Bioactive self-healing hydrogel based on tannic acid modified gold nano-crosslinker as an injectable brain implant for treating Parkinson's disease

Abstract

Background: Parkinson's disease (PD) is one of the most common long-term neurodegenerative diseases. Current treatments for PD are mostly based on surgery and medication because of the limitation and challenges in selecting proper biomaterials. In this study, an injectable bioactive hydrogel based on novel tannic acid crosslinker was developed to treat PD.

Methods: The oxidized tannic acid modified gold nano-crosslinker was synthesized and used to effectively crosslink chitosan for preparation of the bioactive self-healing hydrogel. The crosslinking density, conductivity, self-healing ability, and injectability of the hydrogel were characterized. Abilities of the hydrogel to promote the proliferation and differentiation of neural stem cells (NSCs) were assessed in vitro. Anti-inflammatory property was analyzed on J774A.1 macrophages. The hydrogel was injected in the PD rat model for evaluation of the motor function recovery, electrophysiological performance improvement, and histological repair.

Results: The hydrogel exhibited self-healing property and 34G (~ 80 μm) needle injectability. NSCs grown in the hydrogel displayed long-term proliferation and differentiation toward neurons in vitro. Besides, the hydrogel owned strong anti-inflammatory and antioxidative capabilities to rescue inflamed NSCs (~ 90%). Brain injection of the bioactive hydrogel recovered the motor function of PD rats. Electrophysiological measurements showed evident alleviation of irregular discharge of nerve cells in the subthalamic nucleus of PD rats administered with the hydrogel. Histological examination confirmed that the hydrogel alone significantly increased the density of tyrosine hydroxylase positive neurons and fibers as well as reduced inflammation, with a high efficacy similar to drug-loaded hydrogel.

Conclusion: The new bioactive hydrogel serves as an effective brain injectable implant to treat PD and a promising biomaterial for developing novel strategies to treat brain diseases.

Keywords: Bioactive hydrogel; Conductive hydrogel; Gold nanoparticle; Parkinson’s disease; Self-healing; Tannic acid.

Fig. 1

Preparation and characterization of oxidized tannic acid (OTA) and oxidized tannic acid modified gold nanoparticles (OTA@Au). A The synthetic diagram of OTA@Au nano-crosslinkers. B The IR spectra of tannic acid (TA) and OTA. C The TEM image of OTA. D The TEM image of OTA@Au. E The UV-visible spectra of TA, OTA, and OTA@Au

Fig. 2

Preparation and morphology of the self-healing hydrogels. A The COA hydrogels can be injected through 34-gauge syringe needles with 80 μm internal diameter into the heart-shaped mold (A). An integrated heart-shaped hydrogel with smooth and homogenous appearance formed after self-adaption and self-healing for 30 min at 25 °C. B The integrated hydrogel was chopped into small pieces and then refilled the heart-shaped mold. The heart-shaped hydrogel self-healed for 30 min and recovered into its original shape. C The SEM image for the cross-section of the COA2 hydrogel. D Remaining weights of the CO and COA2 hydrogel in PBS at 37 °C. E Schematic diagram for the possible gelation mechanism of COA hydrogels

Fig. 3

Rheological analyses and time-resolved SAXS experiments of the self-healing hydrogels. A Time-sweep experiments showing the storage moduli (G’) and loss moduli (G”) of the hydrogels against the gelling time at 1 Hz frequency and 1% dynamic strain. B Self-healing experiments showing the G’ and G” values of the equilibrium hydrogels at 1 Hz frequency and alternate continuous damage-healing cycles of 1 and 600% dynamic strains at 25 °C. C The steady shear-thinning properties of the hydrogels determined by measuring the viscosity versus shear rate. D Time-dependent coherent SAXS profiles acquired during the gelation process at 25 °C for investigation of microstructure changes of the hydrogel upon crosslinking. E SAXS profiles for the hydrogel at different temperatures. The SAXS data were obtained from COA2 hydrogel

