Double-Cross-Linked Hydrogel Strengthened by UV Irradiation from a

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Letter Cite This: ACS Macro Lett. 2018, 7, 509−513

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Double-Cross-Linked Hydrogel Strengthened by UV Irradiation from a Hyperbranched PEG-Based Trifunctional Polymer Qian Xu,† Sigen A,*,† Peter McMichael,‡ Jack Creagh-Flynn,† Dezhong Zhou,† Yongsheng Gao,† Xiaolin Li,† Xi Wang,† and Wenxin Wang*,†,§ †

Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin 4, Ireland Institut National Polytechnique - Ecole Nationale Supérieure des Ingénieurs en Arts Chimiques Et Technologiques (INP-ENSIACET), Toulouse, France § School of Mechanical & Materials Engineering, University College Dublin, Dublin 4, Ireland ‡

S Supporting Information *

ABSTRACT: Conventional wound healing materials suffer from low efficiency, poor mechanical strength, and nontunable properties. Responsive hydrogels are appealing candidates for tissue engineering. Herein, we developed a double-cross-linked hydrogel system composed of hyperbranched PEG-based polymer, comprising pre-cross-linked acetal structure and numerous terminal acrylate groups, which can form hydrogels in situ and can be further strengthened by UV irradiation. The hyperbranched glycidyl methacrylate-co-poly(ethylene glycol) diacrylate polymers (HB-GMA-PEGs) were first synthesized via in situ deactivation enhanced atom transfer radical polymerization (DE-ATRP). A series of pre-cross-linked materials were achieved after postfunctionalization. The material can absorb a high amount of water to form hydrogels, and the gel stiffness was evaluated in detail before and after UV irradiation. The in vitro cytotoxicity experiments were conducted with the resulting materials and have demonstrated their good biocompatibility. The results indicate that this type of hydrogel with high water uptake capacity has appealing potential as a responsive biomaterial for wound closure.

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reported a new type of hyperbranched polymer via controlled/ living polymerization of multivinyl monomers.7,8 This type of polymer breaks the long accepted view as F−S mean field theory predicted: polymerization of multivinyl monomer will result in a bulk cross-linked gel.9,10 The hyperbranched polymers with abundant terminal acrylate groups can be postfunctionalized to introduce more functional groups into the polymers on demand.11 Hydrogels possess the abilities that make them appealing candidates for biomedical application. They are three-dimensional polymer networks with high water content, porosity, oxygen and nutrient permeability, and capacity of mimicking the stiffness of natural living tissue. Hydrogels have been utilized in numerous medical applications, especially for tissue engineering. Polymeric hydrogels have been considered to be promising candidates as tissue sealants due to their easy modification and abundant resources.12 PEG-based synthetic hydrogels show good biocompatibility as PEG is an FDA-approved reagent. FocalSeal-L (Genzyme Biosurgery, Inc., Cambridge, MA), which is an FDA-approved product, has been proven to be efficient in clinical use. However, it

ach year, millions of people suffer from the postoperative effects of surgery and traumatic skin wounds which need effective hemostatic and wound closure management.1,2 Suturing is a conventional technique which has been used for thousands of years. Surgeons use suturing methods to stop bleeding and close wounds. While there have been numerous modern suturing needles and threads developed, the efficiency of suturing techniques is low. Besides, suturing will cause inevitable damage of the surrounding tissue, nerves, or vessels. Staples, which are a type of alternative to sutures, show easy and faster operation process during application. However, staples are prone to causing even more damage to surrounding tissues and scar formation. Synthetic biomaterials developed for wound closure have presented promising features during recent years. These synthetic materials can be designed with desirable characteristics, such as mechanical strength, biocompatibility, absorbability, and usability. The most important synthetic wound closure materials include polycyanoacrylates,3 poly(ethylene glycol) derivatives,4 polyurethanes, polyesters, dendrimers, and hyperbranched polymers.1 Hyperbranched polymers have attracted much attention in many important applications since first defined by Kim and Webster in the 1990s.5 The unique three-dimensional structure endows them various superior properties, e.g., low viscosity, high solubility, as well as broad functionality range.6 Wang et al. © XXXX American Chemical Society

