 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-and-release procedure was performed on MCF-7 human breast cancer cells, NIH-3T3 fibroblast cells, and primary human umbilical 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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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 with which 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 yield, 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 bioadhesives,26,27 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 and PBA functional monomers

ynamic 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 using, for example, 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 and 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 © 2017 American Chemical Society

Received: June 26, 2017 Revised: August 13, 2017 Published: August 15, 2017 2235

DOI: 10.1021/acs.bioconjchem.7b00360 Bioconjugate Chem. 2017, 28, 2235−2240

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Bioconjugate Chemistry

the formation of the stable anionic conjugate. This reversible BE formation has been exploited for the development of BAand PBA-functional surfaces with cell capture and release properties.16−23,32 In these systems, the pH is altered between the pKa’s of the cell surface carbohydrates and the competing sugar (glucose). At a specific pH, above the pKa of the cellsurface carbohydrate−PBA complex, stable BE formation occurs, enabling efficient cell capture. Altering the pH destabilizes 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. 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 properties.34 In a simple carbo-diimide coupling reaction, 4-formyl benzoic acid can be conjugated with glycerol ethoxylate to yield a PEGbased tribenzaldehyde cross-linker (A3). The Mitsunobu reaction between PEG-1k and N-hydroxyphthalimide followed by hydrazine deprotection afforded the PEG-1k bis-hydroxylamine monomer (B2) (see the Supporting Information 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 min. In this way, PBA-functionalized, PEGbased hydrogels could be prepared and examined for cellcapture and -release properties (Figure 1). To illustrate the nonfouling 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 h prior to use) to ensure that there was no change in the hydrogel surface properties 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 that healthy cells were adhering to the cell culture surface. Owing to the PEG-like properties of GBA0, very low numbers of cells were found to adhere onto the unfunctionalized hydrogel surface confirming its nonfouling properties (Figure S1A). Fluorescence microscopy was further used to confirm the nonfouling 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 nonfouling properties. To test the viability of the cells grown on GBA0, a replating 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 that the cells cultured

and polymers have been challenging to synthesize and purify in the past31 the use of the highly efficient oxime click reaction allows the facile preparation of PBA functional materials from 3,5-diformylphenyl boronic acid.32,33 Herein, we report a new cell capture-and-release system based on an oxime−PEG hydrogel functionalized with PBA. Simple oxime condensation polymerization allows 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 umbilical vein endothelial cells (HUVECs). Without PBA incorporation, the oxime−PEG hydrogel was found to be nonfouling. 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 and 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.

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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 and BE. It is wellknown that only anionic, tetrahedral (sp3) BE conjugates are hydrolytically stable. Below the pKa of the BE, neutral trigonal planar (sp2) species are produced that 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 Scheme 1. Equilibrium Reactions and General Synthesisa

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(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. 2236


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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.

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 nonfouling and cytocompatible properties of GBA0 and provide the basis for functionalization with PBA to achieve a cell capture-and-release system. The incorporation of 10 or 20 mol % PBA into the PEGbased 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. 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

MCF-7 cancer cells were captured with an efficiency of approximately 40%, which dropped to about 10% in comparison 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. Furthermore, the hydrogel presented here displays higher cellcapture ability than other previously reported boronic-acidfunctionalized 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. To illustrate that cell binding was due to specific interactions between PBA and carbohydrates present on the cell surface, the GBA10 substrates were first incubated with 70 mM glucose before seeding of the MCF-7 cell. In comparison to a GBA10 surface that was not incubated with glucose prior to cell seeding (Figure S2A), preincubation with glucose inhibited almost all cell adhesion (Figure S2B). This confirms that the addition of 70 mM glucose at pH 7.8 acts to inhibit MCF-7 cell adhesion by forming stable glucose−PBA complexes. To study the dual responsive capability of GBA10 to switch between a cell-capture and cell-release state, the pH and glucose concentration were altered simultaneously from pH 6.8 and 0 mM glucose to pH 7.8 and 70 mM glucose (Figure 3). This narrow and very neutral pH range is suitable for cell culture because cells were found to remain viable (green fluorescence, Figure S1 and S2) and effective cell capture and release can be achieved.18−21 As previously determined, cell capture occurred successfully at pH 6.8 with 0 mM glucose due to stable cell-surface carbohydrate BE formation (Figure 3A). Upon the addition of glucose and an increase of pH to 7.8, almost all cells (>90%) were released (Figure 3B). This high cell release number is comparable to some of the best previously reported boronic acid-functional brush polymers (98%).21 Additionally, there was no observable change in the hydrogel (for example further swelling) when the pH was increased from 6.8 to 7.8. This is to be expected because the hydrogel was fully swollen prior to the application of the cells.

