Multiple-Responsive Dendronized Hyperbranched Polymers.

By combining topological structures of hyperbranched polymers with dendronized polymers, a series of hyperbranched poly(acylhydrazone)s pendanted with 3-fold branched dendritic oligoethylene glycol (OEG) units were efficiently prepared through A2 + B3 polycondensation. The constituents of these dendritic polymers can be mediated through dynamic covalent acylhydrazones. Owing to the dense OEG pendants, these dendronized hyperbranched polymers are biocompatible and thermoresponsive, and their cloud points (T cps) can be modulated by the branched architecture, solution pH, and addition of a third component. Cell viability in the presence of these hyperbranched poly(acylhydrazone)s can be maintained above 80%. Based on the unique dendritic architecture with rich acylhydrazine groups, dynamic hydrogels cross-linked via acylhydrazone linkages with good mechanical property were prepared, which inherit the characteristic thermoresponsive behavior of the polymer precursors and also show remarkable self-healing properties. This novel kind of topological polymers and their corresponding hydrogels with dynamic and multiple smart properties may have promising applications as biomaterials.

Introduction

Dendritic polymers such as dendrimers, hyperbranched polymers, and dendronized polymers are highly branched polymers with complex, secondary architectures and well-defined spatial location of functional groups.[1,2] Unlike other dendritic polymers, hyperbranched polymers show the prominent superiority of efficient one-step synthesis and a flexible regulation of structures.[3−6] Although hyperbranched polymers possess irregular branching structure, they have low viscosity and large amount of functional groups at both the linear and terminal units. In recent years, hyperbranched polymers with stimuli-responsive moieties have been intensively investigated due to their promising applications in areas such as targeted drug delivery, catalysis, hybrid materials, light-emitting materials, surface engineering, and biomimetic materials.[7,8] In particular, control of hyperbranched polymers’ architecture and surface groups combined with responsiveness and adaptability make them promising gelators for hydrogel formation. Hyperbranched polymer hydrogels formed from both physical or chemical cross-linking reaction show good mechanical properties and encapsulation ability, which can be used for cell adhesion and biomolecule delivery.[9−16] Dendronized polymers are a class of nanoscopic, rigid polymers with densely packed dendrons as the pendent group. By combining the topological structures from dendronized and hyperbranched polymers, the formed dendronized hyperbranched polymers show a unique architecture and inherit the advantages from both types of polymers. The dendronized hyperbranched polymers possess more possibilities for functionalization compared to a dendron,[5] and their structural defect should be much less than those of normal hyperbranched polymers. For example, by the introduction of low generation of nonlinear optical (NLO) dendrons, a series of dendronized hyperbranched polymers were prepared that possess high NLO coefficient, good stability, and good solubility.[17−20] However, the reported hyperbranched polymers constructed with dendritic units lack responsive properties, which limits their further applications.

Dynamic covalent chemistry has been proved to be an effective strategy for preparing polymers with different architectures and can afford the polymers not only enhanced stability but also constitutional reversible features.[21−23] Recently, we reported dynamic covalent thermoresponsive dendronized polymers prepared through polycondensation of dendritic oligoethylene glycols (OEG) cored with dialdehydes and diacylhydrazine.[24] These polymers, with acylhydrazone bonds at their main chains, were found to show dynamic covalent bond exchange; thus, they can generate new constitutional polymers with different cloud points by the addition of a third dendritic monomer with different hydrophilicity. Other dynamic covalent hyperbranched polymers including hyperbranched polyacylhydrazones,[25] polyoximes,[26] and copolymers with disulfide[27] and Diels–Alder[28] linkages have also been developed. However, dendronized hyperbranched polymers formed through dynamic covalent chemistry at their backbone are rarely reported.

