3D-printed ketoenamine crosslinked polyrotaxane hydrogels and their mechanochromic responsiveness

Abstract

A ketoenamine-based dynamic covalent crosslinker was introduced to α-cyclodextrin-based polypseudorotaxanes, forming viscoelastic hydrogels for direct-ink-write 3D printing. The irreversible enol–keto tautomerization imparts high chemical stability to the extensively crosslinked polyrotaxane hydrogels, which showed mechanochromic responsiveness in the presence of the integrated fluorophores.

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Chenfeng Ke

Chenfeng Ke is an Associate Professor in the Department of Chemistry at Dartmouth College. He obtained his B.S. and Ph.D. from the College of Chemistry, Nankai University, in 2004 and 2009, respectively. He worked with Prof. Anthony Davis at the University of Bristol (2009–2011, Newton fellow, Royal Society), and Sir Fraser Stoddart at Northwestern University (2011–2015), before taking the current faculty position at Dartmouth College (2015 to present). His research focuses on developing supramolecularly designed 3D printing materials and hydrogen-bonded crosslinked organic frameworks (HCOFs). He co-initiated and co-chaired the first Gordon Research Conference on Additive Manufacturing of Soft Materials in 2022.

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Dynamic covalent chemistry (DCC)^1,2 has been integrated into polymer designs as an enabler for self-healing^3–5 and recycling properties.⁵ In these DCC networks, dynamic covalent bonding motifs, including imine,^6–10 oxmine,¹¹ boronic ester,^12,13 Diels–Alder adduct,^14–17 disulfide,^18,19 and hydrazone,^20–23 can form, dissociate, and exchange reversibly upon external stimuli.^24–27 As such, DCC-based polymers have been developed as shape-morphing^4,16 and self-healing materials.^11,12,17,28 Among these DCC reactions, reversible imine condensation has been used to construct direct-ink-write (DIW) 3D printing materials,^22,28 because the reversibility of imine bond formation and dissociation can facilitate the shear-thinning and self-healing properties required for DIW.^29,30 However, the chemical stability of imine linkages in these viscoelastic hydrogels is often concerning due to the abundance of ambient water favoring the equilibrium towards the starting materials.^7,31 On the other hand, α-cyclodextrin (α-CD)-based polypseudorotaxanes have also been introduced as viscoelastic hydrogels for DIW,^32–36 where the micro-crystalline domains formed between the polypseudorotaxanes serve to noncovalently crosslink the network.³⁶ The state-of-the-art polypseudorotaxane hydrogels utilize photo-crosslinking to afford 3D-printed polyrotaxanes, and they exhibited a variety of stimuli-responsive properties such as solvent-,³² pH-,³³ thermo-,³⁴ and moisture-responsiveness.³⁶ While photo-crosslinked hydrogels have been widely studied, hydrogels that can be crosslinked without the need of photo-initiators and light irradiation will simplify the technical prerequisites for deployment, especially in biomedical environments.

In this work, we integrate the dynamic imine network with the polypseudorotaxane hydrogel design, and expect the dual-crosslinked hydrogels to possess improved viscoelasticity for DIW 3D printing while eliminating the need for photo-crosslinking, removing the concern of insufficient crosslinking due to limited light penetration.^32–35 Herein, we introduce two dynamic crosslinkers, namely, 1,3,5-benzenetrialdehyde (BD) and 1,3,5-triformylphloroglucinol (TP) to form DCC networks with α,ω-diamino-polyethylene glycol (PEG_4k-(NH₂)₂). PEG_4k-(NH₂)₂ also serves as the axle for α-CD threading, forming a non-covalently crosslinked polypseudorotaxane network (Fig. 1). Different from the highly reversible imine-crosslinked network formed by BD and PEG_4k-(NH₂)₂, the reaction of TP with PEG_4k-(NH₂)₂ generates a more stable network due to the semi-reversible imine-to-ketoenamine tautomerization.^37–40 In the presence of α-CDs, the polypseudorotaxane hydrogels crosslinked by these dynamic crosslinkers show different viscoelastic features. After extensive crosslinking, we discovered that the ketoenamine-based hydrogels showed good chemical stability against extensive washing, while the imine-crosslinked poly(pseudo)rotaxane hydrogels dissolved completely. After further appending rhodamine B and fluorescein moieties to the PEG axles, the generated ketoenamine-based polyrotaxane hydrogel showed mechanochromic response upon compression as a result of the Förster resonance energy transfer process.^41,42

Fig. 1 Graphical illustration of the construction of 3D-printed polyrotaxane networks connected via dynamic covalent crosslinkages.

