Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release

Journal Pre-proof Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release Heng An, Linmiao Zhu, Jiafu Shen, Wenjuan Li, Yong Wang, Jianglei Qin

PII:

S0927-7765(19)30745-3

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110601

Reference:

COLSUB 110601

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

6 July 2019

Revised Date:

12 September 2019

Accepted Date:

17 October 2019

Please cite this article as: An H, Zhu L, Shen J, Li W, Wang Y, Qin J, Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110601

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Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release Heng Ana#, Linmiao Zhub#, Jiafu Shena, Wenjuan Lib,c, Yong Wangb,c*, Jianglei Qina,c* a College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China. b Medical College, Hebei University, Baoding 071002, China c Key Laboratory of Pathogenesis mechanism and control of inflammatory-autoimmune diseases in

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Hebei Province, Hebei University, Baoding 071002, China. H An and L Zhu contributed equally to this work.

*Correspondence author: Y Wang, E-mail address: [email protected]

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J Qin, E-mail address: [email protected]

Graphical abstract

HN

HN

O

N NH

HN N

OH

Jo

ur

PAEH

PEG DA

H O

O H

na

NH2

H

cumulative drug release(%)

O N H y H O

N H x O

lP

O

O

100 80 60 40 20 0

PEG90 DA cross-linking PEG45 DA cross-linking

0

10

20

30

40

Time(h)

Highlights

 The hydrogel was designed from PAsp with acylhydrazide and ethoxyl groups. 1

 The hydrogels exhibited good mechanical property and self-healing property.  The hydrogels showed good biocompatibility.  The hydrogels have potential in local drug delivery.

Abstract

Self-healing hydrogels were prepared from hydrazide functionalized poly(aspartic acid)

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(PAsp). The polymer succinimide (PSI) was reacted with hydrazine and ethanolamine successively to obtain water soluble poly(aspartic acid) derivatives with hydrazide

functional groups (PAEH). The hydrogel was prepared by cross-linking PAEH with poly(ethylene glycol) dialdehyde (PEG DA) under mild conditions without addition of

-p

catalyst. The rheological property and the self-healing property of the hydrogels were investigated intensively. The in vitro toxicity experiment showed the hydrogels have

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good bio-compatibility and the doxorubicin (DOX)-loaded hydrogels showed controlled release profile. Importantly, the hydrogel can still be degraded based on

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poly(aspartic acid) backbone. The bio-degradable poly(amino acid) based on selfhealing hydrogel could have great potential application in bioscience including tissue repairing, drug loading and release.

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degradability

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Keywords: Self-healing, Hydrogel, Poly(aspartic acid), Dynamic chemical bond, Bio-

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1. Introduction:

Hydrogels are very appealing biomedical materials in applications of bio-medical

areas, ranging from drug delivery to tissue engineering with their higher water content and solid-like mechanical properties [1-4]. The hydrogels can be prepared from either physical cross-linking through intermolecular interaction or a covalent bond with each has its advantages and disadvantages [5-8]. In the past decade, the self-healing hydrogels were prepared based on intermolecular interaction [6,9-13] or reversible 2

covalent bonds like acylhydrazone bond [14-16], oxime bonds [17,18], and boronic ester bond [3,19-23], etc [24-27]. Compared to those hydrogels prepared from carbon chain polymers [10,28,29], the hydrogels prepared from heterochain polymers [14,30] or biodegradable polymers like polysaccharide [16,31], chitosan [26,32], hyaluronic acid [33,34] should have better bio-compatibility and bio-safety. It is understandable the hydrogels prepared from poly(aspartic acid) and their derivatives are very fit for bio-applications without worrying about the by-effect [13,32,35-38]. Based on previous report, the poly(succinimide) (PSI) prepared from aspartic acid can be modified by a variety of amine to form poly(aspartic acid) based hydrogels for bio-medical

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applications [37,39,40]. But the poly(aspartic acid) based self-healing hydrogel has not been studied even if the hydrazide groups, aldehyde groups [35] and catechol units [40] can be easily imported to modified poly(aspartic acid) structures.

In this research, self-healing hydrogel was designed from biodegradable

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poly(aspartic acid) with hydrazide functional groups and ethoxyl groups. The hydrazide group was imported onto the poly(aspartic acid) and partially hydrazide functionalized

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hydroxylethyl grafted poly(aspartic acid) (PAEH) was prepared. Then the PAEH was cross-linked by poly(ethylene glycol) dialdehyde (PEG DA) to prepare self-healing hydrogel with reversible hydrazone bond. The poly(aspartic acid) based hydrogel

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showed good mechanical property and self-healing property. The easy accessibility of the poly(aspartic acid) based PAEH endows this self-healable hydrogel wide potential

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application areas. The in vitro biotoxicity investigation revealed that the PAsp based self-healable hydrogel have good bio-compatibility and have great potential bioscience

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and biotechnology application as drug delivery agent and tissue repairing.