In this work, a novel kind of dendronized hyperbranched poly(acylhydrazone)s with different branched architectures were prepared by traditional A2 + B3 polycondensation of first-generation (G1) OEG-dendron cored with dialdehyde and triacylhydrazine with different molar ratios. The formed dendronized hyperbranched polymers possess unique thermoresponsive behavior. Different from the linear OEG-dendronized polyacylhydrazone reported previously,[24] these hyperbranched polymers possess abundant acylhydrazine end groups, providing chances for further modification to form functional materials. The effect of polymerization conditions on molecular weight and degree of branching was studied, and their thermoresponsive properties were investigated by UV–vis spectroscopy. The dynamic constitutional feature of these dendronized polymers were examined by switching solution pH and also through the addition of competitive monomers with different hydrophilicity to mediate polymer thermoresponsiveness. Moreover, thanks to the surplus peripheral acylhydrazine units, which can be further cross-linked with dialdehyde-terminated poly(ethylene glycol) (PEG) to form hydrogels with thermoresponsive behavior and self-healing ability. The influence of solution pH, gelator concentration, and content of cross-linker on the mechanical property of the hydrogel were evaluated by oscillatory rheology (Figure ).

Figure 1

Schematic synthesis of thermoresponsive dendronized hyperbranched polymers and their corresponding hydrogels from dynamic covalent cross-linking.

Results and Discussion

Synthesis and Characterization of Hyperbranched Polymers

To obtain dynamic covalent hyperbranched polymers, monomers carrying aldehyde and acylhydrazine groups were prepared. Ethoxyl-terminated G1 dendritic OEG cored with dialdehyde is used as an A2 monomer, which also affords polymers characteristic thermoresponsive behavior. Tri(acylhydrazine) amine was used as a B3 monomer. Both A2[24] and B3[29] were synthesized according to previous reports. Dendronized hyperbranched poly(acylhydrazone)s with different branched architecture were prepared by traditional A2 + B3 polycondensation of dialdehyde with triacylhydrazine in a solution of pH 4.0. The detailed polymerization conditions and typical polymerization results are summarized in Table . Three polymers were prepared at the A2/B3 molar feeding ratios of 0.8, 1.0 and 1.5, respectively, and their weight-average molecular weights (Mws) were determined to be in the range of 4.0–11.9 × 104 by gel permeation chromatography with light-scattering detector (GPC-LS) with N, N-dimethylformamide (DMF) as the eluent (Figure S1). All polymers were characterized by 1H NMR spectroscopy (Figure S2). The proton signal assigned to the terminal acylhydrazine group (δ = 9.0) decreases with increase of the dialdehyde monomer as the linear unit. The degrees of branching (DBs) for dendronized hyperbranched poly(acylhydrazones) are calculated based on proton integrations from 1H NMR spectra, which are in the range from 0.60 to 0.30.

Table 1

Conditions for and Results from the Copolymerization

					GPC-LS
samples	A2/B3	[A2] (mmol L–1)	yield (%)	DBa	Mw (×10–4)	Đ	Tcpb (°C)
P1	0.8	6.5	84	0.60	4.0	1.38	47.2
P2	1.0	6.5	68	0.51	6.5	1.38	45.5
P3	1.5	6.5	75	0.30	11.9	1.70	39.4

DBs of hyperbranched polymers were calculated based on proton integrations of the corresponding units according to the equation: DB = (D + T)/(D + T + L), where D, T, and L represent the fractions of the dendritic, terminal, and linear units, which correspond to the proton signals of 11.48, 9.01, and 5.05 ppm, respectively.

Tcps of the polymers in pH 7.0 buffer solutions were determined as the temperature at 50% of the initial transmittance at λ = 700 nm.

