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Self-healing Conductive Injectable Hydrogels with Antibacterial Activity as Cell Delivery Carrier for Cardiac Cell Therapy Ruonan Dong, Xin Zhao, Baolin Guo, and Peter X Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04911 • Publication Date (Web): 16 Jun 2016 Downloaded from http://pubs.acs.org on June 17, 2016

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ACS Applied Materials & Interfaces

Self-healing Conductive Injectable Hydrogels with Anti-bacterial Activity as Cell Delivery Carrier for Cardiac Cell Therapy Ruonan Dong a, Xin Zhao a, Baolin Guo a, *, Peter X. Ma a,b,c,d,e,* a

Frontier Institute of Science and Technology, and State Key Laboratory for

Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an, 710049, China b

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

48109, USA c

Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor,

MI 48109, USA d

Macromolecular Science and Engineering Center, University of Michigan, Ann

Arbor, MI 48109, USA e

Department of Materials Science and Engineering, University of Michigan, Ann

Arbor, MI 48109, USA

* To whom correspondence should be addressed. Tel.: +86-29-83395363. Fax: +86-29-83395131. E-mail: [email protected], [email protected].

1

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Abstract: Cell therapy is a promising strategy to regenerate cardiac tissue for myocardial infarction. Injectable hydrogels with conductivity and self-healing ability are highly desirable as cell delivery vehicles for cardiac regeneration. Here, we developed

self-healable

conductive

injectable

hydrogels

based

on

chitosan-graft-aniline tetramer (CS-AT) and dibenzaldehyde-terminated poly(ethylene glycol) (PEG-DA) as cell delivery vehicles for myocardial infarction. Self-healed electroactive hydrogels were obtained after mixing CS-AT and PEG-DA solutions at physiological conditions. Rapid self-healing behavior was investigated by rheometer. Swelling behavior, morphology, mechanical strength, electrochemistry, conductivity, adhesiveness to host tissue and anti-bacterial property of the injectable hydrogels were fully studied. Conductivity of the hydrogels is ~ 10-3 S·cm-1, which is quite close to native cardiac tissue. Proliferation of C2C12 myoblasts in the hydrogel showed its good biocompatibility. After injection, viability of C2C12 cells in the hydrogels showed no significant difference with that before injection. Two different cell types were successfully encapsulated in the hydrogels by self-healing effect. Cell delivery profile of C2C12 myoblasts and H9c2 cardiac cells showed a tunable release rate, and in vivo cell retention in the conductive hydrogels was also studied. Subcutaneous injection and in vivo degradation of the hydrogels demonstrated their injectability and biodegradability. Together, these self-healing conductive biodegradable injectable hydrogels are excellent candidates as cell delivery vehicle for cardiac repair. Keywords: self-healing, injectable hydrogels, cell therapy, myocardial infarction, conducting polymer 2


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1.

Introduction

Myocardial infarction (MI) has been the enormous cause of death in recent years worldwide.

1-3

There will be a large amount of cell loss when an acute MI happens

and it will lead to heart failure and death of the patients.4 Cell therapy has been developed as a promising strategy to protect and regenerate myocardial tissue during or after MI by delivery of appropriate types of cells to the MI area. 5 Many types of cells have been used in the cell therapy for MI, among which stem cells skeletal myoblasts

8-10

6-7

and

are most widely used. The fraction size and fibrosis will be

reduced by implantation of a certain amount of bone marrow stem cells. 11 Moreover, the adipose derived stem cells have been demonstrated to enhance myocardial function and reduce fibrosis when injected into the MI area.

12-13

The conventional

method of cell implantation is transplanting certain cells through coronary arteries, coronary veins, or peripheral veins as well as the direct intromyocardial injection. However, those delivery methods showed a poor cell retention which leads to the failure of cell therapy. Fortunately, some research showed that using the biomaterials as cell delivery vehicles could enhance cell retention and viability, as well as cell localization. 14-15 Among the biomaterials used as cell delivery vehicles, injectable hydrogels have been widely investigated as an available cell delivery carrier in recent years. Smith et al. 15 reported that the cell retention increased more than 7-folds compared with that in PBS by using a hyaluronan-gelatin hydrogel as a vehicle. Wang et al.

3


16

showed that using


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the chitosan based hydrogel as an injectable cell delivery vehicle could not only enhance the cell retention but also promote the cardiac differentiation of the brown adipose-derived stem cells. Meanwhile, heart as a vital organ has special electrophysiological behavior and the cardiac cells were in an electroactive condition. 17-18

It has been demonstrated that conductive polymers may promote the proliferation

and differentiation of the electrical stimuli responsive cells, such as the stem cells, 19-20

myoblast cells,

21-23

nerve cells

24-25

and the cardiac cells.