2. Experimental

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2.1. Materials

L-Aspartic acid (L-Asp) and 1,3,5-trimethylbenzene were purchased from

Macklin biochemical Co Ltd. Poly(ethylene glycol) (PEG45, Mn=2k; PEG90, Mn=4k) were supplied by Guangfu fine chemical research institute and used to prepare PEG DA according to literature [14]. Tetramethylene sulfone was purchased from Sinopharm Chemical Reageng Co. Ltd. Phosphoric acid was supplied by Huadong Reagent Co. 3

Aminoethanol, hydrazine hydrate (80%), acetone and other solvent including N,NDimethylformamide (DMF), Dimethyl sulfoxide (DMSO) and methanol etc. were supplied by Kermal Chemical Reagent Co. Ltd. and used as received. 2.2. Synthesis of ethanolamine grafted poly(aspartic acid) with partially functionalized hydrazide (PAEH) First, the PSI (Mn=1.2×104) was synthesized by thermal polycondensation reaction of L-Asp catalyzed by H3PO4 according to previous study [35]. Then the PSI was used to react with hydrazine and ethanolamine successively to prepare PAEH with following

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procedures. First, 1.94 g (20 mmol) PSI was dissolved in 10 mL of DMSO in a 50 mL flask, then 0.25 g hydrazine hydrate (80%, 4 mmol) was added into the flask. The flask

was deoxidized and sealed after filled with N2, then the flask was immersed in a 60 oC oil bath for 24 h under continuous stirring. The intermediate was precipitated in

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methanol and washed three times, then the reaction ratio of the intermediate was characterized by 1H NMR after dried in vacuum oven until constant weight.

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The intermediate was dissolved in DMSO again and 3.05 g (50 mmol) 2aminoethanol was added into the flask. The oxygen was removed again and the reaction

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was performed for another 12 h at 60 oC under N2 protection. The solution was precipitated in acetone and washed twice, the product of PAEH was collected after

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dried under vacuum (overall yield: 73%). The composition of the PAEH was characterized by 1H NMR and confirmed by FT-IR. 2.3. Preparation of hydrogels with hydrazone bond linkage

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The hydrogels with PAsp backbone and hydrazone bond linkage were prepared in deionized water. First, the PAEH was dissolved in water to form a solution with 10%

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concentration. Then the cross-linkers of PEG DA with various molecular weight were dissolved in deionized water to form 10% solution. The two solutions were then mixed together with 1:1 group ratio of hydrazide to aldehyde. The hydrogel formation at 25 °C were tracked on rheometer right after the mixing of two solutions. The hydrogels for rheological and self-healing studies were formed directly in a round mould at room temperature without additional stimulus. 4

2.4. Rheological analysis The gelation process and mechanical property of hydrogels were determined on a rheometer (TA AR2000ex) at 25 oC. After the hydrogels were formed and incubated for 24 h to the equilibrium state, the mechanical property was characterized with increasing frequency from 0.1 rad∙s-1 to 100 rad∙s-1. The storage modulus (G′) and loss modulus (G″) were monitored as a function of frequency. The gelation process was carried out right after mixing of PAEH solution and PEG DA solution, the frequency for this characterization was fixed to 1 rad s-1 and the strain was fixed to 1%.

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2.5. Self-healing property of PAsp based hydrogels The hydrazone bond is a dynamic bond that can endow the hydrogels with selfhealing property as reported previously[19,30,41]. The hydrogel in this study also

exhibited self-healing properties due to the dynamic bonds. The hydrogel plate was cut

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into 4 pieces and the pieces were put back into the mould with close contact. The healing result was confirmed by suspended to gravity and under stretching.

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2.6. Gel-sol-gel transition and degradation of the self-healing hydrogels under various conditions

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The reversible characteristics of dynamic covalent bond endows the hydrogels with multi-triggered transition [29], and the PAsp backbone of the hydrogels endowed

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the hydrogel with biodegradability [37]. The pH of the hydrogel was regulated to 3.0 by HCl and shaken to see the gel-sol transition, then the sol was neutralized by N(C2H5)3 to observe the sol-gel transition. The gel-sol-gel transition was confirmed by

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vial leaning and recorded by a digital camera. The PAsp backbone of PAEH is biodegradable, so the hydrogel was expected to be degradable through degradation of PAsp.

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Previous research indicated the PAsp can be degraded slowly under neutral conditions, while mild acid or base can accelerate the degradation rate. In this research, mild base of NaHCO3 and Na2CO3 was used to degrade the self-healable hydrogel. 0.1 g 10% NaHCO3 and Na2CO3 solutions were added onto the PAsp based hydrogel (1 g) in a glass vial and sealed to observe the degradation process. Since the volume of the hydrogel kept intact, the gel-sol transition of the hydrogels indicated the degradation of 5

hydrogel through cleavage of PAsp backbone. The gel-sol transition was confirmed by vial leaning and recorded by a digital camera. 2.7. Microstructure of PAsp based hydrogel The hydrogels were prepared with different cross-linkers of PEG45 DA and PEG90 DA. For the preparation of samples, the hydrogels were lyophilized and broken in liquid nitrogen. The cross-sectional morphology of the hydrogels was observed under a JSM7500 SEM apparatus after coated with Au. 2.8. Thermal stability analysis of the PAsp based hydrogel

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The thermal stability of PAEH polymer, PEG DA cross-linker and hydrogel was determined by TGA analysis. The PAEH polymer, PEG45 DA cross-linker and freeze-

dried hydrogel were put in the crucible and subjected to the TGA analysis. The temperature was increased from 25 oC to 800 oC with a heating rate of 20 oC min-1

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under the N2 protection. The stability was compared according to the TGA curves.