In our design, we chose a medium chain-length PEG_4k (M̄_n = 4000 Da) as the axle because long-chain PEG-based hydrogels are difficult to be filtered out by human kidneys.⁴³ However, when an unfunctionalized PEG_4k axle is mixed with α-CD, white precipitates or brittle hydrogels are formed at different concentrations due to the crystallization of α-CD-threaded polypseudorotaxanes.³⁶ Hence, we introduced DCC networks to improve the viscoelasticity of these polypseudorotaxanes.

To understand the reversible nature of the dynamic networks formed by PEG_4k-(NH₂)₂ and crosslinkers BD and TP in water, time-dependent ¹H NMR studies were performed at the same concentrations for the hydrogel formations in the absence of α-CD (Fig. 2). TP and PEG_4k-(NH₂)₂ were synthesized according to previously reported methods.^33,37 When BD and PEG_4k-(NH₂)₂ were mixed together, the reaction proceeded rapidly (5 min) at room temperature to form imine linkages (Fig. 2a). When the sample was heated to 60 °C, the imine formation was slightly reversed. The reaction equilibrium was re-established when the sample was cooled to room temperature, and the imine-to-aldehyde ratio resumed to that before heating. In contrast, TP was largely unreacted in the mixture of TP and PEG_4k-(NH₂)₂ at room temperature, and the reaction gradually proceeded at 60 °C (Fig. 2b). Two sets of aldehyde proton resonances were observed at 9.75 and 9.66 ppm, attributed to the unreacted and partially reacted TP, respectively. 2D NMR experiments (Fig. S5, S6 and S8–S10†) suggested that the proton resonances observed at 8.2 ppm are attributed to the ketoenamine moieties, and the population of imines is nearly negligible. After 18 h reaction at 60 °C, most TP had been converted to the ketoenamine linkages. These time-dependent ¹H NMR studies suggest that the rate of the reaction between TP and PEG_4k-(NH₂)₂ is much slower than that of BD and PEG_4k-(NH₂)₂, and the ketoenamine network is much less reversible compared to the imine network.

Fig. 2 (a) Temperature-varied time-dependent ¹H NMR spectra of PEG_4k-(NH₂)₂ (10.0 mM) and BD (6.7 mM) in D₂O at 298/333 K, revealing the highly dynamic feature of the imine bonds. (b) Temperature-varied time-dependent ¹H NMR spectra of PEG_4k-(NH₂)₂ (10.0 mM) and TP (6.7 mM) in D₂O at 298/333 K, suggesting the semi-reversible characteristic of ketoename bonds.

In the presence of α-CD, the reactions of PEG_4k-(NH₂)₂ and dynamic crosslinkers BD and TP were heated at 60 °C for 2 h and then cooled down to room temperature, affording a series of viscoelastic hydrogels. Due to the dynamic covalent crosslinking, these hydrogels contain polyrotaxanes and polypseudorotaxanes, some of which are interconvertible. Hence, we name these samples poly(pseudo)rotaxane hydrogels (PpRHs[a,b]), in which a and b are the weight percentages of the PEG_4k-(NH₂)₂ and α-CD, respectively. At different ethylene glycol (EG) repeat unit to α-CD ratios (Tables S1 and S2†), PpRHs possess different viscoelastic properties in the rheological investigations (Fig. S15–S45†). When BD was used as the dynamic crosslinker, the elastic moduli of PpRH_BD hydrogels ranged between 10⁵–10⁶ Pa at a wide range of EG/α-CD ratios (Fig. S33–S45†). For example, PpRH_BD[3,15] possesses an elastic modulus G′ = 9 × 10⁵ Pa in the angular frequency sweep (Fig. 3a), and excellent self-healing features in the step-strain studies (Fig. 3b). In comparison, PpRH[3,15] formed by bare PEG_4k and α-CD has an elastic modulus of 2.5 × 10⁴ Pa with a poor self-healing profile (G′ = 1.1 × 10² Pa after five cycles of step-strain sweeps, Fig. 3a and b). Furthermore, the PEG_4k-(NH₂)₂ and TP reaction mixture at the same concentration as the PpRH[3,15] without α-CD did not yield any hydrogel. These results suggest that the microcrystalline domains of the poly(pseudo)rotaxanes remain the main contributor to the viscoelastic feature of the PpRHs, which is evident in the PXRD studies (Fig. S57†).

