Modular and Injectable Poly(Oligoethylene glycol methacrylate)-Based Hydrogels With Tunable Protein and Cell Interactions

Bakaic, Emilia (McMaster University)
Smeets, Niels (McMaster University)
Hoare, Todd (McMaster University)

Introduction

Poly(ethylene glycol) (PEG) hydrogels have been widely studied as biomaterials, exploiting the hydrophilic, non-immunogenic, and non-cytotoxic properties of PEG. However, the solely chain end-reactivity of PEG polymers limits the mechanical properties and chemical diversity of such hydrogels. In addition, conventional PEG hydrogels are not in situ-gelling (i.e. injectable), as is essential for non-invasive biomedical applications. These limitations can be addressed through the use of poly(oligoethylene glycol methacrylate) (POEGMA). POEGMA is polymerizable by free radical polymerization (enabling facile chain functionalization via copolymerization) but contains an extremely high density of ethylene oxide units owing to its comb-like structure; as such, POEGMA-based materials have been demonstrated to offer many of the advantageous biological properties of PEG. In addition, POEGMA polymers can be designed to be thermoresponsive, with the lower critical solution temperature controlled by free radical copolymerization of OEGMA monomers with varying ethylene oxide chain lengths (n). In this contribution, we present the first reported in situ gelling POEGMA-based hydrogel based on reversible hydrazone bond formation, facilitating facile delivery of the hydrogel (via injection) into the body. In particular, we will compare the physical and biological properties of three POEGMA hydrogels with phase transition temperatures well below (PO0), close to (PO10) and significantly higher (PO100) than physiological temperature.

Materials and Methods

Hydrazide (POxHy) and aldehyde (POxAy) functionalized POEGMA precursors are synthesized from free-radical polymerization of oligo(ethylene glycol) methacrylate monomers with varying ethylene oxide chain lengths (n), offering a flexibility in terms of the (i) degree of reactive functionality (variation of y), (ii) incorporation of additional functionalities (anionic, cationic, hydrophobic), (iii) molecular weight and iv) lower-critical solution temperature (LCST) (variation of x). Reactive precursors are co-extruded from a double barrel syringe to rapidly form hydrogels in situ. Both physical (gelation time, swelling, elastic modulus and thermoresponsivity) and biological (protein adsorption, cell adhesion, and tissue compatibility following subcutaneous injections into BALB-c mice) properties of the resulting hydrogels were assessed.

Results

Control over the LCST of the POEGMA hydrogels (Fig. 1) is achieved by systematically varying the copolymerization ratio (x) of OEGMA monomers with n = 2 and n = 8-9 during precursor synthesis. The PO10 hydrogel (prepared from precursors containing 90 mol% n = 2 and 10 mol% n =8-9 monomers) shows a clear temperature transition around 32-33°C, while the PO0 (100 mol% n = 2 monomer) and PO100 (100 mol% n = 8-9 monomer) hydrogels show corresponding phase transitions of ~24°C and > 60°C respectively. Although the theoretical cross-link density of all hydrogels is similar (all precursors were prepared with 30 mol% reactive aldehyde and hydrazide groups), the mechanical properties vary significantly. The elastic storage modulus of PO¬0 is ~1 order of magnitude higher than that of PO100. Analogously, macroscopic gelation occurs significantly faster for PO0 (~5 s) compared to PO10 (20 s) and PO100 (~20 min). The gelation time and elastic storage modulus of the POEGMA hydrogels can be controlled by varying the concentration of reactive groups and/or the precursor polymer concentration. In vitro MTT assays showed that neither the precursors nor the hydrogels were cytotoxic. Minimal protein adsorption and cell adhesion is observed for PO10 and, in particular, PO100 (Fig. 2). Interestingly, grafting a small density of RGD peptide (1 RGD/chain on the aldehyde-functionalized polymer) to PO100 precursors significantly increased the capacity of the matrix to bind cells Fig.2 without compromising the low non-specific protein adsorption to this hydrogel. In contrast, PO0 hydrogels (phase transition temperature < physiological temperature) do not show any of these favorable biological properties. Correspondingly, in vivo studies on BALB-c mice show mild inflammatory responses at both the acute and chronic time points for PO10 and PO100 but evidence of capsule formation for PO0 (Fig. 3).

Discussion and Conclusion

By tuning the ratio of OEGMA monomers with different side chain lengths used to prepare the hydrogels, materials that behave analogously to both conventional PEG hydrogels (PO100) and the widely-used thermoresponsive polymer poly(N-isopropylacrylamide) (PO10) can be generated based on a POEGMA platform. By varying the functional group densities and polymer concentrations of the reactive precursor polymers, the mechanical properties of the hydrogels (G’ = 0..5– 20kPa) can readily be matched to a range of tissues (e.g. adipose, neural, muscle or cartilage, Analogously, by varying both the phase transition temperature and the composition of the precursors (i.e. whether or not a cell adhesive peptide is incorporated), matrices can be designed with extremely low protein adsorption (performing as well as reported PEGylated surfaces) and virtually no cell adhesion, or moderate cell adhesion but minimal non-specific protein adsorption. Such control over the injectability, mechanics, and biological response of hydrazone cross-linked POEGMA hydrogels makes such materials of interest for drug delivery, tissue scaffold and cell encapsulation applications.

Fig. 1 Thermoresponsive properties of the POEGMA hydrogels. Graph displays the decrease in water content as a function of the temperature (black) PO0, (grey) PO10 and (white) PO100. The photos display the physical appearance of the hydrogels at 20°C, 37°C and 60°C (grid = 5 mm x 5 mm).

Fig. 2 (C-E) Fibroblast adhesion after 7 days to a polystyrene control (C), POEGMA hydrogel (D) and an RGD-functionalized hydrogel (E).

Fig 3. PO10 ,PO100 and PO0 histology following subcutaneous injection (acute = 2 days, chronic = 30 days)

Acknowledgements

Funding from NSERC CREATE-IDEM and 20/20: NSERC Ophthalmic Materials Research Network is gratefully acknowledged.

References

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