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2.9. In vitro release of PAsp based hydrogel loaded DOX·HCl

The in vitro release of drugs from the hydrogels was investigated with different

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cross-linkers. First, 3 mg of DOX·HCl was dissolved in PEG DA solution, then 10% PEAH was added to form an 1 g hydrogel plate and incubated for 12 h. Then the hydrogel was placed into a dialysis bag, and immersed in 200 mL pH 7.4 PBS solution.

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At predetermined intervals, 2 mL PBS was pipette to measure the absorbance of the solutions at 485 nm, and 2 mL fresh PBS solution was added. The following formula

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was used tocalculates the release ratio of DOX·HCl.

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Cumulative DOX release (%) =

n 1

Ve Ci  V0Cn i

m

100%

Where Ve is the volume of released solution collected for each time point (2 mL), Vo is the volume of origin buffer solutions (200 mL), Ci is the DOX·HCl concentration in the release medium at displacement time i, and n is the total times of displacement. Cn data obtained represent the mean value of three replicates with a standard deviation.

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2.10. In vitro cytotoxicity tests of the hydrogels The cytotoxicity was evaluated by determining the viability of cells exposed to the diluted hydrogel solution using a quantitative 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) assay [30]. The hydrogel solutions were subjected to the evaluation. JB6 P+ cell and HeLa cells were seeded into a 96-well microculture plate at a density of 1 × 104 cells per well and incubated for 24 h at 37 °C in a 5% CO2 humidified incubator to obtain a confluent monolayer of cells. Thereafter, hydrogel solutions were pipetted into the wells. Experiments were repeated for three

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times and at least six samples were included in each experiment. After 24 h, the hydrogel solutions were removed following the manufacturer’s instruction. The absorbance of each well was measured at a wavelength of 490 nm using a microplate

reader (Bio-Tek, Synergy H1, USA). H2O was loaded as a negative control and 1%

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triton X-100 as a positive control. The samples with a relative cell viability of less than 70% are deemed to be cytotoxic.

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2.11. Characterizations

The structure and the composition of the polymers were determined by 1H NMR

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characterization, which were carried out on a Bruker 600 MHz spectrometer (Avance III, Bruker) with DMSO-D6 as solvent. The FT-IR spectra of the polymers were

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obtained on a Varian 600 Fourier-transform infrared (FT-IR) spectrometer. Rheological properties of the hydrogels were measured on a TA AR2000ex rheometer with oscillatory mode at 25 oC between a pair of 25 mm parallel aluminium plates. The

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morphology of the hydrogels was observed on a field-emission scanning electron microscope (FE-SEM, JSM-7500), the operating voltage of the SEM was 10 kV and

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the images were recorded by a CCD camera. The hydrogels were freeze dried and broke in liquid nitrogen to preserve the original morphology. The samples were mounted on an aluminum specimen mounts and coated with Au for observations.

3. Result and discussion

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3.1. Preparation of partially hydrazide functionalized ethanol grafted PAsp (PAEH) The preparation of PAEH was carried out through two step ring opening reaction of PSI by hydrazine and ethanolamine successively in DMSO, the design was shown in the Fig. 1(a). By comparing the peak area of the 1H NMR, the ring opening ratio was almost consistent with hydrazine feeding ratio at first step and the excess of ethanolamine can consume all the PSI rings. The 1H NMR spectra of PSI and PAEH with 20% hydrazide ratio are shown in Fig. 1(b). After reacted with hydrazine and ethanolamine successively, the peak at 5.25 ppm represented the PSI ring disappeared

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completely; at same time, new peak appeared at 4.45-4.75 ppm (a′ and a″) proved all the PSI rings were opened by hydrazide and ethanolamine. Based on completely consumption of hydrazine, this sample is named as PAEH-20 for convenience. The

PAEH-6 with 6% ratio of hydrazide groups was also synthesized for comparison. The H NMR of PEG dialdehyde (PEG DA) was shown in Fig. S1.

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1

The structure of the polymers at different stages were also characterized by FT-IR, 1

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as shown in Fig. 1(c). The absorbance of PSI at 1712 cm-1 and the shoulder at 1785 cmdisappeared completely after two step aminolysis; at the same time, new absorbance

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at 1649 cm-1 and 1635 cm-1 appeared proved all the PSI rings have been opened by hydrazine and ethanolamine during the two step reactions. At the same time, because the hydrazide and ethanol pendant groups were imported to the PAsp backbone, the

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PAEH become water soluble and shown brown colour. All above result, proved the ethanol functionalized PAsp with hydrazide group has been prepared successfully and

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ready to be used to prepare self-healing hydrogels with bio-degradability.

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Fig. 1. Synthetic route for the preparation of PAEH (a).1H NMR spectra (b) and FT-IR spectra (c) of PSI and the PAEH-20.

3.2. Preparation of dynamic hydrogels with PEG DA as cross-linkers The hydrazide group on PAEH could react with a variety of carbonyl groups like aldehyde group, ketone group to form dynamic hydrazone bond [28,42-45]. In this study, PEG DA (Fig. S1) was used as cross-linker based on its good bio-compatibility, as shown in Fig. 2(a). The hydrogel was formed in about 50 min when PEG45 DA was added PAEH-20 solution, as shown in Fig. 2(b). The gelation time is too long for injectable hydrogel used in drug loading and tissue engineering, but when gelator concentration was increased to 20%, hydrogel formed within 10 min, as determined by

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rheology study (Fig. S2). Because the PEG45 DA ratio was not too high, the formed hydrogel was still brown. However, the hydrogel was transparent with patterns on the back can be figured out clearly in Fig. 2(c). O

O

N H y O

N H x O

O

H

H

O

PEG DA

HN

HN

O H HN N

NH2

H O

N NH

(c)

OH

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PAEH

lP

(b)

-p

(a)

PEG DA

.