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. The adhesion of normal NIH-3T3 fibroblast was also observed on both GBA10 and GBA20. **P < 0.01; *P < 0.05.

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 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.21,33 2237


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Figure 4. Dual responsive MCF-7 cell capture and release on GBA10. At a constant glucose concentration (70 mM), GBA10 becomes pHsensitive switching between cell capture at pH 6.8 (A) and cell release at pH 7.8 (B). Conversely, at a constant pH (pH 7.8), GBA10 is observed to be sensitive to the presence of glucose switching from (C) a cell capture surface at 0 mM glucose and (D) cell release at 70 mM glucose. Scale bars represent 100 μm.

Figure 3. MCF-7 cell capture and release on GBA10. (A) Efficient cell capture at pH 6.8/0 mM glucose and (B) almost-complete cell release at pH 7.8 with 70 mM glucose. (C) Cell density observations as GBA10 is cycled between a cell-capture and a cell-release state. No significant loss in cell density is observed over the four cycles, indicating the robustness of the prepared surfaces. Scale bars represent 100 μm.

contains between 5 and 50 mM glucose. Interestingly, the surface was found to be cell-capturing at pH 6.8 in the presence or the absence of glucose (Figures 3A and 4A). This indicates that, at pH 6.8, the surface is not responsive to the addition of glucose and remains cell adhesive. However, GBA10 was found to be glucose-responsive at pH 7.8. In the absence of glucose at pH 7.8, GBA10 was found to effectively capture cells (Figure 4C). Increasing the glucose concentration to 70 mM while maintaining the pH at 7.8 led to almost complete cell release (Figure 4D). The response to a single stimulus (pH or glucose) had no observable effect upon the numbers of cells adhered or released in comparison to the response to both a change in pH (6.8 to 7.8) and the addition of glucose (70 mM) (Figure 3). Therefore, GBA10 displays high activity, even in response to a single stimulus, and, in this way, represents a dual-responsive system sensitive to both glucose and pH. To demonstrate the versatility and compatibility of the boronic-acid-functional hydrogels with more-sensitive cell lines, we investigated the capture and release of HUVECs. HUVECs are primary cells and are far less robust than NIT-3T3 fibroblast and MCF-7 cancer cells. However, as this procedure requires very mild operating conditions, effective cell capture and release on GBA10 could be performed (Figure S3). HUVEC capture (at pH 6.8 and 0 mM glucose) and release (pH 7.8 and 70 mM glucose) was performed efficiently with high cell viability, as was indicated by intense green fluorescence (panels A and B of Figure S3, respectively). To confirm the HUVEC viability, the cells, initially captured on GBA10 and released by an increase to pH 7.8 and 70 mM glucose, were transferred to a new TCPS well and imaged by fluorescent microscopy (Figure S3C). Almost all of released cells showed intense green fluorescence indicating high cell viability and confirming the compatibility of the capture and release procedure with more-sensitive primary cell lines, demonstrated here with HUVECs. We report the cell-capture and -release properties of a new PEG-based, PBA-functionalized, oxime hydrogel. This material system addressed several critical problems associated with other reported cell capture-and-release systems (namely, a versatile and facile synthesis). Without PBA incorporation, the unfunctionalized hydrogel (GBA0) was found to be nonfouling owing to its PEG-like properties. Through the incorporation of

