Fabrication of 3D Tubular Hydrogel Materials through On-Site Surface

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Fabrication of 3D tubular hydrogels materials through on-site surface free radical polymerization Shuanhong Ma, Mingming Rong, Peng Lin, Min Bao, Jing Xie, Xiaolong Wang, Wilhelm T. S. Huck, Feng Zhou, and Weimin Liu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b02532 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Chemistry of Materials

Chemistry of Materials (2018-02532q.R1) Article type: (Full Paper) Fabrication of 3D tubular hydrogels materials through on-site surface free radical polymerization Shuanhong Ma, Mingming Rong, Peng Lin, Min Bao, Jing Xie, Xiaolong Wang, Wilhelm T. S. Huck, Feng Zhou*, Weimin Liu

Dr. S.H. Ma, M.M. Rong, Dr. P. Lin, Prof. X. L. Wang, Prof. F. Zhou, Prof. W.M. Liu State Key Laboratory of Solid Lubrication Lanzhou Institute of Chemical Physics 18 Middle Tianshui Road, Lanzhou, China, 730000 E-mail: [email protected] Prof. W. M. Liu. College of Materials Science and Technology College of Materials, Northwestern Polytechnical University 127 YouyiXi Road, Xian, China 710072 M. Bao, J. Xie, W. T. S. Huck Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands

Abstract: Constructing tubular hydrogels materials with desirable structures based on their functional application is a big challenge. Here we report a simple, but effective method to prepare tubular hydrogels with complex geometries by surface radical polymerization, in which an iron wire acts as both catalyst and template for the formation of a gel layer with controllable thickness. The formed hydrogel layer can be easily peeled off from the template after secondary cross linking to obtain hollow hydrogel tubes which exhibit extraordinary and tunable tensile strength, good elasticity and pressure bearing capability. The method can be generalized to construct a series of complex three-dimensional hydrogel tubes with versatile components for building up fluidic channels or biocompatible 3D cell culturing platform for tissue engineering. Such method is a great advance in the field of hydrogels materials. It is anticipated that this innovation would open up the door for developing functional 3D tubular hydrogels materials suitable for multiple applications.

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1. Introduction Structured hydrogels have become a prime candidate for applications including scaffolds in tissue engineering,[1-3] chemical actuators,[4-7] reaction containers,[8-9] medicine transportation and separation,[1011]

as well as microfluidic, [12-13] owing to their extraordinary characteristics such as high water content, [14-

15]

controllable mechanical strength/elasticity,[16-24] self-healing,[25-30] and tunable friction.[31-32] Precise

control over shape and size of target structure units ensures reliable use. Especially, research concerning the development of 3D structural hydrogel scaffolds has been more and more intensified,[33-34] due to its great importance to regulate the cell behavior as proliferation, spreading and differentiation.[35] In order to prepare functional structural hydrogels, many strategies have been developed, including 3D printing technology,[36-37] dynamic external stimuli,[38-40] in situ surface catalysis,[41] template assisted photocrosslinking,[42-43] ionic crosslinking,[44] photocatalytic crosslinking,[45] vapor deposition,[46] phase separation,[47-48] and freezing/thawing technique.[49] However, constructing hollow hydrogels units remains a major challenge, as there are no robust and convenient methods to produce and control the dimensions of hollow hydrogels structures. In addition, the mechanical stability of the hollow hydrogels needs to be improved, especially when considering long term use in biological environments. The preparation of hollow hydrogels tubes that resemble blood vessels, has been a long-standing pursuit in the biomedical engineering field.[50] Meanwhile, tubular hydrogels constructs can be used as ideal extracellular matrix (ECM) for culturing cells so as to mimic the vascular functions perfectly.

[51-55]

Many approaches have

been developed for obtaining functional gel tubes including rolling,[56] template packing,[57] microfluidic molding,[58] and 3D bio-printing technology.[59] However, these approaches face key scientific and technical problems, such as: how to finely control the size and shape of the tubes, how to grant high strength and good elasticity to the tubes, and how to easily construct complex 3D hydrogel tube ensembles. Here we report a simple method to prepare hydrogel tubes with tunable size and mechanical strength, good elasticity and strong pressure bearing properties by on-site surface radical polymerization (SRP) using high purity iron wire as an initiation template. This approach is based on the redox reaction between Fe2+ (dissociated from iron wire) and persulfate at room temperature, which lowers the decomposition


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activation energy for chain initiation process in free radical polymerization,[60-61] leading to the fast formation and molding of a hydrogel around the iron wire surface. The transformation from hydrogel layer to hydrogel tube materials is achieved by strengthening the gel layer via different ways, so that the hydrogel layer can be readily peeled off from the iron wires. Importantly, our method can be used to engineer a series of complex three-dimensional hydrogel tubes arrays, hollow enclosed gel units, and gel tubes embedded functional materials.

2. Experimental Section. 2.1. Preparation of hydrogel tubes PAA-Fe/PAAm hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of AA (acrylic acid) and AAm (acrylamide), which is initiated by KPS (potassium persulfate) in the presence of BIS (N,N'-Methylene bisacrylamide). First, 50 ml pure water in a roundbottom ﬂask was bubbled with N 2 gas for 5 min to remove the dissolved oxygen. Then, 4.26 g AAm (60 mmol), 0.432 g AA (6 mmol), 0.006 g BIS (0.039 mmol) and 0.02 g KPS (0.074 mmol) were added to into the water and mixed by a magnetic bar until total solution. Next, the iron wire (0.2 mm, 0.45 mm, 0.7 mm, 1.60 mm and 4.0 mm) was immersed into monomer solution to allow SRP polymerization. The polymerization was performed statically for 1, 2, 5, 10, 20 and 30 min at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was immersed into Fe3+ (0.03 M/L) solution for 0.5, 1, 2, 5, 10, and 20 h. Then the iron wire was removed to obtain hollow hydrogel tube which was further immersed into water to remove free Fe3+. The effect of relative mass concentration ratio for AA/AAm % was also investigated for 1%, 5%, 10%, 20% and 40%. The hydrogel tubes units and array shown in Figure 2 were prepared by following the same procedure as that of single hydrogel tube. By precisely controlling the gaps among iron wires from d=r to d=2r, in which r is the radius of iron wire, the hydrogel tube channel with each other could be connected or non-connected. The mold was prepared from a commercial 3D MakerBot printer. PVA/PAAm/PAA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of AA and AAm, which is initiated by KPS in the presence of BIS and PVA (polyvinyl



