Chinese Chemical Letters  2018, Vol. 29 Issue (3): 501-504   PDF    
Peptide dendrimer-crosslinked inorganic-organic hybrid supramolecular hydrogel for efficient anti-biofouling
Xiaoqi Liana, Dongxu Shia, Jin Maa, Xiaojun Caia,b, Zhongwei Gua,b    
a National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610065, China;
b College of Materials Science and Engineering, Nanjing Tech University, Nanjing 211816, China
Abstract: We reported a kind of inorganic-organic hybrid supramolecular hydrogel with excellent anti-biofouling capability. The hydrogel was formed via ionic interaction between the negative-charged sodium polyacrylate (SPA) entwined clay nanosheets (CNS) and positive-charged polyhedral oligomeric silsesquioxane (POSS) core-based generation one (L-Arginine) dendrimer (POSS-R). Due to their strong ionic interaction, this kind of hydrogel exhibited a rapid gelation behavior which accomplished even at a low POSS-R concentration about 1% w/v. With the increase of POSS-R concentration, these hydrogels appeared more compact structure, accompanied by remarkable enhanced mechanical strength. In addition, these hydrogels demonstrated rapid thixotropic response and shape-memory capability, as well as good biocompatibility. More importantly, these hydrogels exhibited outstanding anti-biofouling property due to the inherent anti-biofouling capability of SPA. Overall, these findings demonstrated a novel sort of inorganic-organic hybrid supramolecular hydrogel with tunable mechanical strength and excellent anti-biofouling capability, which may have a broad application potential in tissue engineering.
Key words: Peptide dendrimer     Supramolecular hydrogel     Anti-biofouling    

Hydrogels either from naturally derived polymers or synthetic polymers have emerged as a promising platform for various biomedical applications [1, 2], such as gene/drug delivery [3], cell encapsulation [4], tissue engineering [5-7], and medical devices. Bioactive molecules encapsulated in hydrogel matrix could be engineered with a controllable and on-demand release behavior triggered by suitable external or internal stimuli, such as temperature or pH [8, 9]. However, as a kind of implantable biomaterials, hydrogels usually face with severe implant-related infections due to their inherent biofouling property [10]. Undesirable accumulation originating from cell adhesion or protein adsorption lead to biofilm formation upon the implant hydrogels' surfaces, which would not only block the circulation of embedded biomolecules but also cause immune response and inflammation that in turn greatly increase the risk of infection [11]. Therefore, non-toxic and anti-biofouling hydrogels are urgently needed to solve these problems.

Recently, a variety of hydrophilic polymers that could minish the intermolecular forces between biomolecules and hydrogel surface have been reported to develop anti-fouling hydrogels to achieve "stealth" implant. These polymers include PEGylated polymers [12], zwitterionic polymers [13], sodium polyacrylate (SPA) [14, 15], polyacrylates [16] and so on. Among them, SPA demonstrated dramatic bio-adhesion inhibition effect because of its negative charge [17]. It is well recognized that cells have a net negative charge on the cell wall, so that they have an inherent exclusion to polyanionic hydrogels [14]. In addition, compared with the other polymers, SPA is a kind of biocompatible, commercial available, and can be used directly. Thus, they have gained more and more attention for the preparation of antibiofouling hydrogels. However, it is reported that majority of supramolecular hydrogels formed by polymers are generally mechanically too weak to bear constant external mechanical force [18], which may result in the deformation or even totally damage of the hydrogels under slight oppression in vivo.