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Fig. 2. Mechanism of hydrogel preparation (a) and images for preparation of hydrogel form PAEH and PEG45 DA (b), and the transparent brown hydrogel plate (c).

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3.3. Characterization of mechanical properties of hydrogel The formation and the mechanical property of the dynamic hydrogels with PAsp

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segments were characterized by rheological study. Fig. 3(a) shows storage modulus (G′) and loss modulus (G″) of the hydrogel dependant on frequency with various compositions. Both hydrogels prepared from PAEH-20 with PEG45 DA and PEG90 DA showed solid characteristic of G′>G″ in whole frequency range of 0.1 rad/s - 100 rad/s. Moreover, the G′ were almost independent to frequency, indicated high dimensional stability of the hydrogels, as shown in Fig. 3(a). The G′ of the hydrogel with PEG45 DA cross-linking was a little higher than that with PEG90 DA cross-linking because the 9

PEG45DA cross-linked hydrogel had higher cross-linking density. The hydrogel prepared from PAEH-6 cross-linked by PEG45 DA also showed solid characteristic, but the G′ was much lower than that prepared from PAEH-20, as shown in Fig. 3(b). Since the G′ of hydrogel prepared from PAEH-20 was much higher than that from PAEH-6, intensive study was carried out for PAEH-20 hydrogels. The strain scan of the hydrogels was also carried out to investigate the flexibility of the hydrogels, as shown in Fig. 3(c), the G′ of the hydrogel dropped indicated the broken down at above 200% strain, proved good flexibility of the bio-degradable hydrogels. The compression investigation was carried out to confirm the flexibility of the hydrogel, as shown in Fig.

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3(d), when 50 g weight was applied onto PEG90 DA cross-linked elastic hydrogel, the hydrogel can keep its shape with small deformation. Then the hydrogel was pressed gradually to 20% of its original height by additional pressure and kept for 1 min; after

removing the pressure, the hydrogel recovered to its original size and shape without any noticeable crack. The flexibility of the hydrogels ensured its application under

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strain without worrying about breaking of the hydrogel as artificial organs or tissues,

3

G' PEG45 DA

G',G''/Pa

G',G''/Pa

G" PEG45 DA G' PEG90 DA G" PEG90 DA 2

2

10

1

na

10

1 -1

0

10

1

10

10

 /rad S

4

0

10

2

10

I

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10

G' G''

1

10

0

1

10

2

Strain (%)

0

1

10

 /rad S

-1

2

10

10

(d)

3

10

-1

10

-1

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10

1

10

10

G' G" /Pa

3

10

2

10

10

(c)

10

-1

3

10

10

4

(b)10

*/Pa s

4

10

lP

(a)

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etc.

10

10

II

III

Fig. 3. Rheology curves of hydrogel prepared from PAEH-20 (a) and HAEH-6 (b) with PEG45 DA cross-linking; the strain scan of the PAEH-20 cross-linked by PEG90 DA (c) and the compression experiment (d) (I: 50 g compression; II: pressed to 20% height; III: pressure released.).

3.4. Self-Healing property of the hydrogels prepared from PAsp derivatives With dynamic covalent hydrazone bond in the cross-linked network, the hydrogels should have self-healing ability. The hydrogel plates were divided into four parts, then the four parts were put back into the original mould with close contact along the cut line. The graphite oxide was dispersed in some hydrogel plate to indicate the interface of the hydrogel part. After contacted for 24 h, the hydrogel plates were subjected to

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gravity and stretching to see whether the self-healing have occurred. The results of the self-healing are illustrated in Fig. 4. The hydrogels prepared from PAEH-20 and PEG45 DA self-healed successfully with no scar observed, as shown in Fig. 4(a-c). However, the interface with black graphite oxide is clearly figured out, which indicated the

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hydrogel plate was self-healed from four part. The hydrogel bars were also cut into several pieces and contacted in the mould, as expected, the hydrogel pieces self-healing into a whole bar with alternative colors and cannot split along the interface under

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stretching, as shown in Fig. 4(d). The hydrogel prepared from PAEH-20 and PEG90 DA showed the same result, as shown in Fig. 4(e-g). Because of decreased PAEH ratio, the

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colour of the hydrogel become negligible. The mechanism of self-healing through hydrazone exchange is shown in Fig. 4(h). Although low G′ limited its potential application, the hydrogel prepared from PAEH-6 and PEG45 DA can also self-heal

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under neutral conditions without any additional stimulus (Fig. S3). Based on poly(aspartic acid) nature of the PAEH, this kind of hydrogel exhibited improved

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potential applications in areas related to drug loading and controlled delivery, medical

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adhesives and artificial organs, etc.

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Fig. 4. Self-healing of hydrogel prepared from PAEH-20 with PEG45 DA cross-linking (a-c); the

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self-healed hydrogel bar under stretching (d); the self-healing of hydrogel plate with PEG90 DA cross-linking (e-g); and the schematic illustration of hydrazone exchange attributed to the self-

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healing (A) and degradation (B) of the PAEH based hydrogel (h).