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alcohol) polymer chains. 4.26 g AAm (60 mmol), 0.432 g AA (6 mmol), 0.006 g BIS (0.039 mmol) and 0.02 g KPS (0.074 mmol) were added into 50 ml water and mixed by a magnetic bar until total solution (solution A). Next, 20 g PVA powder was dissolved into 100 mL pure water and heated to 100 oC until total solution. After cooling down, the PVA solution was mixed with the solution A by volume ratio as VPVA(1):VHEMA(1). The iron wire (1.60 mm and 4.0 mm) was immersed into mix solution to allow polymerization. The polymerization was performed statically for 20 min at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was fast put into freeze dryer to allow freezing (-40 oC) and thawing (20 oC). After 3 times cycles, the iron wire was removed and the resulted PVA/PAAm/PAA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free PVA polymer chains. PVA/PHEMA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of HEMA (2-hydroxyethyl methacrylate), which is initiated by KPS in the presence of BIS and PVA polymer chains. 6.5 g HEMA (50 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 100 µL AA were added into 50 ml water and mixed by a magnetic bar until total solution (solution A). Next, 20 g PVA powder was dissolved into 100 mL pure water and heated to 100 oC until total solution. After cooling down, the PVA solution was mixed with the solution A by volume ratio as VPVA(1):VHEMA(1.5). The iron wire (1.60 mm) was immersed into mix solution to allow polymerization. The polymerization was performed statically for 20 mins at room temperature (20

o

C). After

polymerization, the hydrogel layer coated iron wire was fast put into freeze dryer to allow freezing (-40 o

C) and thawing (20 oC). After 3 times cycles, the gel layer coated iron wire was immersed into dilute

hydrochloric acid (0.5 M/L) for 1 min and then the wire was removed. The resulted PVA/PHEMA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free PVA polymer chains. POEGMA/PAA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of OEGMA (Polyethylene glycol methacrylate) and AA, which is initiated by KPS in the presence of BIS. 7.5 g OEGMA (Mn: 500, 15 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074


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mmol) and 0.43 g AA (6 mmol) were added into 40 ml water and mixed by a magnetic bar until total solution (solution A). The iron wire (1.60 mm) was immersed into solution to allow polymerization. The polymerization was performed statically for 10 mins at room temperature (20 oC). After polymerization, the wire was removed and POEGMA/PAA hydrogel tube was obtained. The resulted POEGMA/PAA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer. POEGMA/PHEMA/PAA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of OEGMA, HEMA and AA, which is initiated by KPSin the presence of BIS. 7.5 g OEGMA (Mn: 500, 15 mmol), 6.5 g HEMA (50 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 0.43 g AA (6 mmol) were added into 40 ml water and mixed by a magnetic bar until total solution (solution A). The iron wire (1.60 mm) was immersed into solution to allow polymerization. The polymerization was performed statically for 20 mins at room temperature (20 oC). After polymerization, the wire was removed and POEGMA/PHEMA/PAA hydrogel tube was obtained. The resulted POEGMA/PHEMA/PAA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer. POEGMA/PVA/PAA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of OEGMA and AA, which is initiated by KPS in the presence of BIS and PVA polymer chains. 7.5 g OEGMA (Mn: 500, 15 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 0.21 g AA (3 mmol) were added into 40 ml water and mixed by a magnetic bar until total solution (solution A). Next, 20 g PVA powder was dissolved into 100 mL pure water and heated to 100 oC until total solution. After cooling down, the PVA solution was mixed with the solution A by volume ratio as VPVA(1):VA(4). The iron wire (1.60 mm) was immersed into mix solution to allow polymerization. The polymerization was performed statically for 20 min at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was fast put into freeze dryer to allow freezing (-40 oC) and thawing (20 o

C). After 3 times cycles, the wire was removed and POEGMA/PVA/PAA hydrogel tube was obtanied.

The resulted POEGMA/PVA/PAA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free PVA polymer chains.



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POEGMA/PHEMA/PVA/PAA hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of HEMA, OEGMA and AA (acrylic acid), which is initiated by KPS in the presence of BIS and PVA polymer chains. 7.5 g OEGMA (Mn: 500, 15 mmol), 6.5 g HEMA (50 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 0.21 g AAc (3 mmol) were added into 40 ml water and mixed by a magnetic bar until total solution (solution A). Next, 20 g PVA powder was dissolved into 100 mL pure water and heated to 100 ℃ until total solution. After cooling down, the PVA solution was mixed with the solution A by volume ratio as VPVA(1):VA(4). The iron wire (1.60 mm) was immersed into mix solution to allow polymerization. The polymerization was performed statically for 20 mins at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was fast put into freeze dryer to allow freezing (-40 oC) and thawing (20 oC). After 3 times cycles, the wire was removed

and

POEGMA/PHEMA/PVA/PAA

hydrogel

tube

was

obtanied.

