Tuning the Hydrophobicity of a Hydrogel by 2 Self-assembly of Polymer Cross-linkers

: Hydrogels incorporated with hydrophobic motifs have received considerable attention to 11 recapitulate the cellular microenvironments, specifically for the bio-mineralization of a 3D matrix. 12 Introduction of hydrophobic motifs into a hydrogel often results in irregular arrangement of the 13 motifs, and further phase separation of hydrophobic domains, but limited efforts have been made 14 to resolve this challenge in the hydrophobically-modified hydrogel. Therefore, this study presents 15 an advanced integrative strategy to incorporate hydrophobic domains regularly in a hydrogel by 16 self-assembling of polymer cross-linkers, building blocks of a hydrogel. Self-assemblies between 17 polymer cross-linkers were examined as micro-domains to incorporate hydrophobic motifs in a 18 hydrogel. The self-assembled structures in a pre-gelled solution were confirmed with the 19 fluorescence analysis and the hydrophobicity of a hydrogel could be tuned by incorporating the 20 motifs in a controlled manner. Overall, the results of this study would greatly serve to tuning 21 performance of a wide array of hydrophobically-modified hydrogels in drug delivery, cell 22 therapies and tissue engineering.


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Hydrogels have been extensively studied for use in various biomedical applications including 27 drug delivery, tissue engineering and recently, Bio-MEMS(bio-microelectromechanical system) 28 [1][2][3]. The successful use of the hydrogels in these applications greatly relies on their physical and 29 chemical properties for maximizing their functionality [4][5][6][7]. For example, mineralized hydrogel 30 systems are being increasingly studied to understand bio-mineralization processes related to the 31 development, repair, regeneration and remodeling of bone tissue [8,9]. In here, the hydrogels 32 incorporated with hydrophobic motifs are required to recapitulate the hydrophobic/hydrophilic 37 Therefore, we hypothesized that self-assemblies of polymer cross-linkers in a pre-gelled 38 solution would allow us to incorporate hydrophobic motifs regularly as micro-domains in a 39 hydrogel. This hypothesis was examined using a model system for a hydrophobically-modified 40 hydrogel formed from the cross-linking of poly(ethylene glycol) diacrylate (PEGDA). The 41 self-assembled structures of PEGDAs in a pre-gelled solution were confirmed with the fluorescence 42 analysis ( Fig. 1(b)). Then, poly(propylene glycol) methacrylate (PPGMA) with varying of mass 43 fraction was used as model hydrophobic motif (Fig. 1(c)). The effects of the hydrophobic domains 44 incorporated into a hydrogel were studied by measuring swelling ratio and contact angle of a 45 hydrogel. The underlying mechanism by which micro-domains provided by self-assembling of 46 where ρs is the density of water, ρp is the density of polymer and Qm is the swelling ratio, the mass 97 ratio of swelled gel to the dried gel.

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The average pore size (ξ) of hydrogel was calculated from the polymer volume fraction (v2,s) 99 and the unperturbed mean-square end-to-end distance of the monomer unit (ro -2 ) using equation (

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This study presents an effective method to incorporate hydrophobic domains regularly in a 128 hydrogel by self-assembling of polymer cross-linkers. First, the self-assembled structures formed in 129 a pre-gelled solution were confirmed with the fluorescence analysis. The hydrophobic association 130 between the polymer cross-linkers with acrylate groups which are slightly hydrophobic was 131 examined with a ratio of the third-to-first vibrational fine structure (I3/I1) in the fluorescence 132 spectrum of pyrene prove (Fig. 2). Generally, the I1 peak arises from the transition that can be 133 enhanced by the distortion of the π-electron cloud [12,13]. Therefore, as the microenvironment of 134 pyrene becomes more polar, the I1 peak becomes more notable at the expense of other peaks (I3).

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That means the ratio of I3/I1 represents the degree of self-assemblies between acrylate groups linked

Effects of the hydrophobic domains incoported into a hydrogel 150
The hydrophobically-modified hydrogels were prepared via in situ radical polymerization of 151 self-assembled PEGDAs and PPGMAs with varying of mass fraction. As pointed out above, the 152 concentration of polymer cross-linkers used to form hydrogel was higher than the CACs of 153 cross-linkers as shown in Fig. 2(c). Figure 3 shows that in general, the degree of swelling (Q) of 154 hydrogels increases as the concentration of pure PEGDAs decreases due to the increase of average  Table 1, calculated using the eqn. (1). However, 157 interestingly, in case of the hydrophobically-modified hydrogels, the Q of hydrogel was decreased 158 with increasing PPGMA portion from 0 to 0.5 wt%, despite of the decrease of PEGDA concentration.

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And then, the Q was increased with increasing PPGMA from 0.5 to 2.0 wt%. These results indicated  The inner micro-structures of hydrophobically-modified hydrogels (HMHs) were examined 168 with freeze-dried gels by SEM. Figure 4 shows the images of hydrogels depending on the 169 introduced amounts of PPGMAs. Structural difference between pure PEGDA hydrogel (HMH-1) 170 and HMH-2 at 0.5 wt% of PPGMA was not significant and their 3D networked microstructures were 171 both well formed. However, HMH-4 at 2.0 wt% of PPGMAs exhibited relatively large hydrophobic 172 micro-domains (Fig. 4a-3 & 4b-3). It was seem to be taken place of a phase separation between 173 hydrophilic PEGDA and hydrophobic PPGMA molecules. It means that a certain amount of

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PPGMA in hydrogel has functioned as hydrophobic repulsion. Hydrophobic parts, PPGMAs over a 175 certain amount which is likely the loading capacity of the self-assemblies of PEGDAs in pre-gelled 176 solution, caused the phase separation. Also, water contact angles (θw) measured at the surface of 177 hydrogel increased with increasing of incorporating PPGMAs (Fig. 4). As a result, the 178 hydrophobicity of a hydrogel could be tuned by incorporating the hydrophobic motifs into the 179 self-assembled structures in a pre-gelled solution.

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BSA was encapsulated into the HMHs using PEGDA-3400 to evaluate the effects of the 184 hydrophobic motifs on protein release rate. The release rate of BSA from the HMH gel was 185 quantified by measuring the amount of BSA released into the incubation media on a daily basis 186 over ten days (Fig. 5). Increasing the hydrophobicity from 0 to 2 wt% of PPGDAs significantly 187 decreased the amount of BSA initially released from the HMH gels ( Fig. 5(a)). The cumulative mass 188 fraction of BSA released from HMHs over 12 hours, Mt/M∞, was fitted with the Ritger-Peppas 189 equation to calculate a kinetic rate constant (k) (Fig. 5(b)). The k of the HMHs decreased by 30% as