3.5. pH and group ratio triggered gel-sol-gel transition and degradation of the hydrogel

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It was reported the coupling reactions between hydrazide group and aldehyde group are pH-dependent, so the hydrogel can be cleaved by regulating the pH by HCl

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and hydrogel can be re-obtained reversibly at neutral conditions. The pH triggered gelsol-gel transition of the hydrogel with PEG45 DA cross-linking is shown in Fig. 5 (a), when the pH of the hydrogel was regulated to 3.0, the hydrogel degraded gradually into

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a viscous solution; when the N(C2H5)3 was added to regulate the pH of the solution, hydrogel re-obtained again overnight. Since the pH triggered gel-sol-gel is reversible, the gel-sol-gel transition of hydrogel can go several cycles. The hydrogel with reversible cross-linking can be cleaved by mono-functional compounds and gel-sol-gel transition can be regulated by regulating the group ratio of cross-linkers [28,29]. when 3 times excess of PEG45 DA was added into the hydrogel, 12

the hydrogel degraded into a solution overnight; when the PAEH-20 was added into the solution to regulate the group ratio to 1:1, hydrogel re-obtained again, the photograph of this process is also illustrated in Fig. 5(a). Since no any by-product was produced during the group ratio triggered gel-sol-gel transition, this process has more practical application in hydrogel recycling and bio-related application areas. (a)

Acid

PEG DA PAEH

Neutral (b) HN NH3

O

O

H

H

Acid Neutral

H O

O H

N NH

HN N

PEG DA PAEH

O H

H

HN N

O

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O

Fig. 5. The reversible gel-sol transition triggered by pH and group ratio (a); the mechanism of the reversible gel-sol transition (b).

The PAEH is bio-degradable poly(aspartic acid) derivative, so the hydrogel was

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expected to be bio-degradable based on degradability of the PAEH segment. Previous

study indicated the degradation rate of PAsp was very slow and mild base can accelerate

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the degradation reaction. In this research, the hydrogel was expected to be degradable under mild base in observed time scale based on degradability of PAEH, as shown in

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Fig. 4(h). When 0.1 mL Na2CO3 and NaHCO3 solution were added into 1 g of PEG45 DA cross-linked self-healing hydrogel, the Na2CO3 degraded the hydrogel into liquid in 7 days; however, the NaHCO3 did not degrade the hydrogel until about one month,

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as shown in Fig. 4(h) and Fig. S4. This study proved although the PAsp backbone was modified by hydrazine and ethanolamine, the degradability nature of PAsp backbone was not changed, which make this kind of self-healing hydrogel very fit for bio-

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applications.

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3.6. The morphology of the hydrogels The morphology of the self-healable hydrogels was observed by SEM, the

hydrogels were dried by lyophilisation and coated with Au before observation. The porous structure of the hydrogel surfaces can be clearly observed in the Fig. 6. The SEM images of the hydrogels prepared from PAEH-20 and PEG45 DA cross-kinking were shown in the Fig. 6(a), under the magnification of 100 times, the pores of the gel can be clearly observed. Microporous structure of hydrogel provide an ideal place for 13

the drug to be stored and the hydrogel was suitable for transportation of drugs. However, when PEG90 DA is used as the cross-linking agent, the surface of the hydrogel was wrinkled under 200 times magnification, but the pores on the surface of the gel can also be figured out. The wall of the hydrogel with PEG90 DA cross-linking was significantly thinner with similar pore sizes (Fig. S5), this is reasonable because the cross-linking density of the hydrogel was reduced and the result was consistent with rheological study. These properties make the hydrogel versatility in potential bio-application like drug release and cell growth.

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a

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Fig. 6. The SEM images of the hydrogel prepared form PAEH-20 with PEG45 DA (a) and PEG90 DA cross-linking.

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However, with degradable poly(aspartic acid) backbone, the PAEH also showed low thermal stability, which was characterized by thermogravimetric analysis (TGA). The TGA curves of the dry hydrogels compared to PAEH-20 and PEG45 DA are shown

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in Fig. S6. It can be seen that the polymer PAEH has three step weight loss, the weight loss around 100 oC was due to the evaporation of water because the PAEH was very

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hydrophilic. The PAEH began to decompose from 200 oC. After 280 oC the weight loss lasted for pretty large temperature range, which indicated the complicated intermolecular reaction prevented the evaporation of the products. (Fig. S6, black line).

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However, the PEG45 DA showed sharp one step weight loss from 375 oC to 475 oC (Fig. S6, red line). While the hydrogel decomposition was divided into three steps, the first step was consistent with the weight loss process of PAEH. Then the weight loss followed the decomposition curve of PEG45 DA indicated the decomposition products evaporated with that of PEG45. At last step, the curve was almost overlapped with the TGA curve of PAEH, this was because only the PAHy segments reacted with the PEG 14

DA and large part of the PAEH still showed its own decomposition characteristic (Fig. S6.green line). 3.7. In vitro cytotoxicity tests The bio-compatibility is one of the most attractive advantages of poly(aspartic acid) materials, which is very important for potential bio-applications. The cytotoxicity of the hydrogel solution under gelation concentration were evaluated by determining the viability of cells using MTT assay [30]. The sterile solution of 0.1%-1% below gelation concentration was characterized. The cell viability was determined after incubated for 24 and 48 at 37 °C with each dosage was replicated in 6 wells. As shown in Fig. 7, the

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hydrogel solutions are nontoxic to ether JB6 P+ cells or HeLa cells with the viability was higher than 90%, indicated the hydrogels have excellent bio-compatibility and can be used as drug loading and delivery candidate.