The

resulted

POEGMA/PHEMA/PVA/PAA hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free PVA polymer chains. POEGMA/SA-Ca hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of OEGMA, which is initiated by KPS in the presence of BIS and SA (sodium alginate) polymer chains. 7.5 g OEGMA (Mn: 500, 15 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 1 g SA (2%) were added into 40 ml water and mixed by a magnetic bar until total solution. The iron wire (0.7 mm, 1.60 mm) was immersed into solution to allow polymerization. The polymerization was performed statically for 20 min at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was fast immersed into the CaCl2 solution (2%). After 2 h later, the gel layer coated iron wire was taken out and the iron wire was removed. The resulted POEGMA/SA-Ca hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free SA polymer chains. PHEMA/SA-Ca hydrogel tube. The hydrogel layer on the iron wire was formed by radical polymerization of HEMA, which is initiated by KPS in the presence of BIS and SA. 6.5 g HEMA (50 mmol), 0.006 g BIS (0.039 mmol), 0.02 g KPS (0.074 mmol) and 1 g SA (2%) were added into 50 ml


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water and mixed by a magnetic bar until total solution. The iron wire (0.7 mm, 1.60 mm) was immersed into solution to allow polymerization. The polymerization was performed statically for 20 mins at room temperature (20 oC). After polymerization, the hydrogel layer coated iron wire was fast immersed into CaCl2 solution (2%). After 2 h later, the gel layer coated iron wire was taken out and the iron wire was removed. The resulted PHEMA/SA-Ca hydrogel tube was immersed into deionized water for 2 weeks to remove unreacted monomer and free SA polymer chains. 2.2. Mechanical property testing of the hydrogels tubes. Mechanical tensile-stress experiments of high strength gel tubes were performed using a tensile machine at a stretch rate of 100 mm min-1. The tensile experiments were performed at 20% humidity at room temperature. After the gel tubes sufficiently swelling, the outside of the gel tubes was coated with one layer of organic silicone oil to avoid the evaporation of water during the testing. For each sample, at least 5 parallel tests were employed for statistics. The tensile stress is defined as: u=F/A, where F is the applied force on the hydrogel tubes, and A is the calculated cross-sectional area of the hydrogel tubes. In typical case, A=πR2 -π(R-d)2, in which R is the outer radius of hydrogel tube and d is the wall thickness of the hydrogel tube. The tensile strain is defined as: e=(L-Lo)/Lo × 100%，where L is the real-time gauge length of the hydrogel tube, and Lo is the initial gauge length of the hydrogel tube. The Young’s modulus of the hydrogel tube is calculated by the slope of the stress–strain curve at the 5-15 % strain range. The value of tensile strength for the hydrogel tube is recorded at the break peak, while the value of the tensile strain is defined as largest elongation ratio at break peak. The cyclic tensile experiments were performed with 100% tensile strain and 100 mm min-1 stretching rate in a constant humidity condition, and the outside of the gel tube is coated with organic silicon oil. 2.3. Fabrication of 3D PAAm/PAA-Fe hydrogel tubes composites. The iron wire (diameter 0.70 mm) was processed for spring shape with spiral radius 1.5 cm, spiral distance 1.0 cm, total length 10.0 cm. The monomer solution was degassed for 20 min with high purity nitrogen, and then was injected into the mold. The bottom of the mold was processed into hydrophilic by O2 plasma for 1 min, and then was assembled



of 1H,1H,2H,2H-perfluorooctyltrichlorosilane. Next, the spring was immersed into monomer solution to allow polymerization for 20 mins at room temperature (20 oC), and then followed by coordination in Fe3+ (0.03 M/L) solution for 20 h. The iron wire was removed along the rotation direction of the spring and the obtained gel tube was immersed into water to remove free Fe3+. The suspension of magnetic Fe3O4 nanoparticles was injected into the channel of the hydrogel spring tube, and then was driven by a super strong magnet. The “Y” type hydrogel tubes unit and double layer hydrogel tubes array shown in Fig. 7 were prepared by the same experiment procedure. The PDMS was prepared from a commercial silicone elastomer kit (SYLGARD 184 silicone elastomer, base and curing agents, Dow corning, Midland, MI, USA). The base and curing agents of SYLGARD 184 elastomer kit were mixed at 10: 1 ratio (by weight). The PDMS mixtures were transferred into the hydrophobic mold after removing bubbles under gentle vacuum. Then the “Y” type hydrogel tubes unit and array were immersed into the mixture, and then followed a secondary degassing process. After the mixtures flowed flat, incubation was performed in a vacuum drying oven (70 °C) for 2 h.

3. Result and Discussion. 3.1 Mechanism for preparing PAAm/PAA-Fe hydrogel tubes. The method of surface catalyzed fast molding of hydrogels. The mechanism of surface catalyzed radical polymerization and fabrication of hydrogel tubes are schematically shown in Scheme 1. It is well known that potassium persulfate (KPS) can decompose to generate free radicals, a the process that is widely used in aqueous solution free radical polymerization. The process can be accelerated by external triggers, such as amine or aniline accelerators,[62] light,[63] or plasma irradiation.[64] Herein, we used a high purity iron wire as catalyst source to increase the polymerization rate by lowering the decomposition activation energy in chain initiation [60, 65] (Scheme 1a). In a typical case, the Ed (decomposition activation energy) for generating free radical SO4·- from homolysis of S2O82- is ~140 kJ/mol (Ed1), thus requiring heat to initiate the polymerization. However, the redox reaction between Fe2+ and S2O82-, decreases the Ed to