Fig. 4

Proliferation, differentiation, and immunofluorescent staining of neural stem cells (NSCs) encapsulated in the hydrogels. A Cell proliferation of NSCs embedded in the CO and COA2 hydrogels measured by CCK-8 assay during a culture period of 14 days. The proliferation was normalized to the value at day 0 and expressed as the percentage of the cell proliferation (%). B Expression of neural-related genes in NSCs encapsulated in the hydrogels were analyzed by RT-PCR at 14 days. The expression levels normalized to that of housekeeping gene (GAPDH) and represented by the relative ratios of gene expression. Immunofluorescent images of NSCs stained for (C) GFAP and (D) MAP2 at 14 days. Scale bars represent for 100 μm. E Quantification of the fluorescent intensity in GFAP- and MAP2-positive NSCs. *p < 0.05, **p < 0.01, and ***p < 0.001 between the indicated groups

Fig. 5

In vitro antioxidation and anti-inflammatory evaluations of the hydrogels. A Cell viability tests for either healthy or inflamed NSCs. Scatter plots showed changes in propidium iodide (PI) and Vitabright-48 (VB-48) staining in response to the cell treatment with encapsulation by the CO hydrogel and COA2 hydrogel compared to the single cells (mock). B Summary for the distribution of inflamed NSC status after the treatment of CO hydrogel or COA2 hydrogel compared to the single inflamed cells (mock). C Quantification for the rescue rate and dead rate of the inflamed NSCs after the treatment of the CO hydrogel or COA2 hydrogel for 12 h. D Gene expression of the J774A.1 macrophages analyzed by RT-PCR after contacting the CO hydrogel or COA2 hydrogel for 6 h compared to macrophages cultured on the tissue-culture polystyrene plate (TCPS) in vitro. The expression levels were normalized to that of β-actin and represented by the relative ratios of gene expression. *p < 0.05 and **p < 0.01 between the indicated groups

Fig. 6

Behavior tests of Parkinson’s disease (PD) rats treated by hydrogels. A Schematic illustration of the PD rat model. 6-hydroxydopamine (6-OHDA) as a neurotoxin was utilized to induce PD through injection into the brains of rats. After confirmation of PD induction, the hydrogels were further injected into the lesion regions for 14 days, and the efficacy of treatment was evaluated through behavior tests, electrophysiological tests, and immunofluorescent analyses. B, C Quantitative assessment for the functional recovery of different condition treated PD rats (injected by saline, CO hydrogel, COA2 hydrogel, or COA2 hydrogel loaded with curcumin) as compared to the untreated PD rats (0 day), based on the left-side circling speeds (B) and the impaired forelimb contact proportion (C). **p < 0.01, ***p < 0.001, and ****p < 0.0001 between the indicated groups

Fig. 7

Electrophysiological analyses of PD rats treated by hydrogels. A Electrophysiological traces of subthalamic nucleus (STN) spikes in different condition treated PD rats within 10 s at 14 days. B Quantitative overall spike rates of each group from a 30-s period of each recording devoid of significant noise at 14 days. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between the indicated groups

Fig. 8

Biocompatibility evaluation of the hydrogels for treating PD rats. A In vivo immunofluorescent images of CD86 positive (CD86+) M1 macrophages (red) and CD163 positive (CD163+) M2 macrophages (green) for the explanted tissue after implantation in the brain for 14 days. Quantification for (B) the fluorescent intensity of M1 and M2 macrophages and (C) the ratios of M2/M1 in vivo. *p < 0.05, **p < 0.01, and ****p < 0.0001 between the indicated groups

Fig. 9

In vivo immunofluorescent analyses of each animal group for the related markers. The expression of (A) tyrosine hydroxylase positive (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc), (B) TH+ dopaminergic fibers in the striatum, and (C) glial fibrillary acidic protein positive (GFAP+) astrocytes in the PD rat striatum were investigated at 14 days after surgery. Green color represents the indicated marker and blue color represents the nucleus. The fluorescent intensities were quantified and presented as graphics for (D) TH+ dopaminergic neurons in the SNpc, (E) TH+ dopaminergic fibers in the striatum, and (F) GFAP+ astrocytes in the striatum of each group. *p < 0.05, ***p < 0.001, and ****p < 0.0001 between the indicated groups