Received: February 15, 2018 Accepted: April 5, 2018

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DOI: 10.1021/acsmacrolett.8b00138 ACS Macro Lett. 2018, 7, 509−513

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ACS Macro Letters requires a long time to trigger the photoactivation which is a significant drawback of this product.13,14 Current studies for wound closure also possess significant disadvantages which hinder the wound healing process, such as limited range of mechanical properties, potential toxicity, and lack of hemostasis. 15,16 Among these, highly cross-linked hydrogels generated at low polymer concentrations with an enhanced mechanical strength are still challenging. Double-cross-linked hydrogels have been extensively reported in recent years.17−25 Due to the superior mechanical strength of double-cross-linked hydrogels, they have been utilized in biomedical fields such as drug delivery23 and tissue adhesive.25 Responsive hydrogels, as another ideal candidate for tissue engineering and regenerative medicine, exhibit multifarious changes upon external stimuli such as pH response,26,27 thermal response,28−30 and light response.31−33 The highly controllable manner makes these types of hydrogels appealing to meet different requirements. The development of a doublecross-linked hydrogel with external-stimuli property is of great interest as a wound healing material. In this work, a new type of UV-responsive double-crosslinked hydrogel system was developed by a self-cross-linkable multifunctional polymer. Through a postfunctionalization approach, a hyperbranched PEG-based trifunctional polymer bearing terminal acrylate groups, aldehyde groups, and diol groups was synthesized (Scheme 1). Due to the pre-cross-

Scheme 2. Schematic Diagram of the Application of the Double-Cross-Linked Hydrogel As a Wound Closure Material for Skin Wounds and Gel Strengthening Process by UV Irradiation

photoinitiator, the pre-cross-linked material can generate a hydrogel which may be applied to cover the wound bed. The hydrogel stiffness will then be significantly strengthened after UV irradiation. Figure 1A−E and Tables S1−3 show the GPC results of the copolymerization of GMA and PEGDA575 at the molar ratio of 1:2, 1:4, and 1:8 via the in situ DE-ATRP approach. In order to achieve a hyperbranched polymer structure, the ratio of the initiator to divinyl monomer (PEGDA) was fixed to 1:3.7,8 As can be seen from the GPC traces of real-time monitoring of the polymerization (Figure 1A−C), the polymer peaks were growing gradually with the decrease of the monomer peaks during the polymerization process. The polymerizations were stopped before the interchain cross-linking stage (where multiple polymer peaks will show up) in order to achieve an appropriate polydispersity. Purified HB-GMA-PEGs with the Mw between 15 kDa and 20 kDa and PDI below 2.0 were selected for further experiments (Figure 1D). The Mark− Houwink values (α) of the purified polymers were below 0.5 which indicated the branched structure of HB-GMA-PEGs. As can be seen from the NMR spectra (Figure 2A), the three peaks at 5.84, 6.16, and 6.44 were attributed to the protons of the terminal acrylate group. The three peaks at 2.64, 2.85, and 3.23 were attributed to the protons of the epoxy group in the GMA unit. The polymer composition was calculated from the NMR results (Table S4). It is clear that the epoxy content of polymers can be regulated by the feed ratio of monomers. HB-Fs were synthesized by postfunctionalization of HBGMA-PEGs which contain two steps. First, HB-GMA-PEGs were hydrolyzed to afford HB-DIOL-PEGs through the ringopening reaction of an epoxy group in the GMA unit. As can be seen from Figure 2B, the epoxy peaks had disappeared after the first step of the reaction, which means all the epoxy groups were hydrolyzed and transferred into diol groups. Second, sodium periodate was used to oxidize the diol group to achieve the aldehyde group. From Figure 2B, a new peak at 9.62 was presented which was attributed to the proton of the aldehyde group. Moreover, the peaks of terminal vinyl groups were still appearing, indicating that the postfunctionalization process did not affect the acrylate groups in the polymer backbone. As can be seen from Table S5, the aldehyde content and oxidation degree were calculated from the NMR results, which showed a similar trend compared with the GMA feed ratio. These results clearly show that the aldehyde group was successfully