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~50 kJ/mol (Ed2). Minor Fe2+ dissociated from iron wire will accelerate the reaction that takes place at the solid-liquid interface, allowing the Fe wire to act as a template. As shown in Scheme 1b, the polymerization of acrylic acid (AA) and acrylamide (AAm) at room temperature rapidly forms a layer of homogeneous and transparent hydrogel film after immersing the iron wire into a mixed monomer solution containing potassium persulfate (KPS) and N, N'Methylenebisacrylamide (BIS). Since catalytic Fe2+ species are only generated close to the wire surface due to the oxidation of pure iron, the free radical polymerization uniformly occurs around wire surface. As a result, the wire surface was uniformly wrapped with one layer of PAAm/PAAc hydrogel layer. It can be imaged that hollow hydrogel tube may be obtained after removing the iron wire template. However, it is found that the iron wire is difficult to be removed because of low mechanical strength of the formed PAAm/PAAc hydrogel layer (Figure S1a, Supporting information). Even though increasing the crosslinking degree of the hydrogel layer can improve its mechanical strength, it is still impossible to remove the iron wire (Figure S1b, Supporting information). So, transformation from the hydrogel layer to hydrogel tube materials requires significantly strengthening of the gel layer. Soaking in the concentrated Fe3+ (0.03 mol/L) solution leads to the formation of a secondary crosslinked network, which significantly improves the mechanical property of hydrogel layer.[66] The mechanism responsible for this is based on the coordination between carboxyl and Fe3+ with molar ratios of ~3:1. As a result, the iron wire can be easily pulled out due to the slippery nature of the inner surface of the hydrogel layer (Scheme 1c, Figure S2a, Supporting information). Correspondngly, the hollow hydrogel tube is obtained. Next, the asprepared PAAm/PAAc hydrogel tube is immersed into deionized water to remove non-coordinated Fe3+ in the hydrogel network. By contrast, when immersed the PAAm/PAAc hydrogel layer into Ca2+ or Na+ solution, the mechanical strength of the hydrogel layer does not increase that much, so it is still difficult to remove the iron wire and impossible to obtain hydrogels tube (Figure S2b and S2c, Supporting

information).



Scheme 1│ │Schematic depiction of preparing hydrogel tubes with ironII catalyzed SRP method. (a) Growing mechanism of hydrogel layer on iron wire. After immersing into monomer solution (containing initiator: KPS; crosslinker: BIS), the iron wire surface fast forms a layer of transparent hydrogel film based on the K2S2O8-Fe(II) redox reaction. A typical example to prepare PAAm/PAA-Fe hydrogel tube by SRP method. After immersing into AA/AAm monomer solution (containing initiator: KPS; crosslinker: BIS), the iron wire surface fast forms a layer of transparent hydrogel film. Then followed by immersing into Fe3+ solution (b) and remove iron wire (c), we obtain high strength hollow hydrogel tubes. (Monomer mass concentration ratio: AA/AAm: 1%, 5%, 10%, 20%, 40%; initiator mass concentration ratio: KPS0.01%; crosslinker mass concentration ratio: BIS-0.01%; Fe3+ concentration: 0.03 mol/L). 3.2 Preparation of PAAm/PAA-Fe hydrogel tubes. The polymerization kinetics is characteristic of free radical polymerization and usually very fast. Unless special instruction, the polymerization time was kept constant at 10 mins to ensure the surface flatness. Figure 1a shows that within 10 min polymerization on ϕ1.6 mm iron wire, apparent PAAm/PAA-Fe hydrogel tubes are obtained. The tubes wall thickness can however be controlled by the subsequent coordination time of PAAm/PAA with Fe3+, both the outer diameter D and tube wall thickness d decrease with the coordination time because the coordination interaction will dramatically shrink the hydrogel network (Figure 1b). When the coordination time was extended to 40 h, the size didn’t change


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any more, indicating a saturated physical crosslinking network was formed between PAA and Fe3+. Polymerization on variety iron wires of with different radius results hydrogel tubes with different diameters (Figure 1c). The experimental results showed that large radius iron wire led to hydrogel tubes with larger thickness at the same polymerization time (Figure 1d). As reported, increasing the concentration of iron will accelerate the degradation of persulfate, resulting in a larger value of first-order reaction rate constant (k).[67] In our experiment, iron wire with large radius possesses larger surface

area based on the higher catalyst concentration than that of small radius (Figure S3, Supporting information), resulting in a large value of first-order reaction rate constant (k). So, under the same polymerization time, iron wire with larger radius can generate larger thickness hydrogel layer. Further, it was found that the size for the as-prepared PAAm/PAA-Fe hydrogel tubes could also be well controlled by adjusting the polymerization time in hydrogel layer formation (Figure 1e). Both the outer diameter D and tube wall thickness d increase with the polymerization time. When the coordination time exceeded 30 h, it is found that the surface of the formed hydrogel layer became uneven because catalyst diffusion is limited. Moreover, adjusting the relative mass concentration ratio of AAc/AAm can also affect the size for the resulted PAAm/PAA-Fe hydrogel tubes (Figure 1f). After systematically investigating, it was found that the surface morphology of the as-prepared hydrogel tube at AAc/AAm 10 % kept perfectly flatness and uniform size distribution (Figure S4, Supporting information). In a word, the size of the as-prepared hydrogel tubes can be finely controlled by adjusting the coordination time in Fe3+ solution, iron wire diameter, polymerization time and mass concentration ratio of AAc/AAm.