26-29

Therefore,

injectable hydrogels with conductivity as cardiac cell delivery vehicle are highly anticipated for cardiac repair. Although conventional injectable conductive hydrogels showed a great deal of advantages such as the promotion of cell proliferation,

30

they

could not be long-term used with a stable function due to their non-self-healing property. Besides, the cell delivery vehicles would tolerate a considerable mechanical load after injection due to the contraction behavior of the heart,

31-32

making the

vehicle be damaged easily. When the damage of the vehicle happened during the service period, the self-healing capacity of the hydrogels could prolong the lifetime of the vehicle. 33 Therefore, the development of the injectable conductive hydrogels with self-healing ability would be highly beneficial for cardiac cell therapy, and it is an ongoing challenge. Herein, we developed a series of self-healing conductive injectable hydrogels based on chitosan-graft-aniline tetramer (CS-AT), and demonstrated their potential as cell delivery vehicle for cardiac cell therapy. The hydrogels were synthesized by mixing the solution of CS-AT and benzaldehyde groups capped poly (ethylene glycol) under 4


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physiological condition. The conductivity of those hydrogels (about 2×10-3 S cm-1) was quite similar with that of native cardiac tissue. These hydrogels showed fast self-healing capacity, good antibacterial activity, adhesiveness and in vivo degradability. They also showed a good biocompatibility that could support the proliferation of the C2C12 cells and prevent cells from injury when injected into the body. C2C12 cells and H9c2 cells were encapsulated in the hydrogel, and they show a controlled linear-like release profile. All these results suggest that these injectable self-healing conductive hydrogels are ideal candidates as cell delivery carries for cardiac repair applications.

2.

Materials and methods

2.1 Materials N-phenyl-1,4-phenylenediamine, butanedioic anhydride, ammonium persulfate, chitosan (Mw=100000-300000), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC•HCl), N-hydroxysuccinimide (NHS), poly(ethylene glycol) with Mn of 2000 (PEG2000), 4-formylbenzoic acid, N,N′-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino) pyridine (DMAP) were all purchased from Sigma Aldrich and used as received. Tetrahydrofuran (THF), dimethylformamide (DMF) were stored at a nitrogen atmosphere and dried before use. The carboxyl-caped aniline tetramer was synthesized according to reference 34. All other organic solvents were analytical grade. 2.2 Synthesis of chitosan-graft-aniline tetramer Chitosan-graft-aniline tetramer (CS-AT) was synthesized according to our previous 5



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report. 35 We synthesized CS-AT with different AT contents, feed ratio ranging from 8% to 20%. Take the CS-AT8 (the feed ratio of AT in the system is 8%) as a typical example: 0.5 g of chitosan was first dissolved into 50 mL of 0.05 M HCl to obtain a 1 wt% chitosan solution. Second, a mixture of EDC·HCl (0.0916g), NHS (0.0550g) and aniline tetramer (0.044g) was dissolved in 5 mL of DMF, and stirred at room temperature to activate the carboxyl group of aniline tetramer. After 24 h, the mixture was added dropwise into the 1 wt% chitosan solution, and the reaction was continued for another 24 h. Then, the product precipitated by dropping 3 M NaOH into the solution and was then filtered. The precipitate was re-dissolved in a 1% (v/v) acetic acid aqueous solution and then centrifuged to remove the insoluble parts. After the centrifugation, 3 M NaOH was added to the supernatant until the product precipitated. Distilled water was used to wash the product until the pH was neutral. Finally, the product was dried in a vacuum oven for 48 h. 2.3 Synthesis of dibenzaldehyde-terminated poly (ethylene glycol) (PEG-DA) PEG-DA polymers were synthesized using a reported method. 36 2.0000 g of PEG2000, 0.3000 g of 4-formylbenzoic acid and 0.6108 g of DMAP were first dissolved in 10 mL of dry THF. Then, 1.0318 g of DCC was added into the solution under nitrogen atmosphere. The system was then put at room temperature with stirring. After 72 h of reaction, the solution was centrifuged and the supernatant was precipitated in diethyl ether. The precipitate was re-dissolved in THF and centrifuged again. The dissolution and precipitation step was repeated for 3 times in order to obtain the product. The final product was dried in a vacuum oven at room temperature. 6


