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Home > Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release
Self-healing PEG-poly(aspartic acid) hydrogel with rapid shape recovery and drug release
Heng Ana, Linmiao Zhub, Jiafu Shena, Wenjuan Li, Yong Wang, Jianglei Qin
1.Introduction
Hydrogels are very appealing biomedical materials in applications of biomedical 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 covalent bonds like acyl hydrazone 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 biocompatibility 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 the 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 biomedical 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 poly(aspartic acid) with hydrazide functional groups and methoxyl groups. The hydrazide group was imported onto the poly(aspartic acid) and partially hydrazide functionalized hydroxyl ethyl 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 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 application areas. The in vitro biotoxicity investigation revealed that the PAsp based self-healable hydrogel has good bio-compatibility and has great potential bioscience and biotechnology application as drug delivery agent and tissue repairing.
2.Experimental
2.1.Materials
L-Aspartic acid (L-Asp) and 1,3,5-trimethyl benzene 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 Reagent Co. Ltd. Phosphoric acid was supplied by Huadong Reagent Co. Aminoethanol, hydrazine hydrate (80 %), acetone and other solvents including N, N-Dimethylformamide (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 the previous study [35]. Then the PSI was used to react with hydrazine and ethanolamine successively to prepare PAEH with the following 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 °C oil bath for 24 h under continuous stirring. The intermediate was precipitated in 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.
The intermediate was dissolved in DMSO again and 3.05 g (50 mmol) 2-aminoethanol was added into the flask. The oxygen was removed again and the reaction was performed for another 12 h at 60 °C under N2 protection. The solution was precipitated in acetone and washed twice, the product of PAEH was collected after 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
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 a 10 % concentration. Then the cross-linkers of PEG DA with various molecular weights were dissolved in deionized water to form a 10 % solution. The two solutions were then mixed together with a 1:1 group ratio of hydrazide to aldehyde. The hydrogel formation at 25 °C was tracked on the rheometer right after the mixing of two solutions. The hydrogels for rheological and self-healing studies were formed directly in a round mold at room temperature without additional stimulus.
2.4.Rheological analysis
The gelation process and mechanical property of hydrogels were determined on a rheometer (TA AR2000ex) at 25 °C. 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 %.
2.5. Self-healing property of PAsp based hydrogels
The hydrazone bond is a dynamic bond that can endow the hydrogels with self-healing 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 into 4 pieces and the pieces were put back into the mold with close contact. The healing result was confirmed by suspended to gravity and under stretching.
2.6.Gel-sol-gel transition and degradation of the self-healing hydrogels under various conditions
The reversible characteristics of the dynamic covalent bond endow the hydrogels with multi-triggered transition [29], and the past backbone of the hydrogels endowed 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 vial leaning and recorded by a digital camera. The past backbone of PAEH is bio-degradable, so the hydrogel was expected to be degradable through the degradation of PAsp. 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, the 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 hydrogel through cleavage of the 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 JSM-7500 SEM apparatus after coated with Au.
2.8.Thermal stability analysis of the PAsp based hydrogel
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 °C to 800 °C with a heating rate of 20 °C min−1 under the N2 protection. The stability was compared according to the TGA curves.
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 cross-linkers. First, 3 mg of DOX·HCl was dissolved in PEG DA solution, then 10 % PEAH was added to form a 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. 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.
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)-2,5-diphenyl-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 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 % Triton X-100 as a positive control. The samples with a relative cell viability of less than 70 % are deemed to be cytotoxic.
2.11.Characterizations
The structure and the composition of the polymers were determined by 1H NMR 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 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 °C between a pair of 25 mm parallel aluminum plates. The 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 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.
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