100

24 h 48 h

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100

-p

(b) 150 Cell Viability (%)

24 h 48 h

50

0

50

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Cell Viability (%)

(a) 150

0

Control

10

100

Triton

Control

10

100

Triton

Gelator Concentration (μg/mL)

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Gelator Concentration (μg/mL)

Fig. 7. In vitro cytotoxicity of hydrogel solutions to JB6 P+ cell (a) and HeLa cells (b).

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3.8. In vitro DOX release test of the hydrogel As potential application in bioscience and biotechnology, the drug loading and

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release property of the hydrogels with PEG45 DA and PEG90 DA as cross-linkers were investigated with DOX∙HCl as the model drug because of its anticancer applications. Figure 8 shows the cumulative drug release profile of the hydrogels at pH 7.4. The drug released fast in first 12 h and then the release rate decreased. The release rate of the PEG90 DA cross-linked hydrogel (Fig. 8, black line) was higher than PEG45 DA crosslinked hydrogel (Fig. 8, red line) possibly because of lower cross-linking density. The controlled release behavior lasted for about 36 h, which means this kind of self-healable 15

hydrogels have enhanced application in drug release. With good bio-compatibility and bio-degradability, this hydrogel have great potential application in drug delivery and tissue engineering.

80 60 40 20

PEG90 DA cross-linking PEG45 DA cross-linking

0

10

20

Time (h)

30

40

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0

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cumulative drug release(%)

100

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Fig. 8. In vitro DOX release of hydrogels with PEG90 DA (black) and PEG45 DA (red) cross-linking

4. Conclusions

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New kind of self-healing hydrogels were prepared based on bio-degradable PAsp derivatives through hydrazone bond. The hydrogels formed and self-healed without any additional stimulus. Water soluble PAsp derivative bearing hydrazide groups was

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synthesized through two step ring opening of PSI and then used to prepare self-healable hydrogels. This kind of self-healable hydrogels showed good bio-compatibility and

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biodegradability, which endow the hydrogel with great potential application in bioscience and bio-technology including artificial organs and drug loading and delivery,

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etc.

Acknowledgement This research was funded by the Natural Science Foundation of Hebei Province (B2017201019, B2018201140, H2019201084); the Department of Education, Hebei Province (No. QN2017014) and Hebei University (2017011, 2017014); Post-graduate’s Innovation Fund Project of Hebei University (hbu2019ss010). 16

References [1] T. R. Hoare, D. S. Kohane, Hydrogels in drug delivery: Progress and challenges, Polymer 49 (2008), pp.1993-2007. [2] W. Wang, Y. Zhang, W. Liu, Bioinspired fabrication of high strength hydrogels from noncovalent interactions, Prog. Polym. Sci.71 (2017), pp.1-25 [3] Y. Guan, Y. Zhang, Boronic acid-containing hydrogels: synthesis and their applications, Chem. Soc. Rev. 42 (2013), pp. 8106-8121. [4] V. Yesilyurt, M.J. Webber, E.A. Appel, C. Godwin, R. Langer, D.G. Anderson, Injectable Self-Healing Glucose-Responsive Hydrogels with pH-Regulated Mechanical Properties, Adv. Mater. 28 (2015), pp. 86-91.

ro of

[5] Z. Wei, J.H. Yang, J. Zhou, F. Xu, M. Zrinyi, P.H. Dussault, Y. Osada, Y.M. Chen, Selfhealing gels based on constitutional dynamic chemistry and their potential applications, Chem. Soc. Rev. 43 (2014), pp. 8114-8131.

[6] A. Phadke, C. Zhang, B. Arman, C.-C. Hsu, R.A. Mashelkar, A.K. Lele, M.J. Tauber, G.

4383-4388.

-p

Arya, S. Varghese, Rapid self-healing hydrogels, P. Natl. Acad. Sci. USA 109 (2012), pp.

[7] Y. Yang, X. Ding, M.W. Urban, Chemical and physical aspects of self-healing materials,

re

Prog. Polym. Sci. 49-50 (2015), pp. 34-59.

[8] D.Y. Wu, S. Meure, D. Solomon, Self-healing polymeric materials: A review of recent developments, Prog. Polym. Sci. 33 (2008), pp. 479-522.

lP

[9] M. Kohei, N. Masaki, T. Yoshinori, H. Akira, Self-Healing, Expansion-Contraction, and Shape-Memory Properties of a Preorganized Supramolecular Hydrogel through Host and Guest Interactions, Angew. Chem. Int. Edit. 54 (2015), pp. 8984-8987.

na

[10] M. Zhang, D. Xu, X. Yan, J. Chen, S. Dong, B. Zheng, F. Huang, Self-Healing Supramolecular Gels FormedbyCrown Ether Based Host–Guest Interactions, Angew. Chem. Int. Edit. 124 (2012), pp. 7117-7121.

ur

[11] Q. Chen, L. Zhu, H. Chen, H. Yan, L. Huang, J. Yang, J. Zheng, A Novel Design Strategy for Fully Physically Linked Double Network Hydrogels with Tough, Fatigue Resistant, and Self-Healing Properties, Adv. Funct. Mater. 25 (2015), pp. 1598-1607.