Figure 1 │ Polymerization formation of the PAAm/PAA-Fe hydrogel tubes. (a) Optical image showing the as-prepared PAAm/PAA-Fe hydrogel tubes with increasing coordination time in Fe3+ solution (0.03M/L) (iron wire: 1.6 mm, polymerization: 10 min) and (b) the effect of coordination time on the size of hydrogel tubes. (c) Optical image showing the as-prepared PAAm/PAA-Fe hydrogel tubes by iron wires with different diameters (0.2 mm, 0.45 mm, 0.7 mm, 1.6 mm and 4.0 mm; coordination time: 10 h; polymerization: 10 min) and (d) corresponding sizes of hydrogel tubes. (e) Effects of polymerization reaction time on the size of the PAAm/PAA-Fe hydrogel tubes with 10 h coordination in Fe3+ solution (0.03M/L) on 1.6 mm iron wire. (f) Effects of relative mass concentration ratio of AAc/AAm% on the size of PAAm/PAA-Fe hydrogel tubes with 10 h coordination in Fe3+ solution (0.03M/L) based on 10 min polymerization reaction. 3. 3. Mechanical property testing for PAAm/PAA-Fe hydrogel tubes. Subsequently, the mechanical strength of the as-prepared PAA/PAA-Fe hydrogel tube was investiagted. Firstly, the effect of coordination time for hydrogel layer in Fe3+ solution on strength of the as-prepared hydrogel tube was investigated. As demonstrated in Figure 2a and Figure S5 (Supporting information), the hydrogel tube prepared with 15 mins Fe3+ coordination shows low tensile strength of less than 0.1 MPa and low strain of 170 %. With the increasing of coordination time to 5h, the hydrogel tube demonstrates high tensile strength of 1.0 MPa and large strain of close to 275 %. To further increase the


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coordination time to 10 h, it is found the tensile stength can reach to 1.6 MPa while the strain decreases obviously to 160 %. With 20 h Fe3+ coordination, the prepared hydrogel tube shows high tensile strength of 2.5 MPa, but only with low strain of 140 %. When the coordination time was extended to 40 h, it was found that both the tensile strength and strain kept unchanged, indicating a saturated physical crosslinking network formed between PAA and Fe3+. Meanwhile, it was found that the elastic modulus of the prepared hydrogel tube increased obviously with the extending of coordination time (Figure S6, Supporting information). As demonstrated in Figure 2b and Figure S7(Supporting information), the tensile strength of the hydrogel tubes with different sizes nearly keeps similar value in the range of 1.6~2.0 MPa but presents slight increasing, while their strain increases obviously with extending the sizes. This result is completely different from the traditional hydrogel system, for which its strength and strain is independent of thickness. The possible reason responsible for this can be analyzed as below. The larger iron wire possesses larger surface area, which would provide more adequate space volume to allow the reaction. Under the same polymerization time, the iron wire surface with larger size would generate thicker and more uniform hydrogel network. By contrast, the iron wire surface with smaller size would generate thinner and nonuniform hydrogel network because of reaction space limitation. Especially for the iron wire with smaller size, once its surface was covered by one layer of hydrogel flim. Subsequent increasing of the hydrogel layer thickness would become difficult because of its small reaction space. The speculative mechanism can be further confirmed by the SEM characterization of the hydrogel tubes network (Figure S8, Supporting information). As shown in Figure S9 (Supporting information) and Figure S10 (Supporting information), it was found that the tensile strain of the formed gel tubes increased from 60 % (polymerization time: 1 min) to 350 % (polymerization time: 30 mins), while the tensile strength achieved maximum at reaction time of 10 mins (1.60 MPa) and then decreased with the polymerization time. As analyzed, prolonging the polymerization time would increase the thickness of the hydrogel layer but weaken its flatness and uniformity. Further, it was found that reducing the mass ratio of AAc in monomer solution would weaken the tensile strength but improve the strain of the formed gel tubes (Figure S11 and Figure S12, Supporting information). By contrast, too high mass ratio of AAc in monomer solution leads



to excessive cross-linking coordination of Fe3+ with COO- in gel network, which makes the gel tubes become hard and brittle. In a typical cases, the hydrogel tube prepared at AAc/AAm 5.0 % shows low tensile strength of 0.28 MPa but high strain of 680 %, while the hydrogel tube prepared at AAc/AAm 40 % shows strain of only 20 % and while tensile strength of 0.48 MPa. At the mass concentration ratio of AAc/AAm 10.0 %, the tensile strength of the as-prepared gel tube achieves maximum, and presents considerable strain of 170%. Furthermore, the macroscopic demonstration experiments were performed to evaluate the elasticity of the as-prepared hydrogel tube. When the hydrogel tube was filled with water and stretched ~5 times, it was observed that the tube wall became uniformly transparent but without water leakage (Figure 2c), indicating a highly compact microstructure. The good elasticity of the as-prepared hydrogel tube was also characterized by the radial tensile test. As demonstrated in Figure 2d, the tube could be easily stretched from 5.09 mm to 19.28 mm and recovered to 5.89 mm in less than 2 seconds, after which the tube could be stretched again to 23.96 mm. Importantly, no damage to the gel tube is observed after getting through continuous stretching cycles, which indicates its good shape memory, elasticity and self-recovery properties (Figure S13, Supporting information). Moreover, continuous stretching test was employed to evaluate the fatigue-resistance property of the hydrogel tube. As shown in Figure 2e, with extending the stretching times, the strength of gel tube (template: 1.6 mm iron wire; polymerization: 10 mins; coordination: 20 h) reduced gradually. After 100 cycles at 100 % strain, the tensile strength of gel tube levels off at 0.87 MPa. Then the tube was immersed into pure water to allow 5 hours recovery, followed by 100 times loading and unloading cycles with 100 % strain. The mechanical strength could also recover to the original level.