These polymers are well soluble in water at room temperature but precipitated out, and their solution becomes turbid when heated at elevated temperature. Temperature-varied UV–vis spectroscopy was used to follow thermally induced transition of the dendronized hyperbranched poly(acylhydrazone)s and detect their cloud points (Tcps). Figure a shows the transmittance of polymers at 0.25 wt % in pH 7.0 buffer solutions at a constant heating rate of 0.5 °C min–1, and Tcps for P1, P2, and P3 were determined to be 47.2, 45.5, and 39.4 °C, respectively. Although containing hydrophilic acylhydrazine moieties, phase transition regions of these polymers span a narrow temperature range (less than 2.5 °C). This sharp transition indicates that dendritic OEGs provide efficient shielding to the hyperbranched polymers with prominent thermoresponsive properties. Furthermore, Tcp decreases from 47.2 to 43.9 °C with DB from 0.60 to 0.30, suggesting that dendron coverage dominates their thermoresponsive behavior. The concentration effect on the phase transition of P1 was checked. Turbidity curves of P1 with concentration ranging from 0.1 to 3 wt % are shown in Figure S3a, and the dependence of cloud point on its concentration is plotted in Figure S3b. It was found that the cloud points decrease gradually from 49.9 to 40.8 °C with increase of concentration, which may be due to the enhanced aggregation kinetics at higher concentration. Although when the solution concentration is increased to above 2 wt %, the cloud points tend to become similar to each other. The effect of heating rate on the cloud point was also checked. As shown in Figure S4a, a minor influence on the cloud points was observed by the heating rate lower than 2 °C min–1. However, when heating rate is very high, such as in the case of 4 °C min–1, delayed kinetics during the thermally induced aggregation results in an apparent higher Tcp. A hysteresis effect (Figure S4b) was observed between heating and cooling, which may be related to large amount of hydrazone bonds that form a strong hydrogen bonding between the polymer chains. All polymers exhibit a pH-dependent thermoresponsive behavior, and their Tcps are varied at various pH conditions. As shown in Figure b, the Tcps of polymers decrease efficiently with the decrease of the solution pH. The turbidity curves of all three polymers are shown in Figure S5. P1 was taken as the example. Its Tcp decreases from 47.2 to 41.6 °C when the solution pH decreases from 7 to 2. We suspect this significant effect may be due to the acid-induced decomposition of acylhydrazone, which releases more hydrophobic acylhydrazone moieties. For clarifying, the 1H NMR spectra of a model acylhydrazone compound from tri(acylhydrazine) amine with a monoaldehyde at different pH conditions were recorded (Figure S6). At solution pH of 2.0, a clear proton signal corresponding to acylhydrazine appears, indicating decomposition at strong acidic condition. The remarkable characteristics of dynamic covalent polymers relay on their structural variability upon the addition of third components through the exchange. Therefore, dialdehyde monomers with different hydrophilicities were added to form the hyperbranched polymers to examine the tunability of their structures as Tcp. Glutaraldehyde and methoxyl-terminated dendritic OEG cored with dialdehyde (A2′) were selected as additive monomers. The former is more hydrophobic, whereas the latter is more hydrophilic, and their influence on polymer Tcp is shown in Figure c. Considering the time-consuming process of exchange reaction for acylhydrazone, mixture of P1 with competitive monomer in a buffer solution with pH 2.0 was kept at room temperature for 5 days. After the addition of 1.1 equiv hydrophobic glutaraldehyde, Tcp of P1 decreases from 41.5 to 39.2 °C. However, after the addition of 1.1 equiv hydrophilic A2′, Tcp of P1 increases from 41.5 to 43.8 °C. These results indicate that the Tcp of dendronized hyperbranched poly(acylhydrazone)s can be tuned by constitutional dynamics.

Figure 2

(a) Plots of transmittance vs temperature for P1, P2, and P3 in pH 7.0 buffer solutions. (b) Influence of pH on Tcps of P1, P2, and P3 in aqueous solutions. (c) Plots of transmittance vs temperature at pH 2.0 for P1 and P1 with glutaraldehyde and P1 with methoxyl-terminated first-generation dendritic OEG cored with dialdehyde A2′ in pH 2.0 buffer solutions. Polymer concentration = 0.25 wt %, heating rate = 0.5 °C min–1.