Scheme 1. Schematic Diagram of the Synthetic Pathway of Double-Cross-Linked Hydrogel As a Wound Healing Materiala

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HB-GMA-PEG polymers were synthesized via an in situ DE-ATRP approach. Post-functionalization process was conducted, and the product was purified by dialysis and freeze-drying.

linking reaction between the aldehyde group and diol group, the polymer can form a cotton-shaped foam that possesses high water uptake capacity. The remaining terminal acrylate groups can be further polymerized to generate a double-cross-linked structure, resulting in a mechanical robust hydrogel. Moreover, the functional group density, swelling profile, and mechanical strength can be easily adjusted by tuning the feed ratio of raw monomers. This study also investigated the biocompatibility of the newly designed double-cross-linked hydrogel. Specifically, hydrogels with a relatively low ratio of GMA content showed a better biocompatibility. The application of this type of material as wound healing material is shown in Scheme 2. After immersing in water with a certain amount of dissolved 510

DOI: 10.1021/acsmacrolett.8b00138 ACS Macro Lett. 2018, 7, 509−513

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ACS Macro Letters

Figure 1. Characterization of the copolymerization of GMA and PEGDA575 at different ratios using GPC. (A)−(C) GPC traces of the copolymerization. (D) GPC curves with single peaks for the purified polymers. (E) Mark−Houwink values (α) of the purified polymers.

Figure 2. 1H NMR spectra of the intermediate products and final HB-GMA-PEG polymers. (A) 1H NMR spectra of the three purified polymers obtained with different ratios of GMA and PEGDA575. (B) Representative 1H NMR spectra of the intermediate products with proton peaks for the HB-GMA-PEG1:2 polymer.

Figure 3. Rheological measurements of the hydrogels of HB-F formed by 30 s UV curing. (A)−(C) Storage (G′) and loss moduli (G″) values of all three hydrogels with the final concentration of 5% (A), 10% (B), and 20% (C). (D)−(F) Strain sweep (from 0.1 to 1000%) of all three hydrogels after UV irradiation with the final concentration of 5% (D), 10% (E), and 20% (F).

groups and the remaining diol groups. HB-Fs can be immersed into a high amount of water to achieve hydrogels (HBACETAL-PEGs). Due to the terminal acrylate groups in the

introduced into the polymer backbone. After purification and freeze-drying, the final polymers formed a pre-cross-linked structure, which is due to the reaction between the aldehyde 511

DOI: 10.1021/acsmacrolett.8b00138 ACS Macro Lett. 2018, 7, 509−513

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PEG1:4 and over 90% cells when cocultured with HB-ACETALPEG1:8 at both time points. A live/dead staining method was performed to observe the cell living status with HB-ACETAL-PEG1:8 at 24 h for both cell lines (Figure 4A and B). Fluorescent images showed that both cell lines showed normal cell morphology and a high degree of viability, verified by abundant living cells (green color). The cytotoxicity tests provided the evidence of material biocompatibility. In our study, these results emphasized that the HB-ACETAL-PEG1:2 hydrogels with higher GMA feed ratio and potentially higher aldehyde residual components may cause biological harm. In other words, with the increase of PEGDA as the cross-linking point, the hydrogels show elevated biocompatibility. Three UV-responsive double-cross-linked hydrogels were successfully generated by postfunctionalization of HB-GMAPEG polymers. The resulting materials were pre-cross-linked by acetal groups and possess an adjustable swelling ratio by changing the amount of GMA unit in the precursor polymer. Moreover, the numerous acrylate groups in the terminal chain endow these types of materials UV-curable property which can further enhance the gel strength. The swelling ratio of the precross-linked materials ranged from 400% to 800%. As for the gel strength, before UV irradiation, the G′ ranged from hundreds of Pascal to 27 kPa, whereas after UV irradiation, the G′ was significantly increased from thousands of Pascal to over 65 kPa. HB-ACETAL-PEG 1:4 and HB-ACETAL-PEG 1:8 showed a high cell viability of NHK and 3T3. The above results indicated that this type of material can be used as a promising wound healing material bearing a UV-responsive enhancing property.