Inorganic-organic hybrid materials have been widely used in biomedical applications for their excellent characteristics [19], such as improving the mechanical strength of hydrogels [20]. Clay nanosheets (CNS) is a kind of hydrophilic and biocompatible inorganic nano-composite with negative charge on each face and positive charge along the edges [21]. Polyhedral oligomeric silsesquioxane (POSS)-based dendrimers are kind of inorganic– organic nano-compounds bearing a rigid inorganic inner core surrounded with eight-armed amino acids [22]. It is reported that POSS core-based crosslinkers could make outstanding contribution to the mechanical strength of the hydrogels [23]. In addition, peptide dendrimers are ideal elements for the preparation of hydrogels because of their well-defined size, architectures, and multiple functional groups which endow this kind of monodisperse dendritic macromolecular great potential to fabricate 3D cross-linking networks [24]. More importantly, their abundant peripheral functional groups could further provide multiple crosslinking sites that could potentially improve the mechanical strength of the hydrogels. Thus, the masterly integration of SPA and POSS core-based peptide dendrimers into hydrogels may be capable of establish a sort of mechanical strength controllable inorganic-organic hydrogel for successful antifouling.

Herein, we reported a novel POSS-R-crossed inorganic-organic hybrid hydrogel that had excellent anti-biofouling capability. The hydrogels were fabricated via the ionic interaction between SPAwrapped CNS and POSS-R. The content of POSS-R showed significant effect on the construction and mechanical properties of the hydrogel. The hydrogel also possessed remarkable biocompatibility and excellent anti-biofouling capacity which would make the supramolecular hydrogel a promising biomedical material for various kinds of implant-related biomedical applications.

The synthesis route for POSS-R was shown in Scheme S1 in Supporting information. In brief, POSS was synthesized firstly (Fig. S1 in Supporting information), then mixed with Boc-Arg(Pbf)-OH, HOBT and HBTU to synthesize protected POSS-R in anhydrous DMSO. Following treatment with TFA for 12 h, guanidine group ended POSS-R was obtained. The successful synthesize of POSS, protected POSS-R and deprotected POSS-R were characterized by 1H NMR spectrum and MALDI-TOF-MS analysis. As shown in Fig. S2a in Supporting information, the representative peaks of Pbf could be observed at 2.43 ppm in the 1H NMR spectrum of protected POSS-R. After deprotection, the disappearance of Pbf peaks in the 1H NMR spectrum of POSS-R demonstrated the successful synthesis of POSS-R (Fig. S3a in Supporting information). The MALDI-TOF-MS results ([M+H]+ = 2129.80) further confirmed the structure of POSS-R which was consistent well with theoretical value of 2129.09 (Fig. S3b).

As illustrated in Scheme 1, our supramolecular hydrogel was formed via the following procedures. First, as the CNS is layered structure with negative charge on each face and positive charge along the edges [25], thus, the anionic SPA could surround on the edges of CNS to form a homogenously inorganic–organic solution. Next, the aqueous solution of POSS-R was added into the resultant dispersion. After stirring by a vortex mixer for several seconds, POSS-R rapidly adsorbed onto the CNS-SPA mixture to form the desired supramolecular hydrogel by ionic interaction.

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Scheme 1. The formation of supramolecular hydrogel.

To validate the formation mechanism of the hydrogels, we first investigated the zeta potential of CNS, SPA, POSS-R by a Malvern Zeta-sizer Nano ZS. As shown in Fig. 1a, the zeta potential of CNS, SPA and POSS-R were -37.9, -44.6 and +14.1 mV, respectively. The relatively high positive zeta potential of POSS-R was attributed to its abundant peripheral guanidine groups. Besides, the dramatical difference in zeta potential between CNS-SPA and POSS-R confirmed the feasibility to form the desired supramolecular hydrogels via the electrostatic interaction. This conclusion was further confirmed by the tilt test. As depicted in Fig. 1b, the mixture of CNS and SPA could not form hydrogels. However, with the presence of POSS-R, the solution lost its fluidity within several seconds and appeared to be transparent hydrogels, suggesting the sol-to-gel transition. It is indicated that the cationic binder POSS-R functioned as "molecular glue" could intercalate into the interlayer of the anionic CNS by electrostatic interaction and formed the supramolecular hydrogels [26].