Considering their potential application as biomaterials, we examined the in vitro cytotoxicity of these dendronized hyperbranched polymers. Cell-counting kit-8 (CCK-8) assay was used to investigate viability of HeLa cells in the presence of these polymers. As shown in Figure S7, no obvious decrease in cell survival rate was observed when the concentration of all these polymers was below 50 μg mL–1. Even when the concentrations of P1 and P2 reached 100 μg mL–1, all cells kept their viability. However, when the concentration of P3 reached up to 50 μg mL–1, the cell viability showed an obvious decrease. These results indicate that all polymers show good biocompatibility at low concentration, which may pave the way for their bioapplications.

Hyperbranched Poly(acylhydrazones) Hydrogels via Acylhydrazone Chemistry

Due to abundant peripheral acylhydrazine groups, dendronized hyperbranched poly(acylhydrazone)s can be further cross-linked through reaction with dialdehyde. Dialdehyde-terminated PEG (Mn = 1000) (PEGDA) was selected as the cross-linker because PEG shows good biocompatibility and well solubility in buffer solutions. Dendronized hyperbranched poly(acylhydrazones) hydrogels were formed from P1 via acylhydrazone cross-linking, and the hydrogel formation was visually recognized by the reversed vial test method. Hydrogels with different pH solution, gelator concentration (wt %), and content of cross-linker were prepared (Table ). The optimal condition for hydrogel formation was determined, and the results are summarized in Table S1. All conditions, including solution pH, concentration, and P1/PEGDA ratios, affect the formation and mechanical properties of the hydrogels. Very fast gelation occurred at pH 2.0. As shown in Figure a, the hydrogel GEL1 from pH 2.0 buffer solution of 15 wt % P1 (with acylhydrazine/aldehyde ratio = 2.10:1) was formed in 15 min and can withstand various deformations such as elongation and folding. Gelation also occurred at intermediate pH 4.0 but required significantly longer reaction periods (more than 2 h). The enhanced gelation efficiency at low pH is understandable, since hydrazone formation can be catalyzed by acid. However, with gelator concentration of 10 wt %, the mixture of P1 and PEGDA can form hydrogels in the pH 2.0 buffer solution but not in the pH 4.0 buffer solution. The lowest concentration to form hydrogels from P1 via PEGDA was also confirmed through the inversion method. It was found that the lowest concentration for hydrogel formation at pH 2.0 and 4.0 is about 10 and 15 wt %, respectively. In addition, feed ratios of cross-linker can also take effect on the hydrogel formation. At the same pH condition and gelator concentration (pH 2.0, 10 wt %), when the molar ratio between the remaining acylhydrazine groups from P1 and the aldehyde groups from PEGDA decreases from 2.10:1 to 1.05:1, only viscous liquid was obtained. However, hydrogel was formed slowly and showed low mechanical strength when increasing the molar ratio to 3.15:1. Thus, we assume that the optimal ratio of acylhydrazine/aldehyde is about 2.10:1 to form the necessary cross-linking density for gelation.

Table 2

Conditions of Gelation from P1 and PEGDAa,b

entry	GEL1	GEL2	GEL3	GEL4
pH	2.0	2.0	4.0	4.0
gelator concentration (wt %)	15	10	15	20
G′ (kPa)	4.5	3.0	12.0
G″ (kPa)	0.55	0.48	1.32

P1/PEGDA (wt/wt) = 2:1, mole ratio of hydrazine/aldehyde = 2.1:1.

ω = 1 rad s–1, γ = 0.1%, 25 °C.

Figure 3

(a) Photographs of sol–gel transitions (up) for P1 after the addition of PEGDA (15 wt % P1, in pH 2.0 buffer) and hydrogel GEL1 stored for 15 min after the addition of PEGDA (down). (b) Fourier transformed infrared (FTIR) spectra of P1, PEGDA, and GEL1. (c) scanning electron microscopy (SEM) image of lyophilized GEL1. (d) Rheological properties of GEL1, GEL2, and GEL3.