Jo

[12] R. Tepper, S. Bode, R. Geitner, M. Jaeger, H. Goerls, J. Vitz, B. Dietzek, M. Schmitt, J. Popp, M.D. Hager, U.S. Schubert, Polymeric Halogen-Bond-Based Donor Systems Showing Self-Healing Behavior in Thin Films, Angew. Chem. Int. Edit. 56 (2017), pp. 4047-4051.

[13] G. Li, J. Wu, B. Wang, S. Yan, K. Zhang, J. Ding, J. Yin, Self-Healing Supramolecular Self-Assembled Hydrogels Based on Poly(l-glutamic acid), Biomacromolecules 16 (2015), pp. 3508-3518.

17

[14] G.H. Deng, F.Y. Li, H.X. Yu, F.Y. Liu, C.Y. Liu, W.X. Sun, H.F. Jiang, Y.M. Chen, Dynamic Hydrogels with an Environmental Adaptive Self-Healing Ability and Dual Responsive Sol-Gel Transitions, ACS Macro Lett. 1 (2012), pp. 275-279. [15] R. Chang, X. Wang, X. Li, H. An, J. Qin, Self-Activated Healable Hydrogels with Reversible Temperature Responsiveness, ACS Appl. Mater. Inter. 8 (2016), pp. 2554425551. [16] Z. Wei, J.H. Yang, Z.Q. Liu, F. Xu, J.X. Zhou, M. Zrínyi, Y. Osada, Y.M. Chen, Novel Biocompatible Polysaccharide-Based Self-Healing Hydrogel, Adv. Funct. Mater. 25 (2015), pp. 1352-1359. [17] S. Mukherjee, M.R. Hill, B.S. Sumerlin, Self-healing hydrogels containing reversible oxime crosslinks, Soft Matter 11 (2015), pp.6152-6161.

ro of

[18] J. Collins, M. Nadgorny, Z. Xiao, L.A. Connal, Doubly Dynamic Self-Healing Materials

Based on Oxime ClickChemistry and Boronic Acids, Macromol. Rapid Commun. 38 (2017), pp.1-25

[19] O.R. Cromwell, J. Chung, Z. Guan, Malleable and Self-Healing Covalent Polymer

Networks through Tunable Dynamic Boronic Ester Bonds, J. Am. Chem. Soc. 137 (2015),

-p

pp. 6492-6495.

[20] C.C. Deng, W.L.A. Brooks, K.A. Abboud, B.S. Sumerlin, Boronic Acid-Based Hydrogels

re

Undergo Self-Healing at Neutral and Acidic pH, ACS Macro Lett. 4(2) (2015), pp. 220-224. [21] R.W. Guo, Q. Su, J.W. Zhang, A.J. Dong, C.G. Lin, J.H. Zhang, Facile Access to Multisensitive and Self-Healing Hydrogels with Reversible and Dynamic Boronic Ester and

lP

Disulfide Linkages, Biomacromolecules 18 (2017), pp. 1356-1364. [22] L. He, D.E. Fullenkamp, J.G. Rivera, P.B. Messersmith, pH responsive self-healing

7499.

na

hydrogels formed by boronate-catechol complexation, Chem. Commun. 47 (2011), pp. 7497-

[23] M. Vatankhah-Varnoosfaderani, S. Hashmi, A. GhavamiNejad, F.J. Stadler, Rapid selfhealing and triple stimuli responsiveness of a supramolecular polymer gel based on boron-

ur

catechol interactions in a novel water-soluble mussel-inspired copolymer, Polym. Chem. 5 (2014), pp. 512-523.

[24] T.C. Tseng, L. Tao, F.Y. Hsieh, Y. Wei, I.M. Chiu, S.H. Hsu, An Injectable, Self-Healing

Jo

Hydrogel to Repair the Central Nervous System, Adv. Mater. 27(23) (2015), pp. 3518-3524.

[25] A. Chao, I. Negulescu, D. Zhang, Dynamic Covalent Polymer Networks Based on Degenerative Imine Bond Exchange: Tuning the Malleability and Self-Healing Properties by Solvent, Macromolecules 49 (2016), pp. 6277-6284.

[26] Y. Zhang, C. Fu, Y. Li, K. Wang, X. Wang, Y. Wei, L. Tao, Synthesis of an injectable, selfhealable and dual responsive hydrogel for drug delivery and 3D cell cultivation, Polym. Chem. 8 (2017), pp. 537-544. 18

[27] T. Kakuta, Y. Takashima, M. Nakahata, M. Otsubo, H. Yamaguchi, A. Harada, Preorganized Hydrogel: Self-Healing Properties of Supramolecular Hydrogels Formed by Polymerization of Host-Guest-Monomers that Contain Cyclodextrins and Hydrophobic Guest Groups, Adv. Mater. 25 (2013), pp. 2849-2853. [28] X. Wang, G. Bian, M. Zhang, L. Chang, Z. Li, X. Li, H. An, J. Qin, R. Chang, H. Wang, Self-Healable Hydrogels with Cross-Linking Induced Thermo-Responsiveness and MultiTriggered Gel-Sol-Gel Transition, Polym. Chem. 8 (2017), pp. 2872-2880. [29] R. Chang, H. An, X. Li, R. Zhou, J. Qin, Y. Tian, K. Deng, Self-healable polymer gels with multi-responsiveness of gel-sol-gel transition and degradability, Polym. Chem. 8 (2017), pp. 1263-1271. [30] M. Shan, C. Gong, B. Li, G. Wu, A pH, glucose, and dopamine triple-responsive, self-

ro of

healable adhesive hydrogel formed by phenylborate-catechol complexation, Polym. Chem. 8 (2017), pp. 2997-3005.