polymer backbone, the hydrogel possesses a UV-responsive property. To prove this, the HB-Fs were immersed into a solution of I2959 (0.5 wt %) to achieve hydrogels with three different concentrations (5%, 10%, and 20% w/v). As can be seen from Figure 3A−C and Figure S4, the stiffness of the resulting hydrogels before UV cross-linking showed both crosslinking-dependent and concentration-dependent behaviors and the storage modulus ranging from 1.3 to 26 kPa, indicating an easily controlled stiffness profile. After 30 s of UV irradiation, the stiffness was dramatically increased 2-fold, resulting in a double-cross-linked hydrogel. The stiffness of the hydrogels ranged from 3.6 to 66 kPa, which was in line with the strength of soft to hard human tissue. Strain sweep tests were also conducted in order to determine if the critical strain value will be affected by UV irradiation. As can be seen from Figure 3D− F and Figures S1−S3, the critical strain value did not change significantly after UV irradiation (Figure S4). In order to determine the highest water uptake, the swelling ratio of HB-F foams was tested (Figure S5). The swelling ratio of HB-F1:2, HB-F1:4, and HB-F1:8 was around 400%, 710%, and 800%, respectively. It is clear that there is a GMA content dependent behavior. A higher GMA content resulted in a higher acetal cross-linking degree and tougher structure that shows poorer swelling rate. To evaluate the biocompatibility of hydrogels, an indirect contact method was conducted in a semipermeable transwell system. NHK and 3T3 cells were first seeded on 6-well plates, and then the hydrogels were added into the insets (n = 4 per group). The viabilities of both cells were evaluated after 24 and 48 h with alamarBlue assay and live/dead staining (Figure 4).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00138. Materials, instrumentation, experimental procedures, supporting figures and tables, and characterization (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

Figure 4. Assessments of cytotoxicity of all three HB-Fs using NHK and 3T3 cell lines. (A) and (B) Representative live/dead staining images on day 1 for NHK (A) and 3T3 (B) cell lines. Live cells are stained green (calcein AM), and dead cells are stained red (ethidium homodimer-1). Scale bar: 100 μm. (C) and (D) Quantitative cell viability evaluated by alamarBlue assay using cells without hydrogels as a positive control group for NHK (C) and 3T3 (D) cell lines. (mean ± SD, n = 4).

ORCID

Wenxin Wang: 0000-0002-5053-0611 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by Science Foundation Ireland (SFI) Principal Investigator Award (13/IA/1962), Investigator Award (12/IP/1688), Health Research Board (HRA-POR-2013-412), and Irish Research Council CAROLINE Fellowship (CLNE/ 2017/358).

In the sample of hydrogels formed by HB-ACETAL-PEG1:2, survival of both cell types was poor at 24 and 48 h. Typically, relative cell viability for NHK is 41% at 24 h and 35% at 48 h (Figure 4C and D). With the increase of PEGDA content in the hydrogels of HB-ACETAL-PEG1:4 and HB-ACETAL-PEG1:8, the cell survival rates increased at both 24 and 48 h. Typically, over 75% cells survived when cocultured with HB-ACETAL-

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