To verify the acylhydrazone bond formation within hydrogels, the IR spectra of P1, PEGDA, and hydrogels were recorded by attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectrometry (Figure b). For PEGDA, absorption band at 1683 cm–1 was attributed to the stretching vibration of the aldehyde group. Appearance of the C=N characteristic band at 1674 cm–1 in the spectrum of GEL1 confirmed the cross-linking reaction between the acylhydrazine groups from P1 and the aldehyde groups from PEGDA to form hydrazone bonds. Also compared with the spectrum of P1, the characteristic peak ascribed to the amino group weakened in the spectrum of GEL1, demonstrating the acylhydrazine groups were consumed. These results indicate that the hydrogels were formed through hydrazone cross-linking reaction. Internal morphology of the hydrogels was analyzed by SEM. As shown in Figures c and S8, the SEM image revealed that GEL1 and GEL3 composed of 3D porous interconnected microstructure with pore sizes in the range from 20 to 50 μm.

To further investigate the viscoelastic behavior of these hydrogels, dynamic rheology measurements at both frequency sweep and strain sweep modes were performed on a rotational rheometer. In the frequency sweep test, G′ and G″ of hydrogel samples were measured with a range of frequencies from 0.1 to 100 rad s–1 at controlled regular strain of 10%. The rheological curves of G′ and G″ of GEL1 and GEL2 from pH 2 buffer, as well as GEL3 from pH 4 buffer, are shown in Figure d. G′ exceeds G″ by about 1 order of magnitude, and G′ is independent of oscillatory frequency, exhibiting a plateau in a wide frequency region. These are characteristics for strong polymer gels with covalent cross-linked networks. For hydrogels GEL1 and GEL2 with different gelator concentrations (15 and 10 wt %, respectively), G′ of the former was 4.5 kPa, which is larger than that of the latter (3.0 kPa), indicating that a high gelator concentration enhances the mechanical performance. In addition, G′ of GEL1 (pH 2.0) and GEL3 (pH 4.0) formed from the same gelator concentrations but in different pH make even large difference. The latter is 12.0 kPa, in contrast to the former (4.5 kPa), indicating too acidic condition (pH 2.0) is not favorable for acylhydrazone formation, which weakens the cross-linking network. As shown in Figure S9 in the strain sweep, the breaking shear stain of GEL3 (70%) is higher than that of GEL1 (100%), suggesting that GEL3 showed better toughness. All these could be attributed to the enhanced cleavage of acylhydrazone linkages at strong acidic condition.

A key characteristic of dynamic covalent networks is their self-healing ability. The self-healing properties of dendronized hyperbranched hydrogels were investigated by using two hydrogels colored differently. As shown in Figure , two disks of hydrogels were cut into two halves and placed together at room temperature. After 30 min, the two pieces of the gels rejoined into one piece, and the crack at the cutting site disappeared. The healed hydrogel could be lifted against its own weight. These support the excellent self-healing ability of the dynamic covalent hydrogels, which should have originated from the dynamic nature of the acylhydrazone exchange between the acylhydrazine and aldehydes within the hydrogel network. Acylhydrazone bonds can dynamically break and regenerate, with the dissociation rate constant controlling the acylhydrazone exchange reaction, resulting in healing of the gel.

Figure 4

Self-healing of GEL1 at room temperature. One of them is colored with disperse red 1 (DR1) for clarity.