Additionally, the absence of any pH-responsive chemical groups, which could cause further swelling or a change in electrochemical potential due to the protonation or deprotonation of responsive groups, means that there is no change in the mechanical or chemical properties of the hydrogel during the increase from pH 6.8 to 7.8. This indicates that the surface properties of the hydrogels do not change with the increase in pH and, therefore, would have no effect upon the cell binding and release properties of the hydrogel. These results confirm that as the pH is increased the cell-surface carbohydrate BE complexes become hydrolytically unstable and are replaced by stable glucose BE complexes, which form under neutral and slightly basic conditions. The fundamental responsive mechanism of the dual responsive surfaces between cell capture and release has been studied by Liu et al.21 using quartz crystal microbalance (QCM) analysis. They showed that the competitive binding between PBA and sialic acid and between PBA and glucose at different pH values contributes to the dual response of the PBA-containing surfaces. The robustness of GBA10 as a cell capture and release system was tested by repeated cycling between pH 6.8 and 0 mM glucose and pH 7.8 and 70 mM glucose (Figure 3C). GBA10 was switched between cell capture and cell release for at least four cycles with negligible loss in cell adhesion or release number. These results indicate that GBA10 is able to reversibly capture and release cells over multiple cycles in response to both pH and glucose. To investigate the capability of GBA10 to switch between the cell-capture and cell-release state, the cell response to pH and glucose was investigated separately. In the presence of 70 mM glucose, GBA10 effectively captures cells at pH 6.8 and releases them at pH 7.8 (Figure 4A,B). This indicates that GBA10 is pHresponsive in the presence of 70 mM glucose. Importantly, as cell capture and release can be achieved via a small pH change in the presence of glucose, this platform is applicable for cell culture using commercially available cell culture media, which 2238


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(5) Collins, J., Nadgorny, M., Xiao, Z., and Connal, L. A. (2017) Doubly Dynamic Self-Healing Materials Based on Oxime Click Chemistry and Boronic Acids. Macromol. Rapid Commun. 38, 1600760. (6) Mukherjee, S., Bapat, A. P., Hill, M. R., and Sumerlin, B. S. (2014) Oximes as reversible links in polymer chemistry: dynamic macromolecular stars. Polym. Chem. 5, 6923−6931. (7) Mukherjee, S., Hill, M. R., and Sumerlin, B. S. (2015) Self-healing hydrogels containing reversible oxime crosslinks. Soft Matter 11, 6152−6161. (8) Liu, B., Chen, H., Li, X., Zhao, C., Liu, Y., Zhu, L., Deng, H., Li, J., Li, G., Guo, F., et al. (2014) pH-responsive flower-like micelles constructed via oxime linkage for anticancer drug delivery. RSC Adv. 4, 48943−48951. (9) Karimi, F., Mckenzie, T. G., O’Connor, A. J., Qiao, G. G., and Heath, D. E. (2017) Nano-scale clustering of integrin-binding ligands regulates endothelial cell adhesion, migration, and endothelialization rate: novel materials for small diameter vascular graft applications. J. Mater. Chem. B 5, 5942−5953. (10) Shirbin, S. J., Karimi, F., Chan, N. J.-A., Heath, D. E., and Qiao, G. G. (2016) Macroporous Hydrogels Composed Entirely of Synthetic Polypeptides: Biocompatible and Enzyme Biodegradable 3D Cellular Scaffolds. Biomacromolecules 17, 2981−2991. (11) Zhang, B., Digby, Z. A., Flum, J. A., Chakma, P., Saul, J. M., Sparks, J. L., and Konkolewicz, D. (2016) Dynamic Thiol−Michael Chemistry for Thermoresponsive Rehealable and Malleable Networks. Macromolecules 49, 6871−6878. (12) Hall, D. G. (2006) Structure, Properties, and Preparation of Boronic Acid Derivatives. Overview of Their Reactions and Applications. In Boronic Acids, pp 1−99, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, FRG. (13) Brooks, W. L. A., and Sumerlin, B. S. (2016) Synthesis and Applications of Boronic Acid-Containing Polymers: From Materials to Medicine. Chem. Rev. 116, 1375−1397. (14) Deng, C. C., Brooks, W. L. A., Abboud, K. A., and Sumerlin, B. S. (2015) Boronic Acid-Based Hydrogels Undergo Self-Healing at Neutral and Acidic pH. ACS Macro Lett. 4, 220−224. (15) Wu, X., Li, Z., Chen, X.-X., Fossey, J. S., James, T. D., and Jiang, Y.-B. (2013) Selective sensing of saccharides using simple boronic acids and their aggregates. Chem. Soc. Rev. 42, 8032. (16) Saito, A., Konno, T., Ikake, H., Kurita, K., and Ishihara, K. (2010) Control of cell function on a phospholipid polymer having phenylboronic acid moiety. Biomed. Mater. 5, 54101. (17) Ivanov, A. E., Eccles, J., Panahi, H. A., Kumar, A., Kuzimenkova, M. V., Nilsson, L., Bergenståhl, B., Long, N., Phillips, G. J., Mikhalovsky, S. V., et al. (2009) Boronate-containing polymer brushes: Characterization, interaction with saccharides and mammalian cancer cells. J. Biomed. Mater. Res., Part A 88, 213−225. (18) Ivanov, A. E., Panahi, H. A., Kuzimenkova, M. V., Nilsson, L., Bergenståhl, B., Waqif, H. S., Jahanshahi, M., Galaev, I. Y., and Mattiasson, B. (2006) Affinity adhesion of carbohydrate particles and yeast cells to boronatecontaining polymer brushes grafted onto siliceous supports. Chem. - Eur. J. 12, 7204−7214. (19) Ivanov, A. E., Galaev, I. Y., and Mattiasson, B. (2006) Interaction of sugars, polysaccharides and cells with boronatecontaining copolymers: from solution to polymer brushes. J. Mol. Recognit. 19, 322−331. (20) Ivanov, A. E., Kumar, A., Nilsang, S., Aguilar, M. R., Mikhalovska, L. I., Savina, I. N., Nilsson, L., Scheblykin, I. G., Kuzimenkova, M. V., and Galaev, I. Y. (2010) Evaluation of boronatecontaining polymer brushes and gels as substrates for carbohydratemediated adhesion and cultivation of animal cells. Colloids Surf., B 75, 510−519. (21) Liu, H., Li, Y., Sun, K., Fan, J., Zhang, P., Meng, J., Wang, S., and Jiang, L. (2013) Dual-Responsive Surfaces Modified with Phenylboronic Acid-Containing Polymer Brush To Reversibly Capture and Release Cancer Cells. J. Am. Chem. Soc. 135, 7603−7609. (22) Ulrich, S., Boturyn, D., Marra, A., Renaudet, O., and Dumy, P. (2014) Oxime ligation: A chemoselective click-type reaction for

