Influence of Hydrophobic Cross-Linkers on Carboxybetaine Copolymer Stimuli Response and Hydrogel Biological Properties

Abstract

Poly­(carboxybetaine) (pCB) hydrogels do not elicit a foreign body response due to their low-fouling properties, making them ideal implantable materials for in vivo drug and cell delivery. Current reported pCB hydrogels are cross-linked using cytotoxic UV-initiated radical polymerization limiting clinical and in vivo translation. For clinical translation, we require in situ and biorthogonal cross-linking of pCB hydrogels that are both low-fouling and low-swelling to limit nonspecific interactions and minimize tissue damage, respectively. To this end, we synthesized carboxybetaine (CB) random copolymers (molecular weight (MW): ∼7–33 kDa; Đ: 1.1–1.36) containing azide (pCB-azide) or strained alkyne (Dibenzocyclooctyne (DBCO); pCB-DBCO) that rapidly cross-link upon mixing. Unlike CB homopolymers and other CB copolymers studied, high DBCO content pCB-DBCO30 (30% DBCO mole fraction) is thermoresponsive with a upper critical solution temperature (UCST; cloud point of ∼20 °C at 50 g/L) in water due to electrostatic associations. Due to the antipolyelectrolyte effect, pCB-DBCO30 is salt-responsive and is soluble even at low temperatures in 5 M NaCl, which prevents zwitterion electrostatic associations. pCB-azide and pCB-DBCO with 0.05 to 0.16 cross-linker mole fractions rapidly formed 10 wt % hydrogels upon mixing that were low-swelling (increase of ∼10% in wet weight) while remaining low-fouling to proteins (∼10–20 μg cm–2) and cells, making them suitable for in vivo applications. pCB-X31 hydrogels composed of pCB-azide32 and pCB-DBCO30 formed opaque gels in water and physiological conditions that shrunk to ∼70% of their original wet weight due to pCB-DBCO30’s greater hydrophobicity and interchain electrostatic interactions, which promotes nonspecific protein adsorption (∼35 μg cm–2) and cell binding. Once formed, the electrostatic interactions in pCB-X31 hydrogels are not fully reversible with heat or salt. Although, pCB-X31 hydrogels are transparent when initially prepared in 5 M NaCl. This is the first demonstration of a thermo- and salt-responsive CB copolymer that can tune hydrogel protein and cell fouling properties.

Modulating the Thermoresponse of Polymer-Protein Conjugates with Hydrogels for Controlled Release

“…As we have previously demonstrated that 10 wt% pCB hydrogels formed upon crosslinking copolymers of pCB-azide and pCB-DBCO that resulted in injectable, low-fouling hydrogels, we synthesized the relevant pCB-azide, pCB-DBCO copolymers and precursors ( Table S1 and Figure S5 ) and studied their influence on P(D x T y ) LCSTs [ 17 ]. We synthesized pCB-Az and pCB-DBCO copolymers with 3.5 and 3.9 mol% of azide and DBCO moieties, respectively, resulting in a hydrogel with ~3.5 mol% of crosslinks, which was previously shown to be low-fouling and enabled protein diffusion.…”

“…3-(3-dimethylaminopropyl)-1-ethyl-carbodiimide hydrochloride (EDC-HCl) was obtained from Chem-Impex International Inc. (Wood Dale, IL, USA) Alexa Fluor 647 NHS ester, Alexa Fluor 488 NHS Ester, Fetal bovine serum (FBS)DMEM, Alamar blue was purchased from Thermo Fisher Scientific (Burlington, ON, Canada). O-(6-azidohexyl)-O’-succinimidyl carbonate (NHS-azide), O-[1-(4-chlorophenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidylcarbonate (NHS-AzCl) and carboxybetaine monomer were synthesized as previously described [ 15 , 16 , 17 ].…”

“…Poly(carboxybetaine) (pCB) hydrogel synthesis. pCB hydrogels were synthesized in a similar manner as previously described [ 15 , 17 , 18 ]. Copolymers of pCB-APMA, pCB-Azide (pCB-Az), pCB-AzCl and pCB-DBCO were synthesized as described below.…”

Fabrication of Hollow Structures in Photodegradable Hydrogels Using a Multi-Photon Excitation Process for Blood Vessel Tissue Engineering

“…We reported that the turbidity of the solution of DBCO-PC-4armPEG crosslinker increased at temperatures above 30 °C [ 41 ], indicating polymer condensation due to hydrophobic interactions between DBCO–PC moieties. In general, hydrophobic groups induce shrinkage in hydrogels due to their hydrophobic interactions [ 56 ]. During higher modification rate of azide-gelatin, the concentration of counter DBCO moieties was high, which presumably reduced the hydrogel swelling and induced hydrogel shrinkage due to the hydrophobic properties of DBCO–PC moieties.…”

Controlled degradation of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) hydrogels

“…7A), indicating the gels are low-fouling towards cells. PCBMAA gels formed through in situ PCBMAA-AZ and PCBMAA-DBCO (6 mol% AZ/DBCO) crosslinking were included as controls; PCBMAA gels are known to resist non-specic cell binding 36 (Fig. 7A).…”