Furthermore, inheriting from the dendronized hyperbranched poly(acylhydrazone) precursors, these hydrogels also show thermoresponsive properties. They are transparent at room temperature but become opaque when heating to elevated temperature, which was followed by using temperature-varied UV–vis spectroscopy. Typical transmittance curves are shown in Figure . The Tcps for GEL1, GEL2, GEL3, and GEL4 are 35.8, 36.4, 37.2, and 37.0 °C, respectively, which are much lower compared to their corresponding polymer precursors in aqueous solutions. This decrease is attributed to the high polymer concentration within hydrogels and also high concentration of OEG dendrons in vicinity, which enhances the aggregation kinetics for collapsed dendrons. Furthermore, the hydrophilic peripheral acylhydrazines were consumed during the cross-linking reaction, resulting also in more hydrophobic polymer networks. However, the phase transition span for the hydrogels (ΔT > 5 °C) is much wider than that of the corresponding polymers (ΔT < 2 °C). This should be because the network within the hydrogels inhibits the mobility of polymer chains, resulting in lower aggregation kinetics of collapsed OEG dendrons compared to free polymer chains in solutions. Notably, Tcps of these hydrogels were not affected obviously by pH or concentration, suggesting that the collapsed dendritic OEGs fixed in the vicinity within the cross-linked network dominate efficiently the aggregation of polymer chains. All hydrogels show Tcp around physiological temperature, which make them suitable candidates for bioapplications.

Figure 5

Plots of transmittance vs temperature for GEL1, GEL2, GEL3, and GEL4. Inset: photographs of GEL1 below and above Tcp, respectively.

It was reported that the mechanical properties of the thermoresponsive hydrogels were tuned by temperature.[30,31] The temperature-dependent rheological properties of these hyperbranched polymers were investigated. GEL1 is taken as an example, and its G′ and G″ as functions of temperature from 25 to 50 °C are plotted in Figure S10. At low temperatures, G′ shows a minor change with decrease of temperature, whereas a significant decrease starts at about 33 °C, which is close to the volume transition temperature of the corresponding hydrogel. At 40 °C, G′ decreased to ∼570 Pa. We suppose that this is due to the thermally induced dehydration of the hydrogel. When the temperature was higher than the volume transition temperature of the hydrogel, the hydrogel became heterogeneous and the water moved to the surface of the hydrogels through oscillation. Similar phenomenon has also been reported with other thermoresponsive dynamic hydrogels.[32]

Conclusions

The novel dendronized hyperbranched poly(acylhydrazones) were efficiently prepared through A2 + B3 one-step strategy. These polymers exhibit characteristic constitutional dynamics, thermoresponsive behavior, and low cytotoxicity. Their thermally induced phase transitions can be tuned not only by solution pH but also by the branched architecture and addition of a third component. The surplus acylhydrazine units within the polymer periphery provide a chance for further modification to form functional materials. Cross-linking of these hyperbranched polymers with dialdehyde cross-linkers via dynamic covalent chemistry leads to the formation of hydrogels with macroporous morphology. These hydrogels exhibit excellent mechanical properties and self-healing ability, as well as temperature and pH responsive behaviors. The phase-transition temperatures of these biocompatible hydrogels are in the vicinity of physiological temperature, which makes them promising candidates for biomedical applications.

Experimental Section

Materials

Hydrazine hydrate, nitrilotriacetic acid, and glutaraldehyde were purchased from TCI (Japan). Thionyl chloride (SOCl2) and methanol (MeOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. Other reagents and solvents were purchased at reagent grade and used without further purification. Merck KGaA TLC Silica gel 60 F254 were used for thin-layer chromatography (TLC) analysis. Silica gel 60 M (Macherey-Nagel, 0.04–0.063 mm, 200–300 mesh) was used as the stationary phase for column chromatography. A2 and A2′ monomer bearing dendritic oligoethylene glycol (OEG) units[24] and the cross-linker poly(ethylene glycol) dibenzyl aldehyde (PEGDA)[33] were synthesized according to our previous works.