[31] X. Yang, G. Liu, L. Peng, J. Guo, L. Tao, J. Yuan, C. Chang, Y. Wei, L. Zhang, Highly

Efficient Self-Healable and Dual Responsive Cellulose-Based Hydrogels for Controlled Release and 3D Cell Culture, Adv. Funct. Mater. 27 (2017), pp. 1703174.

-p

[32] F.-Y. Hsieh, L. Tao, Y. Wei, S.-h. Hsu, A novel biodegradable self-healing hydrogel to induce blood capillary formation, NPG Asia Materials 9 (2017), pp. 363.

re

[33] H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Injectable in situ forming biodegradable chitosan–hyaluronic acid based hydrogels for cartilage tissue engineering, Biomaterials 30 (2009), pp. 2499-2506.

lP

[34] H.-K.T. Liang, X.-S. Lai, M.-F. Wei, S.-H. Lu, W.-F. Wen, S.-H. Kuo, C.-M. Chen, W.Y.I. Tseng, F.-H. Lin, Intratumoral injection of thermogelling and sustained-release carboplatin-loaded hydrogel simplifies the administration and remains the synergistic effect

na

with radiotherapy for mice gliomas, Biomaterials 151 (2018), pp. 38-52. [35] C. Lu, X. Wang, G. Wu, J. Wang, Y. Wang, H. Gao, J. Ma, An injectable and biodegradable hydrogel based on poly(α,β-aspartic acid) derivatives for localized drug delivery, J. Biomed.

ur

Mater. Res. Part A 102 (2014), pp. 628-638. [36] S. Roy, A. Baral, A. Banerjee, An Amino-Acid-Based Self-Healing Hydrogel: Modulation of the Self-Healing Properties by Incorporating Carbon-Based Nanomaterials, Chem. Mater.

Jo

19 (2013), pp. 14950-14957.

[37] D. Juriga, K. Nagy, A. Jedlovszky-Hajdu, K. Perczel-Kovach, Y.M. Chen, G. Varga, M. Zrinyi, Biodegradation and Osteosarcoma Cell Cultivation on Poly(aspartic acid) Based Hydrogels, ACS Appl. Mater. Inter. 8 (2016), pp. 23463-23476.

[38] T. Feng, H. Tian, X. Dong, M.H.-W. Lam, H. Liang, X. Chen, pH-sensitive OEIpoly(aspartic acid-b-lysine) as charge shielding system for gene delivery, J. Control. Release 213 (2015), pp. 104. 19

[39] R. Zhang, Z. Huang, M. Xue, J. Yang, T. Tan, Detailed characterization of an injectable hyaluronic acid-polyaspartylhydrazide hydrogel for protein delivery, Carbohyd. Polym. 85 (2011), pp. 717-725. [40] C. Gong, C. Lu, B. Li, M. Shan, G. Wu, Injectable dopamine-modified poly(alpha,betaaspartic acid) nanocomposite hydrogel as bioadhesive drug delivery system, J. Biomed. Mater. Res. Part A 105 (2017), pp. 1000-1008. [41]J.J. Cash, T. Kubo, A.P. Bapat, B.S. Sumerlin, Room-Temperature Self-Healing Polymers Based on Dynamic-Covalent Boronic Esters, Macromolecules 48 (2015), pp. 2098-2106. [42] J. Kalia, R.T. Raines, Hydrolytic Stability of Hydrazones and Oximes, Angew. Chem. Int. Edit. 47 (2008), pp. 7523-7526. [43] N. Kuhl, S. Bode, R.K. Bose, J. Vitz, A. Seifert, S. Hoeppener, S.J. Garcia, S. Spange, S.

ro of

van der Zwaag, M.D. Hager, U.S. Schubert, Acylhydrazones as Reversible Covalent Crosslinkers for Self-Healing Polymers, Adv. Funct. Mater. 25 (2015), pp. 3295-3301.

[44] Z. Guo, W. Ma, H. Gu, Y. Feng, Z. He, Q. Chen, X. Mao, J. Zhang, L. Zheng, pHSwitchable and self-healable hydrogels based on ketone type acylhydrazone dynamic covalent bonds, Soft Matter 13 (2017), pp. 7371-7380.

-p

[45]N. Yuan, L. Xu, H. Wang, Y. Fu, Z. Zhang, L. Liu, C. Wang, J. Zhao, J. Rong, Dual

Physically Cross-Linked Double Network Hydrogels with High Mechanical Strength,

Jo

ur

na

lP

Inter. 8 (2016), pp. 34034-34044.

re

Fatigue Resistance, Notch-Insensitivity, and Self-Healing Properties, ACS Appl. Mater.

20