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Fig. 1. (a) The zeta potential of CNS, SPA and POSS-R (means ± SD, n = 3). (b) Gelation behaviour of POSS-R crosslinked hydrogels. CNS + SPA: the mixture of 2.5% CNS and 0.3% SPA. 1%, 3%, 5%, 10%: the mixture of CNS and SPA with POSS-R concentration of 1%, 3%, 5%, 10%.

The supramolecular hydrogel with different POSS-R concentrations were characterized by scanning electron microscopy (SEM). As shown in Fig. 2, the cross-sectional morphology and porosity were dependent on POSS-R concentration. Hydrogels with 1% POSS-R presented highly uniform macropore structure (Fig. 2a), while smaller pores were observed inner the hydrogels when the POSS-R concentration increased to 3% (Fig. 2b). In addition, when the POSS-R concentration raised to 5% and 10% (Figs. 2c, d), the structures of the hydrogels tended to be denser, besides, more and more apparent filamentous structures were found regularly distributed in the inner walls of the micrometer-size pores which was the POSS-R. The POSS-R played an important role in the formation and structure of hydrogels. More importantly, the more and more compact structure may significantly increase the mechanical strength of the hydrogels.

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Fig. 2. SEM images of hydrogels with POSS-R concentration of (a) 1% (b) 3% (c) 5% (d) 10%.

As mentioned above, POSS-R is inorganic–organic compound with abundant periphery guanidine groups that can provide multiple cross-linking sites. The distinctive molecular structure endowed POSS-R great potential to improve the mechanical strength of the hydrogels. To validate it, we first investigated the viscoelastic characteristics of the hydrogels with different POSS-R concentrations (ranging from 1% to 10%). As shown in Fig. 3, in the frequency sweep, the storage modulus (G') and loss modulus (G") were shown as functions of frequency ranging from 0.01 Hz to 10 Hz at a fixed strain (0.5%). The results exhibited a highly POSS-R concentration-dependent manner. All hydrogels had single plateau region in their dynamic modulus. Besides, the G' values were always larger than that of the G" values over the entire range of frequencies, indicating a substantial elastic response. Notably, the increase of POSS-R concentration led to a remarkable increasing of G' and G" values. Hydrogels with 1% POSS-R approached their plateau G' at ~260 Pa and G" at ~36 Pa, while hydrogels with 3% POSS-R have a G' around 430 Pa and G" around 30 Pa. Interestingly, when the POSS-R concentration increased to 5%, the G' and G" value has respectively increased to 2800 Pa and 570 Pa. The G' and G" value even soared to ~8780 Pa and ~1015 Pa respectively for 10% hydrogels. Overall, these results clearly demonstrated that POSS-R could remarkably improve the mechanical strength of hydrogels, which implied that our supramolecular hydrogels could meet the needs of different applications via a simple adjusting the POSS-R concentration.

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Fig. 3. Rheological Characterization. (a) G' and G" of the hydrogels with POSS-R concentration of 1%, 3%, 5% and 10%. (b) Continuous step strain tests at γ = 0.5 and 100%.

In addition, strain sweep cycles of the hydrogels with 10% POSSR were conducted and the hydrogel showed extraordinary resistance to strain-induced deformation, as shown in Fig. 3b. The G' was about 9000 Pa at 0.5% strain. However, under a large amplitude oscillatory force (100% strain), the G' rapidly dropped to 20 Pa (< G"), which indicated a gel-to-quasi liquid transition. When the amplitude decreased back to 0.5% strain, G' quickly recovered back to original value without any loss. This recovery behavior was totally reversible during the cyclic tests.

Encouraged by the rheological characterization results, we further quantified the mechanical strength of the hydrogels prepared at different POSS-R concentrations. The storage modulus was presented as functions of frequency at a fixed strain of 0.5% (Fig. S5 in Supporting information). The supramolecular hydrogels displayed frequency-dependent modulus behavior. In detail, the storage modulus of POSS-R (10% concentration) was 38.5 ± 5.8 kPa at frequency of 0.1 Hz and increased to 74.1 ±12.7 kPa at frequency of 10 Hz. The other three POSS-R concentrations (1%, 3%, 5%) hydrogels also presented similar tendency. Moreover, the storage modulus of the hydrogels that crosslinked by POSS-R with varied concentrations were significant different at the same sweep frequency, indicating that the dendrimer cross-linker led to stronger hydrogel network when POSS-R concentration increased. It's important to determine the mechanical properties of the hydrogels under dynamic conditions when applying the hydrogels to tissue engineering.