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Figure 2│ │ Mechanical tensile properties of the as-prepared PAAm/PAA-Fe hydrogel tubes. (a) Tensile strain-stress curves of the PAAm/PAA-Fe hydrogel tubes with increasing coordination time in Fe3+ solution (0.03M/L) using 10 mins polymerization reaction on iron wire (1.6 mm) and (b) tensile strain-stress curves of the PAAm/PAA-Fe hydrogel tubes with 10 h coordination time in Fe3+ solution (0.03M/L) using 10 mins polymerization reaction on iron wires with different sizes (0.2 mm, 0.45 mm, 0.7 mm, 1.6 mm and 4.0 mm). (c) Optical image showing the hydrogel tubes with good stretching elasticity along the longitudinal direction. (d) Optical image showing the hydrogel tubes with extraordinary elasticity and fast self-recovery along radial direction. (e) 100 times loading-unloading cycles test with 100 % tensile strain at 100 mm min-1; after recovery in water for 6 h, the hydrogel tubes show good strength and elasticity as before. Especially for the tubular hydrogel material, evaluating its pressure resistance property both in liquidfilled and gas inflated state is very necessary. So, we tested the anti-pressure property of the hydrogel tube under very demanding conditions. As shown in Figure 3a, the as-prepared gel tubes were filled with water, the two sides were closed, and then compressed by a 4.0 kg steel block. The gel tube remained intact



without leaking water. Further, we investigate the gas-pressure resistance of the as-prepared hydrogel tube by gasing high purity nitrogen. As shown in Figure 3b, the hydrogel tube with 30 min coordination (template: 1.6 mm iron wire; polymerization: 10 mins) could support ~0.065 MPa (489 mm Hg) N2 pressure, which is far more than that of systolic and diastolic blood pressure for blood vessels (below 150 mm Hg). By contrast, the hydrogel tube with 20 h coordination time could support ~0.1 MPa (752 mm Hg) N2 pressure without obvious elastic inflation. Such results indicate that the gas-pressure resistance property of the hydrogel tube is highly tunable. Very interestingly, we found that the expansion of the hydrogel tube with weak iron cross-linking (30 min coordination) is highly reversible at controllable gaspressure condition. As shown in Figure 3c, one side of the as-prepared hydrogel tube (Polymerization:10min; coordination:0.5 h; iron wire:1.6 mm) was enclosed while the other side of tube was connected to nitrogen source, the tube can realize continuous/switchable expansion and shrinkage by intermittently feeding the N2 flow (Figure 3c, Video S1). Meanwhile, it was also found the expanded gel tube wall was still structural compactness. For example, when we injected water into the expanded hydrogel tube and then followed the violent shaking, no water was observed and found on outside surface of the tube (Figure 3d, Video S2). All these results show that the as-prepared PAAm/PAA-Fe hydrogel tube has very good pressure resistance property, along with its tunable elasticity.


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Figure 3｜｜Anti-pressure and expansion property of the as-prepared PAAm/PAA-Fe hydrogel tubes. (a) Optical image showing the water filled hydrogel tubes are strong to support high pressure under rolling crush by 4.0 kg stainless steel block without destroying. (b) Optical image showing high elastic expansion of the hydrogel tube (Polymerization: 10 mins; coordination: 0.5 h; iron wire:1.6 mm) as a balloon under 0.065 MPa nitrogen flow, and low elastic expansion of hydrogel tube (Polymerization: 10 mins; coordination: 20 h) under 0.1 MPa nitrogen flow. (c) Continuous/switchable expansion and shrinkage process of the hydrogel tube (Polymerization: 10 mins; coordination: 0.5 h; iron wire:1.6 mm) with the assistance of N2 flow. (d) Injecting water into the expanded hydrogel tube and then following the violent shaking.

3.4. Building up complex 3D PAAm/PAA-Fe hydrogel tubular structures. 17 ACS Paragon Plus Environment


In addition to the easy preparation of single hydrogel tube, the current method is also highly available for construction of PAAm/PAA-Fe hydrogel tubes with complex 3D structures. A series of complex gel tubes units may be well prepared by using different iron templates arrangements. As shown in Figure 4a, by placing iron wires side by side, multichannel hydrogels could be prepared, whereas stacked multichannels can be obtained from stacked iron wires (Figure 4b). Importantly, hydrogel tubes remain unconnected even when using twisted two iron wires (Figure 4c). We expanded the multichannel idea by combining 27 iron wires into an ordered spaced 3D network (Figure 4d). As no gaps exist among these iron wires, the formed hydrogel tubes in one layer directly connect with those in another layer. By increasing the gaps between iron wires from d = 0 to d = 2r (Figure S14, supporting information), these 3D hydrogel multichannel tubes became disconnected. The flexibility and robustness of our approach was further demonstrated by the fabrication of more complex hydrogels such as branched hydrogel tubes (Figure 4e) which can be used for liquid transportation and building up reaction circuits that allow different kinds of liquids to mix and react each other (Video S3, Figure S15, Supporting information). We can even form a 3D hydrogel tube spring (Figure 4f). Due to the elastic nature of the gel tube, the asprepared gel spring could be stretched along the axial direction over 10 times without losing the original shape. By injecting magnetic iron oxide suspension into the channel of the gel spring, the stretching could be continuously and reversibly operated by applying super strong magnet (Figure S16, Supporting information). Furthermore, by placing iron wires with different sizes together, highly branched hydrogel tubes system was well engineered (Figure 4g), providing a new inspiration for constructing blood capillaries or complex channels. By placing different iron wires vertically oriented, integrated hydrogel tubes array were obtained (Figure 4h).


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Figure 4│ │ Schematic preparation and optical image of complex PAAm/PAA-Fe hydrogel tubes units, arrays and channels. (a) Parallel but disconnected hydrogel tube arrays. (b) Multiple hydrogel tube channels. (c) Twisted disconnected hydrogel tubes. (d) Large-scale three-dimensional interlinked hydrogel tubes array. (e) Branched hydrogel tubes. (f) Magnetic hydrogel tube spring. (g) Blood vessel like linked and interlinked hydrogel tube network with different diameters (from 0.5 mm to 5.0 mm). (h) Threedimensional disconnected hydrogel tubes array.