Fig. 3 (a) Angular frequency sweeps and (b) step-strain sweeps of PpRH[3,15] (black), PpRH_TP[3,15] (red), PpRH_BD[3,15] (blue). (c) Images of 3D-printed cubic and pyramidal lattices printed with PpRH_TP[3,15]. (d) Images of the degradation process of PpRH_BD[3,15] in excess water. (e) Images of the kPH-1 monoliths after extensive crosslinking. Scale bars: 2 mm. (f) FT-IR spectra of (i) TP, (ii) kPH-1, (iii) dehydrated PpRH_BD[3,15], and (iv) BD.

When TP was used as the crosslinker, the elastic modulus of the PpRH_TP[3,15] was recorded as 1.1 × 10⁴ Pa in the angular frequency sweep (Fig. 3a), which is 100 times smaller than that of PpRH_BD[3,15]. This result is consistent with the time-dependent ¹H NMR studies, in which the effective crosslinking density in the PpRH_TP[3,15] is smaller than PpRH_BD[3,15]. PpRH_TP[3,15] also showed good self-healing properties in the step-strain studies as the elastic moduli fully recovered after five cycles of step-strain sweeps (Fig. 3b). When PEG_4k-(NH₂)₂, α-CD, and TP were heated for 18 h at 60 °C and then cooled down, the obtained hydrogel showed decreased elastic modulus (G′ = 1.3 × 10³ Pa) and poorly self-healing properties (Fig. S16†). In comparison, the PpRH_BD[3,15] hydrogel did not show any reaction-time-dependent rheological variations (Fig. S33†). These results revealed that both the imine and ketoenamine networks effectively promote the viscoelasticity features of PpRHs. However, as a result of the effective imine crosslinking, the viscosity of PpRH_BD hydrogels reached 10³–10⁴ Pa·s (Fig. S33†), making them difficult to be extruded. In comparison, the viscosity of PpRH_TP[3,15] hydrogels is 350 Pa·s (Fig. S16†), which is suitable for DIW 3D printing. For example, 2 × 2, 4 × 4, and 5 × 5 piled grids and pyramids (40 layers) were 3D-printed using PpRH_TP[3,15] (Fig. 3c).

The PpRH samples were heated to 70 °C overnight, cooled down, and immersed in excess water to examine their chemical stability. As a result of the high reversibility of the imine-crosslinked network, the PpRH_BD[3,15] sample dissolved completely in water over 48 h (Fig. 3d). The soluble species were also confirmed in the ¹H NMR experiment (Fig. S55†). In contrast, the 3D-printed PpRH_TP[3,15] samples were extensively crosslinked. The polypseudorotaxanes in PpRH_TP[3,15] were converted to ketoenamine-based polyrotaxane hydrogels (kPH-1, Fig. 3e), and they are stable in excess of water (Fig. S56†). IR spectra showed that the kPH-1 possesses a C=C stretching band at 1246 cm⁻¹ (Fig. 3f), compared to the C=N stretching band of the PpRH_BD[3,15] at 1644 cm⁻¹ (Fig. 3f). In a NaOD/D₂O solution (5 w/v%), the kPH-1 was hydrolyzed completely after being heated at 60 °C for 2 h. ¹H NMR spectrum (Fig. S47†) showed that 10 α-CDs per PEG were mechanically interlocked in the kPH-1, corresponding to 22% of the PEG axles covered by α-CDs. Macroscopically, the 3D-printed kPH-1 showed good chemical stability and structural integrity in DMSO and water (Fig. 3e).