Instrumentation and Measurements

1H and 13C NMR spectra were recorded on a Bruker AV 500 (1H: 500 MHz, 13C: 125 MHz) spectrometer, and chemical shifts are reported as values (ppm) relative to internal Me4Si. Gel permeation chromatography (GPC) measurements were carried out on a Waters GPC e2695 instrument with 3 column set (Styragel HR3 + HR4 + HR5) equipped with a refractive index detector (Waters 2414) and N,N-dimethylformamide (DMF) (containing 1 gL–1 LiBr) as eluent at 45 °C. Multiangle light-scattering detector (Wyatt Technology Corporation, Dawn EOS 243-E) was used for the measurements. The calibration was performed with poly(methyl methacrylate) standards in the range of Mp = 2580–981 000 (Polymer Standards Service-USA Inc.). UV–vis turbidity measurements were carried out on a PE UV–vis spectrophotometer (Lambda 35) equipped with a thermocontrolled bath. Polymer aqueous solutions were placed in a spectrophotometer (path length 1 cm) and heated or cooled at different ramping rates. Absorptions of the solution at λ = 700 nm were recorded per 5 s. Cloud point (Tcp) is determined to be the one at which the transmittance at λ = 700 nm reached 50% of its initial value. The pH was measured with a Mettler-Toledo SevenCompact 220 pH meter and an InLab Flex-Micro semi-microelectrode (after 3 points calibration at pH 7.00, 4.01, and 2.00). Rheological experiments were performed on a Discovery HR-3 rheometer from TA Instruments with 12 mm diameter parallel plates at room temperature. The hydrogels disks (12 mm in diameter and polymer concentration at 10 and 15 wt %, respectively) were measured with the frequency test. The SEM studies were carried out by using a HITACHI SU-1510 instrument. After rendering conductive with a platinum coated on the cross section of lyophilized hydrogels, the morphologies of the samples were detected.

Synthesis of Dendronized Hyperbranched Poly(acylhydrazones)

Condensation polymerization of monomers with different molar ratios was carried out in a pH 4 buffer solution at room temperature. The mixture was vigorously stirred for 24 h and then dialyzed against deionized water for 5 days using a dialysis bag (MWCO, 8–14 kDa), followed by lyophilization to give the polymer in a white solid.

Preparation of Hydrogels

P1 stock solution with a certain concentration was prepared and the cross-linker PEGDA was added at room temperature to afford a hydrogel. In a typical procedure for the preparation of GEL1, a 15 wt % P1 stock solution was obtained by dissolving 30 mg of P1 in 0.2 mL pH 2 buffer solution in a 4 mL vial. A 15 wt % PEGDA stock solution was obtained by dissolving 15 mg of the cross-linker PEGDA in 0.1 mL of pH 2 buffer solution. Then, the PEGDA solution was added to the P1 solution and a transparent gel with a bright yellow color was obtained after several minutes. The self-healing properties of the hydrogels were then investigated by taking the GEL1 as an example. Two hydrogel disks, with one of them colored with disperse red 1 (DR1), were prepared. A drop of DR1 solution was added to P1 stock solution and the cross-linker PEGDA was then added at room temperature to afford a colored hydrogel.

In Vitro Cytotoxicity

Cytotoxicity of dendronized hyperbranched poly(acylhydrazones) to HeLa cell lines was determined by cell-counting kit-8 (CCK-8) assay (Dojindo Laboratories, Japan). These cells were seeded in two 96-well tissue culture plates, respectively, at a concentration of 1000 cells/well in 100 μL culture medium. The cells were cultured overnight at 37 °C in a humidified atmosphere of 5% CO2. Then, the cells were incubated with different concentrations of hyperbranched poly(acylhydrazones) aqueous solution (2 μL) for 48 h. Ten microliters of a CCK-8 solution was added to each well and the cells were incubated for another 2 h in a tissue culture incubator. The absorbance was then measured at 450 nm using a microplate reader. Relative cell viability = [(As – Ab)]/[(Ac – Ab)] × 100%. (As: sample, Ac: control, Ab: blank).