3.5. Constructing hydrogel tubes embedded PDMS composite as fluidic device. Although

many

approaches

have

been

developed

for

constructing

porous

PDMS

(polydimethylsiloxane) system,[68] it is still difficult for constructing hydrophilic network channels in such low surface energy hydrophobic system, owing to lacking of effective technologies. Fortunately, our current method allows to easily embed high strength hydrogel tube or tubes array in the PDMS piece. In a typical case, after polymerization and coordination, the gel layer wrapped iron wires were coated with



organic silicone and then embeded into PDMS monomer solution for heat-polymerization. Followed by removing the iron wires after PDMS solidification, we engineered hydrogel tubes embeded PDMS composite. As expected, we could prepare parallel (Figure 5a), “Y” type (Figure 5b) and hydrogel tubes array (Figure 5c) embedded PDMS composites. Meanwhile, due to hydrophilic nature of hydrogel, the gel tube can be used as functional channel for transporting liquid. As for the “Y” type gel tube embeded composite, different kinds of liquid could mix or react in such device (Video S4). Especially for the hydrogel tubes array embeded PDMS composite, the injected liquid can be freely transported in multiple directions among hydrophilic gel channels (Video S5 and Video S6). So simply integrating such hydrogel tube or tubes array into hydrophobic PDMS substrate enables to the successful construction of functional fluidic device. Meanwhile, the cavity of the hydrogel tube or tube arrays can act as hydrophilic channels for directly transporting and mixing liquid, while PDMS works as flexible support substrate. Even though the size (outer diameter) of the embeded hydrogel tubes is relatively large in Figure 5, the channel size of the embeded hydrogel tubes in PDMS is in micron scale. It can be speculated that hydrogel tube with smaller channel size may be available only by employing iron wires template with samller diameters (Figure 1d). Such results well expand the route for developing functional PDMS microfluidic devices.[68]


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Figure 5｜｜Preparation of hydrogel tubes embedded PDMS composites and used as fluidic devices. From the top to the bottom, optical image showing the cross-section, the front and the flow transportation experiments of the hydrogel tubes embedded PDMS composites: as parallel to transport water (a), “Y” type to mix liquid A and liquid B (b), and tubes array to transport liquid B (c). (Liquid A and liquid B were prepared by adding two different kinds of water-soluble dyes) 3.6. Universality of the SRP method for the preparation of other kinds hydrogel tubes and evaluating their bioactivity. Polymer hydrogel scaffolds have been widely researched as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue. Despite tremendous effort on creating micro/nano structures to improve cell adhesion for tissue engineering, such as porous hydrogels or electro-spun fibrous hydrogels,[69-70] fabrication of macroscopically shaped hydrogels for combining tissue replacement and tissue engineering remains a challenge. The method we presented here, allows us



to design multicomponent hydrogels with complex shapes. Figure 6a1 shows hydrogel tubes made by SRP of hydroxylethylmathacrylate (HEMA) in the presence of alginate, resulting in PHEMA/SA hydrogel layers that can be further crosslinked by calcium ions. In a similar way, we can introduce PVA during SRP of HEMA or copolymerization of acrylamide and acrylic acid (AAm/AA). PVA/PHEMA and PVA/PAAm/PAA hydrogels are not mechanically strong, but after several freeze-thaw cycles to allow PVA crystallization, hydrogel tubes can be peeled off (Figure 6a2-6a3). By using oligoethylene glycol methacrylate (OEGMA), we were able to produce POEGMA/PAA, POEGMA/PHEMA/PAA, POEGMA/PVA/PAA, POEGMA/PHEMA/PVA/PAA, POEGMA/ SA-Ca hydrogel tubes without further toughening (Figure 6a4-6a8). These hydrogel tubes are potentially biocompatible and can be used as scaffold in tissue engineering. Moreover, the mechanical property of these hydrogel tubes were investigated (Figure S17-S24, Supporting information). Meanwhile, PVA/PHEMA hydrogel tube showed good tensile stress and strain than that of other systems. Upon desirable arrangement of iron wires template, it is also possible to engineer complex PVA/PHEMA hydrogel tubes system (Figure S25, Supporting information). Especially, when immersed in aqueous cell culture media (Figure S26, Supporting information) and saline (Figure S27, Supporting information) for as long as 12 days, or encountered 12 hours of induced flow at physiological Reynolds (Figure S28-S31, Supporting information), the PVA/PHEMA hydrogel tube can still maintain its stability without obvious swelling and dissolution problems found by others.[71-72] This result showed the as-prepared PVA/PHEMA hydrogel tube was a good candidate system for performing the biological experiment. To demonstrate the suitability of PVA/PHEMA hydrogel tube for cell culture, diploid human fibroblasts (WI-38) and mouse embryo fibroblast cells (NIH3T3) were cultured on the matrix surface, after coating the hydrogel with a collagen layer (soaking in 50 µg/mL solution) to improve cell adhesion. To explore the potential for forming stable vasculature of the PVA/PHEMA hydrogel tubes, we also cultured pulmonary artery endothelial cells (PAECs) on the inner side surfaces of tubes. All three types of cells could spread and maintain their phenotype after incubating for 3 days (Figure 6b). We fabricated a simple tissue of channels lined with PAECs which were incubated in the growth medium [DF plus 10% (vol/vol) FBS], and subsequently