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2.4 Preparation of CS-AT hydrogel CS-AT polymer was dissolved in 1% (v/v) acetic acid aqueous solution to form a 3 wt% solution. The PEG-DA solution was prepared with different concentrations to obtain different molar ratios (R=0.75, 1, 1.5 and 2) of –NH2 and –CHO in the hydrogel. For example, 0.35 g of CS-AT solution was then mixed with 0.15 g of PEG-DA solution, and the mixture was placed at 37 oC. The mixture turned into hydrogel less than 1 min. Hydrogels formed by different CS-AT polymers were named as CS-ATX hydrogel. For example, CS-AT10 hydrogel meant that the hydrogel was made from CS-AT10 polymer with a 1:1 molar ratio of –NH2 to –CHO. 2.5 Characterization A Bruker Avance 400 MHz NMR instrument was used to record the 1H nuclear magnetic resonance (1HNMR) spectra of CS-AT20 and PEG-DA with DMSO-d6 and CDCl3 as solvents respectively at room temperature. The FT-IR spectra of PEG, PEG-DA, chitosan, CS-AT20 polymer and CS-AT20 hydrogel were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Scientific Instrument) with a resolution of 4 cm-1. 0.5 wt% of CS-AT20 polymer solution (3.5 mL) and 2 wt% of PEG-DA (1.5 mL) were mixed together, and the solution was recorded by UV-vis spectrometer after doping with 1 M HCl. 0.35 g of 3 wt% of CS-AT20 polymer solution and 0.15 g of PEG-DA solution were mixed together, daubed onto an ITO glass, and dried at room temperature. Then the dried gel was used as working electrode accompanied with a platinum wire as 7



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auxiliary electrode, and an Ag/AgCl as reference electrode to conduct the cyclic voltammetry (CV) on an Electrochemical Workstation (CHI 660D). The swelling ratio of these hydrogels was also measured. Briefly, different groups of hydrogels with similar size and shape were immersed into the Dulbecco phosphate-buffered saline (DPBS) at 37 oC and taken out at a regulate time interval. After the excess water was removed with a filter paper, the hydrogels were weighted. The swelling ratio of the hydrogels was calculated using Equation.1, where wt represented the hydrogel’s weight after swelling and wo meant the hydrogels weight before swelling. Swelling ratio =

( )

× 100%

(Equation.1)

The morphology of those hydrogels before and after swelling was observed using a field emission scanning electron microscope (Quanta 250 FEG, FEI). The hydrogels were divided into two groups. One group was freeze dried directly as prepared and another group was freeze dried after swelling equilibrium state was reached. All the samples were sputter-coated with gold before observation. 2.6 Conductivity of the hydrogel Hydrogels with different AT contents were used to study the conductivity of the hydrogels. 7 g of CS-AT solution and 3 g of PEG-DA solution were vortex oscillated to form a homogenous mixture and the mixture was put at 37 oC for 2 h to obtain completely cross-linked hydrogel. After gelation, the hydrogel was transferred into a cylinder and the conductivity of the hydrogel was detected using a pocket conductivity meter (HANNA8733). 8


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2.7 Rheological properties of the hydrogels The mechanical properties of hydrogels with different concentrations of PEG-DA were measured using a TA rheometer (DHR-2) instrument with a parallel-plate configuration (plate diameter of 20 mm, gap of 1000 µm). A dynamic frequency sweep test with frequency ranging from 1 to 100 rad·s-1 at a shear amplitude of 0.1% was employed to perform the measurement. The hydrogels with different molar ratios -NH2 to –CHO (0.75, 1, 1.5 and 2) and AT contents were made as disks (diameter of 20 mm and height of about 1 mm). 2.8 Self-healing behavior of the hydrogels The self-healing behavior of the hydrogel was tested with both microscopic and quantitive methods using the CS-AT10 hydrogel as an example. In the microscopic way, the hydrogel was first injected through a syringe into a heart shape mould and piled up at 37 oC in a humidified atmosphere. And then the hydrogel was cut into two pieces. Subsequently, the two pieces of hydrogels were put close to each other at 37 o

C in a humidified atmosphere. The results of the self-healing test were photographed

in a certain time. In the quantitive way, the hydrogels were prepared as a disk with a 20 mm in diameter and a 1 mm in height. Using the strain amplitude sweep method (γ changed from 1% to 500%), the value of the critical strain region was detected. And then a new hydrogel with the same size was used to test the self-healing behavior by using the alternate strain sweep test at a fixed angular frequency (1 rad·s-1). Amplitude oscillatory strains were switched from small strain (γ = 1.0%, 100 s for each interval) 9