To study the mechanical performance of kPH-1, we performed tensile, cyclic (un)loading, and compression tests at room temperature (Fig. 4a and b). The Young's modulus and the compressive modulus of kPH-1 were measured as 214 kPa and 194 kPa, respectively (Fig. 4a). The strain at break of kPH-1 was measured as 260% in the tensile test. kPH-1 was deformed to a strain of 93% in the compression test without breaking (Fig. 4a), showing good mechanical performance. In the cyclic (un)loading tests (Fig. 4b), the hysteresis of kPH-1 decreased gradually, suggesting the crystalline domains in the kPH-1 were disrupted during the elongation process but not fully reformed in each cycle.

Fig. 4 (a) Uniaxial tensile and compression stress–strain profiles of kPH-1. Tensile strain rate: 1.5 mm min⁻¹, compression strain rate: 1 mm min⁻¹. (b) Multi-cyclic tensile (un)loading stress–strain profiles of kPH-1. Tensile strain rate: 1.5 mm min⁻¹. The profile is offset by 100% in each cycle. (c) Images of kPH-1 with doped PEG_4k-fluorescein and PEG_4k-rhodamine FRET pairs at different compression strains under white light (top) and 450 nm light irradiation (bottom). (d) Fluorescence emission spectra of kPH-1 with doped FRET pairs at different compression strains. (e) The FRET efficiency (I₅₈₂/I₅₃₃) at different compression strains was calculated from (d).

To probe the structural change upon mechanical loading at the molecular level, we introduced a Förster resonance energy transfer (FRET) pair^44,45 to the kPH-1. Experimentally, a mixture of fluorescein and rhodamine-modified PEG axles (modification ratio = 1% for fluorescein/PEG and rhodamine/PEG) were used to construct the kPH_FRET (see the ESI, section 9†). A set of orange-colored hydrogels were sandwiched between two glass slides, compressed to determined strains, and subjected to fluorescent measurements. At 0% strain, the hydrogel emitted at 533 and 582 nm upon irradiation (λ_ex = 450 nm, Fig. 4d, black line). Upon compression, the emission at 582 nm decreased, and the emission at 533 nm increased (Fig. 4c and d). The decreased I₅₈₂/I₅₃₃ values suggest that the averaged distances between two neighboring FRET pairs are increasing (Fig. 4e), corresponding to larger distances between PEG axles. Hence, the crystalline domains connecting the polyrotaxanes in kPH-1 were disrupted, which explained the decreased hysteresis upon cyclic loading.

Conclusions

In summary, we have successfully developed mechanically robust 3D-printed polyrotaxane hydrogels by integrating the dynamic covalent network with the noncovalently connected polypseudorotaxane network. We discovered that the highly reversible imine crosslinkages significantly improved the viscoelasticity of the poly(pseudo)rotaxane network, but the product gradually dissolved in excess water. In comparison, the ketoenamine crosslinkages improved the viscoelasticity of the poly(pseudo)rotaxane network for direct-ink-writing and provided good chemical stability after extensive crosslinking. Tensile and compression tests showed that the ketoenamine-crosslinked polyrotaxane hydrogels could be stretched to 260% and compressed to more than 93% with Young's modulus of 214 kPa. Furthermore, when Förster resonance energy transfer pairs were introduced to the polyrotaxane network, the hydrogel showed strain-dependent emissions, revealing the micro-environment changes upon compressing.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the Department of Energy the Basic Energy Sciences DE-SC0022267. In addition, C. K. thanks the support of the Cottrell Scholar program from the Research Corporation for Science Advancement and the Beckman Young Investigator program from the Arnold and Mabel Beckman Foundation.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3py00337j

‡ These authors contributed equally to this work.

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