PBA, efficient cell capture and release was afforded due to the dynamic properties of boronic acids and boronic esters. Stable boronate ester formation with cell surface carbohydrates and PBA under slightly acidic conditions (pH 6.8) is described. MCF-7 cancer cells, normal NIH-3T3 fibroblast cells, and HUVECs were shown to efficiently adhere to the PBA surface. The cell capture−release state was cycled from 0 mM glucose at pH 6.8, where efficient cell capture was observed, to 70 mM glucose at pH 7.8, where cell release was observed. Finally, the response to glucose and pH alone was investigated with successful conversion from a cell-capture to a cell-release surface when changing from pH 6.8 to 7.8 in the presence of glucose (70 mM) and when changing from 0 to 70 mM glucose at pH 7.8. The facile synthesis and the successful cell-capture and -release properties of the presented system indicate potential applications in the biomedical field for cell diagnostics and cell-culture applications.

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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.bioconjchem.7b00360. Synthetic and cell culture procedures and supplementary figures showing NMR and assay analysis. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +61-3-90353578. Fax: +61-3-8344-4153. ORCID

Luke A. Connal: 0000-0001-7519-977X Author Contributions ∥

F.K. and J.C. contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Victorian Endowment for Science Knowledge and Innovation (LAC). The authors thank the Research Training Program (RTP), the Endeavor IPRS, and the Australian Postgraduate Awards (APA) for providing financial support to this project. We thank the Particulate Fluids Processing Center (PFPC) and the Materials Characterization and Fabrication Platform (MCFP).

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REFERENCES

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