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to large strain (γ = 250%, 60 s for each interval), and 6 cycles were carried out in the test. The self-healing behavior under a cyclic high and low stress was taken out to measure the self-healing ability of the hydrogel under a native cardiac contractile environment. The high stress was set as 400 Pa and 800 Pa (60 s for each interval) respectively and the low stress was 1 Pa (100 s for each interval). 2.9 Cell encapsulation and proliferation in the hydrogel CS-AT10 polymers were both dissolved in 1% (v/v) acetic acid solution (100 µL of acetic acid in 10 mL of DMEM solution) to obtain a 3 wt% CS-AT10 polymer solution. Meanwhile, the PEG-DA polymer was dissolved in Dulbecco's Modified Eagle Medium (DMEM) to form a 23.4 wt% solution. Then the CS-AT10 and PEG-DA solution were sterilized. Before encapsulated in the hydrogel, the C2C12 cells were treated with CellTracker GreenTM (5 µM) for 30 min. To encapsulate the cells, first, 150 µL of PEG-DA solution were added into the 24 well plate. Then, 5 µL of cell suspension was added into the mixture and finally 350 µL of CS-AT10 polymer solution. After gelation for 1 h at 37 oC, 500 µL of DMEM with 10% FBS was added into each well and the plate was incubated at 37 oC in a humidified atmosphere incubator containing 5% CO2. After 24 h, 48 h and 72 h cultivation, the hydrogels were taken out of the wells carefully. Then they were observed under a confocal laser microscope (FV1200, Olympus) and analyzed using NIH ImageJ software. The thickness of scanning was 60 µm for each sample. ADMSCs proliferation in the CS hydrogel and CS-AT10 hydrogel was also measured. 10


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ADMSCs were incubated with CellTrackerTM Green (5 µM) for 30 min before encapsulated into the hydrogels. The cells were encapsulated in the hydrogels as described above. After culturing for 24, 48 and 72 h, the hydrogels were taken out of the well and observed under a confocal laser microscope (FV1200, Olympus). The results were analyzed using NIH ImageJ software and the thickness of scanning was 50 µm for each sample. 2.10

Cell viability in hydrogel after injection

After cells were encapsulated in the hydrogels, the hydrogels were divided into two groups. One of them was incubated directly in the well for another 24 h, and another group was injected using a 22-gauge needle. After injection, the hydrogels were incubated at 37 oC for 2 h to make it self-heal and 500 µL of DMEM were then added into each well. After 24 h incubation, the hydrogels encapsulating cells and the injected hydrogels were observed under the confocal laser microscope (FV1200, Olympus). The picture of each sample was merged of 60 slides and analyzed using NIH ImageJ software. 2.11

Two types of cells co-culture in the hydrogels

ADMSCs were incubated with CellTrackerTM Red (5 µM) for 30 min before encapsulated into the hydrogels. At the same time, C2C12 cells were tracked with the CellTrackerTM Green (5 µM). These two types of cells were encapsulated into hydrogels individually and incubated for 12 h. After incubation, hydrogels encapsulating with C2C12 cells and hydrogels with ADMSCs were mixed together by injection. Furthermore, two pieces of the hydrogels with different cells were put 11



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together to make it integrate into a construct. After 24 h, the samples were observed under the confocal laser microscope (FV1200, Olympus). 2.12

In vitro cell delivery property of the hydrogels and in vivo cell retention test

To test the cell delivery ability of this hydrogel, a cell delivery test was conducted. C2C12 cells were marked and encapsulated into CS-AT10 hydrogel in a 48 well plate with a density of 1×106 cells mL-1, and 250 µL of DMEM with 10% FBS were added into each well. After cultured for 24 h, the DMEM in each well was transferred into a new well respectively, and 250 µL of DMEM with 10% FBS was added into the hydrogel’s well. The cell number in the transferred solution was calculated using the NIH ImageJ software after observing under inverted fluorescence microscopy (IX53, Olympus). The released cell number was analyzed from 1 to 7 days. The in vivo cell retention was measured using the in vivo optical bioluminescence imaging. ADMSCs were incubated with CellTrackerTM Deep Red (5 µM) before encapsulated into the hydrogel or suspended with DPBS. About 1× 106 of cells were encapsulated into 100 µL of hydrogel (or 100 µL of PBS as control), and injected into the mouse subcutaneously immediately. The fluorescence intensity was correlated with the cell number.16,

37

The relative fluorescence intensity was calculated by

Equation 2, in which FIt and FI0 mean the fluorescence intensity at t day (t=3, 7) and 0 day. The bioluminescence image of the mouse with the cells was taken using the IVIS® Spectrum instrument (Xenogen, USA) at 1, 3, and 7 days after injection. &'

!"#$%$ = &'