Shape-memory hydrogels represent an interesting class of smart materials that can memorize temporary shapes and recover their original shapes in response to external stimulus [27]. We proposed that our supramolecular xerogel could revert to their initial shape after water molecules fill the interstitial sites of the entwined components. To demonstrate it, we investigated their shape memory capabilities. A piece of heart-shaped hydrogel (Fig. 4a) was air-dried at room temperature to obtain a translucent, shrunken resultant xerogel (Fig. 4b). After soaked in water at 20 ℃ for 4 h, the xerogel swelled and recovered its heart-like shape and dimensions compared with the original hydrogel (Fig. 4c).

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Fig. 4. Shape memory profiles of the hydrogel with POSS-R concentration of 5%: (a) the original hydrogel, (b) dried xerogel, (c) hydrogel by re-swelling in water.

To testify the hydrogel's excellent antifouling performance against cell attachment, we investigated the cells adherence behavior on the hydrogel covered microwell dishes by fluorescence imaging. MC3T3 cells were selected as model cells in our experiment. At first, in order to exclude possible components toxicity induced anti-cell adhesion, we studied the in vitro cytotoxicity by CCK-8 assay [11]. As shown in Fig. S6 in Supporting information, the cell viabilities were about 100%, which demonstrated that the materials possessed good biocompatibility within the experimental concentrations.

After cultivating MC3T3 cells for 24 h and 72 h on hydrogels' surface, we stained the cells by FDA which selectively stained metabolically active cells to visualize the living MC3T3 cells (green) under fluorescence microscope. As shown in Fig. 5, after cultivating for 24 h, the seeded cells on the glass bottom plate formed dense cell layer. In contrast, the hydrogels showed exceptional cell adhesion resistance as less green fluorescence points could be found in the fluorescence microscopy images. Rational explanation should be attributed to the presence of the electronegativity component of antifouling polymer SPA, which tended to repel adhesion of the cells bearing the same surface charges. When the incubation time was prolonged to 72 h, there were almost no adhered cells on the hydrogels.

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Fig. 5. CLSM images of control and supramolecular hydrogels with POSS-R concentration of 1%, 3%, 5% and 10% coated microwell dishes after exposure to MC3T3 cells for 24 h and 72 h. (scale bar, 500μm).

In summary, we have developed a novel bio-inspired inorganicorganic hydrogel that simultaneously possess tunable mechanical strength and anti-biofouling capability. The robust hydrogels were prepared via the ionic interaction between SPA-wrapped CNS and POSS-R. The hydrogels demonstrated uniform porous structures under SEM observations. The density and porosity of hydrogels could be greatly strengthened by adjustment of the content of POSS-R. Moreover, the mechanical strength of the hydrogels performed a highly POSS-R concentration-dependent manner which made it possible to control the mechanical strength of the hydrogel according to the application requirements. The noncovalent crosslinking strategy for formation of the supramolecular hydrogels endowed the hydrogels with rapid thixotropic response and shape memory functions. It has also been demonstrated that the hydrogels realized significantly antifouling efficacy, without causing toxicity to cells at the same time. Overall, the inorganic– organic anti-fouling hydrogels would offer alternative opportunities for various biomedical applications (e.g., medical devices, wound healing, drug delivery).

Acknowledgments

The authors are thankful for the financial support from the National Natural Science Foundation of China (Nos. 51133004, 81361140343, 81621003, and 51503131), the Joint Sino-German Center for Research Promotion (No. GZ905), the International Science and Technology Cooperation Program of China (No. 2015DFE52780).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.08.014.

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