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formed a confluent monolayer that was similar to walls of blood vessels (Figure S32, Supporting information). To confirm the biocompatibility of matrix, cells were stained by calcein AM and ethidium homodimer-1 after 3 days incubation. The confocal image shows that the majority of PAECs were live (Figure S33, Supporting information) which can be compared to the results of MTT assay. Cell proliferation was measured by the MTT assay at 1, 3, 5 and 7 days culturing time, with the TCP culture plate as control group (Figure 6c). The OD values of PAEC cultured on the PVA/PHEMA matrix increased sharply with time, and cells proliferated quickly at the initial stages of incubation and reached the highest speed at 3 days, leveling off after 5 days. Similar experiments for NIH3T3 cells showed smaller differences in OD values between PVA/PHEMA hydrogel tubes and TCP

(Figure S34,

Supporting information). Overall, the MTT assay data and live/dead staining image confirmed the good cytocompatibility of PVA/PHEMA hydrogel tubes. For PAECs, the changing of morphologies with incubation time extending also further confirmed the cells proliferation on the matrix. The cells dispersed sparsely and shrank themselves meanwhile exhibited less filopodias when incubated just for 16 h (Figure 6d). As for 40 h, not only did the cells proliferate and congregate into pieces of monolayer, but also their morphology obviously changed into elongated spindle-like shapes and stretched into larger areas with numerous filopodia extended on the cells surfaces (Figure 6e). As for longer period 7 days the cells can connect to each other to form an unoriented monolayer with actins extended to exhibit typical “cobble stone” morphology (Figure 6f). When coming to 12 days, the F-actin of PAECs developed into much clearer and finer, and even exhibited fibril-like skeletons (Figure 6g). Alpha smooth-muscle actin (α-SMA) is one of mammalian cytos[73] and a feasible marker for characterizing the matrix maturation stage. Endothelial cells in some situations can transdifferentiate into the smooth muscle-like cells which can express α-SMA,[74-75] and the activity of SMA is greatly enhanced during vascular tissue maturation. A successful vascular scaffold must demonstrate the support for enhanced SMA expression. From Figure 6f, it is seen that after 7days and 12 days, the cells all expressed some α-SMA which located near the nuclei and F-actin and the rising of α-



SMA was partly due to the accumulation of some cells that can express α-SMA such as smooth muscle cells, fibroblasts and pericytes. Taken together, these results demonstrate that our material possess a greater ability to support functional expression of vascular-related protein. Collectively, all the above biological data demonstrated that the material fabricated by SCIP method is suitable for the application of biology. They have low cytotoxicity for multi cells and can provide long time supports for cells spreading, elongation and proliferation. With the expression of α-SMA, it demonstrates potentials for developing into blood vessels, which will be extensively studied in the future.


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Figure 6│ │Universality of the SRP method for the preparation of other kinds hydrogel tubes and evaluating their bioactivity. (a) Photographs of the prepared hydrogel tubes, (a1) PHEMA/SA-Ca, (a2) PVA/PAAm/PAA, (a3) PVA/PHEMA, (a4) POEGMA/PAA, (a5) POEGMA/PHEMA/PAA, (a6) POEGMA/PVA/PAA, (a7) POEGMA/PHEMA/PVA/PAA and (a8) POEGMA/SA-Ca by SRP. (b) WI38, NIH3T3 and PAEC cells growing on the inner surface of PVA/PHEMA hydrogel tubes (Scale bar: 50 µm). (c) Cells viability assay of PAEC inside PVA/PHEMA hydrogel tubes after incubating over one-week period. Fluorescent image of stained PAECs on the inner surface of PVA/PHEMA hydrogel tubes with



incubated for 16 h (d) and 40 h (e) (Scale bar: 50 µm), cell skeleton was stained by phalloidin (red) and nuclei were stained with DAPI (cyan). Immunocytochemical analysis of protein expression of α-SMA (blue) after cells were incubated for 7 d (f) 12 d (g) (Scale bar: 10 µm).

4. Conclusions In summary, we have shown here how surface catalytic radical polymerizations rapidly initiates polymerization to produce very thick hydrogel layers that homogeneously cover the catalytic substrate. Additional formation of a coordinatively-crosslinked network or through post-treatment produces a series of tubular hydrogel objects with different chemical components. Tube diameters ranged from a few hundred microns to a few millimeters, while the tube wall thickness could be tuned from dozens of microns to a few hundred microns. The as-prepared hydrogel tubes can exhibit various applications including microfludic device and biological experiment. Meanwhile, the excellent stability and biocompatibility of PVA/PHEMA hydrogel tubes opens up new avenues for fabricating tubular hydrogel systems for biomedical applications including the design of vascular-like hydrogel extracellular matrix for tissue engineering, and engineering bioinspired functional composite materials.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Details for performing biological experiments, photographs/optical microscope photos/SEM images of the hydrogels tube, additional results from mechanical property, bio-relative experiments; PartⅠand PartⅡ: Figure S1-S34 (PDF).

Acknowledgements We gratefully acknowledge support from the National Key Research and Development Program of China (2016YFC1100401), the National Natural Science Foundation of China (21434009, 51705507) and CAS (QYZDY-SSW-JSC013). Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))


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The table of contents entry: We report a generalized method to engineer single or complex 3D hydrogels tube system, based on the interfacial redox reaction between Fe2+ and S2O82-. The as-prepared hydrogel tubes show tunable mechanical strength, toughness and elasticity while the size is highly controllable. Such hydrogel tubes can be successfully used as fludic device and vascular-like extracellular matrix for tissue engineering.

Shuanhong Ma, Mingming Rong, Peng Lin, Min Bao, Jing Xie, Xiaolong Wang, Wilhelm T. S. Huck, Feng Zhou*, Weimin Liu

Fabrication of 3D tubular hydrogels materials through on-site surface free radical polymerization TOC (8.25 cm×4.45 cm)


Fabrication of 3D Tubular Hydrogel Materials through On-Site Surface

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