2.13

Tissue adhesive test of the hydrogels 12


Equation 2

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In order to investigate the adhesive ability of the hydrogel to the host tissue, a lap-shear test with fresh porcine skin as adherend was conducted. Briefly, the fresh porcine skin was cut into rectangle pieces with 2 ×1 cm size and then immersed into DPBS before use. 150 µL of mixture containing 105 µL of CS-AT (3 wt%) and 45 µL of PEG-DA (23.4 wt%) was added onto the porcine skin and placed at 37 oC for 1 h to make the gelation complete. Subsequently, adhesion properties were tested using the lap-shear test on an Instron Materials Test system (MTS Criterion 43, MTS Criterion) equipped with a 50 N load cell at a rate of 5 mm/min. All the tests employed more than 3 samples. 2.14

Antibacterial properties of the CS-AT polymers and hydrogels

Minimum inhibitory concentrations (MICs) of the CS-AT polymers for both E. coli (ATCC 8739) and S. aureus (ATCC 29213) were investigated using a reported method 38

. Briefly, CS-AT copolymers were dissolved in 1% (v/v) acetic acid solution to

obtain 3 wt% copolymer solutions. Subsequently, these 3 wt% copolymer solutions were diluted with MH broth using the twofold dilution method in a 96-well microplate and a series of copolymer solutions were obtained (100 µL in each well). 100 µL (104–105 CFU mL-1) of bacterial suspension was added into each well and mixed repeatedly to acquire a homogeneous solution. The gradiently diluted copolymer solutions with no inoculum were chosen as the negative control and MH broth with inoculum served as the positive control. The 96-well plate was incubated at 37 oC for 18 h, and the absorbance of the solutions at 600 nm was read using a microplate reader (Molecular Devices) at the end of time. The minimum 13



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concentration of the copolymer that could inhibit the bacterial growth was intended to be the MICs of the copolymers. To further evaluate the antibacterial activity of the hydrogels, surface antibacterial assay of the hydrogels was conducted. Hydrogels were prepared as previously described – 140 µL of CS-AT solution (3 wt%) and 60 µL of PEG-DA solution (23.4 wt%) were mixed homogeneously, and then 200 µL of mixture was added into each well in a 48-well plate. The plate was placed at 37 oC for complete gelation, and 5 µL of bacterial suspension (in PBS, 105 CFU mL-1) was spread onto the surface of each hydrogel subsequently. Afterward, the hydrogels were incubated at 37 oC in a humidified atmosphere for 4 h. Any bacterial survivors were re-suspended by adding 1 mL of PBS into each well then. 1 mL of PBS solution with 5 µL bacterial suspension (in PBS, 105 CFU mL-1) served as the control. After incubated at 37 oC for 12-24 h, the colony-forming units (CFU) on the Petri dish were counted. Each test was repeated for 3 times and the kill% was used to measure the antibacterial activity of the hydrogels. Kill% = 2.15

()** (+,-. +/ (+-.0+*1,0232+0 (+,-. +- )45)036)-.7* 89:0+;)*1 ()** (+,-. +/ (+-.0+*

×100%

Subcutaneous injection, in vivo degradation of CS-AT hydrogels and

histopathological analysis Female Harlan Sprague–Dawley rats (200-220 g) were anesthetized with 10% chloral hydrate and shaved. The CS-AT solution and the PEG-DA solution were sterilized. The hydrogels with different AT contents (CS-AT8, CS-AT10 and CS-AT20) were prepared and then injected into the shaved rats subcutaneously with the same volume 14


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(200 µL) on the dorsum using a 22-gauge needle in a sterile condition. 15 min later, the rats were sacrificed by cervical dislocation and the hydrogels were taken out to photograph. For in vivo degradation test, the hydrogels were injected under the dorsum of the rats as described above and were taken out from the dorsum at 5, 10, 15, 20 and 45 days and photographed. The degradation properties of the hydrogels in vivo were evaluated by measuring the size of them under the dorsum. CS-AT10 hydrogels were injected into the SD rats subcutaneously on the dorsum after sterilized. After 7 days, the tissue near the hydrogel was excised and frozen in O.C.T medium. The frozen section was cut into 20 µm in thickness. The sections were stained with hematoxylin–eosin (H&E) stains to evaluate the inflammatory response of the hydrogels. All experiments were performed in compliance with the relevant laws and institutional guidelines, and had been approved by the Animal Ethics Committee of the University. 2.16 Statistic analysis All the experiments were conducted using more than 3 samples, and the data were expressed as mean ± standard. The statistical significance was analyzed using the student t-test, and it was considered to be a statistically significant difference when p

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