Biocompatible Self-Healing Hydrogels Based On Boronic Acid-Functionalized Polymer And Laponite Nanocomposite Applied For Water Pollutant Removal

The problem of global water pollution is becoming more and more severe, among which organic dyes and heavy metal ions are two typical types of the most common pollutants. The adsorption method for water purication and wastewater treatment is widely studied and applied. Hydrogel has unique advantages in the eld of adsorption due to its three-dimensional porous structure. In this paper, a new type of self-healing hydrogels based on reversible covalent bond were prepared by mixing poly(vinyl alcohol) (PVA) and 2-aminophenylboronic acid modied polyacrylic acid (PAA-2APBA). In addition, the introduction of laponite nanoparticles into the hydrogel can increase both the mechanical strength and adsorption eciency. This low-cost PAA-2APBA/PVA/laponite nano-composite hydrogel could eciently remove the organic dyes and heavy metal ions in model waste water through simple immersion, which makes it have application prospects in the elds of both biomedical and environmental engineering.


Introduction
Organic dyes and heavy metal ions are two of the most common water pollutants. Many types of organic dyes are biologically toxic and di cult to degrade, especially that some cationic dyes are more harmful to hydrosphere (Bae and Freeman 2007). Compared to organic dyes, heavy metal ions such as copper and cadmium are more di cult to eliminate, which can be easily accumulate in organisms (Sekhar, Chary et al. 2004). Heavy metal ions are completely non-degradable, and always cause irreversible pollution (Nagajyoti, Lee et al. 2010). It can be enriched in organisms and spread along the food chain, causing heavy metal poisoning at various levels as well as large scales. For these two types of pollutants, the adsorption method for water puri cation and wastewater treatment is widely studied and applied (Barry, Mane et al. 2017).
Hydrogel is composed of a cross-linked network formed by water-soluble polymers, and a large amount of water swells in this quasi-steady network (Ahmed 2015), which gift it suitable properties to serve as an adsorption material. Due to their special mechanical properties and excellent hydrophilicity, hydrogels are widely used in the preparation and modi cation of tissue engineering scaffold, water puri cation materials, oil-water separation membranes, sensors, adsorption materials, and some other industrial devices (Deepthi,  In different application scenarios, speci c working conditions such as temperature, moisture content, and pH etc. are always required speci cally for certain types of hydrogels in order to maintain their stability (Zhou, Chen et al. 2019). However, in the practical application scenarios like wastewater treatment, the changing of the conditions and factors are often unpredictable and uncontrollable. One solution to adapt the environmental change is to employ some special dynamic covalent bond. One common type of reversible covalent bonds is the B-O bond between boronic acids and 1,2-or 1,3-diols, which is often used as a synthetic self-healing material (Tarus, Hachet et al. 2014). However, the formation of B-O always requires relatively high pH (pH > pKa), and most B-O bond-based self-repairing behaviors occur in high pH range (pH ~ 8.5) which is sometimes di cult to maintain. 2-aminophenylboronic acid (2APBA) can change its structure to form intramolecular coordination under different pH, which has unique advantages in adapting to special pH environments (Yang, Lee et al. 2006, Deng, Attalla et al. 2018, Smithmyer, Deng et al. 2018. We have previously synthesized 2-aminophenylboronic acid modi ed hyaluronic acid (HA-2APBA), and prepared HA-2APBA/poly(vinyl alcohol) (PVA) plus alginate/calcium interpenetrating network (IPN) hydrogels, which possessed both self-healing properties and good biocompatibility (Deng, Attalla et al. 2018 Based on the previous work, simpler raw materials -polyacrylic acid (PAA), PVA and laponite, and simple method could be used to prepare a nano-composite hydrogel that has various excellent properties while adapting to a wide range of pH changes. Herein, we report a new type of hydrogel prepared by mixing 2APBA-conjugated PAA and PVA, as well as the introduction of laponite to form a nano-composite hydrogel (NC gel). The hydrogel is versatile, biocompatible, injectable, and self-healable, which could gel at a wide range of pH and could e ciently remove the organic dyes and heavy metal ions in model waste under neutral or alkaline conditions. In this project, a modi ed PAA (PAA-2APBA) was synthesized by using an amide bond reaction between -COOH on PAA and -NH 2 on 2APBA (Scheme 1a and Figure S1). In this way, 2APBA acts as a "bridge" which connects to PAA through an amide bond and combines with PVA through the reversible covalent bond of B-O (Scheme 1a). 2APBA can change its own conformation to accommodate a wider pH range (Scheme S1), which enables it to adapt to more complex environments ( Nano-composite hydrogels were subsequently formed by simple mixing of PAA-2APBA + laponite and PVA solutions at neutral, acidic, or alkaline pH via coextrusion through a double-barrel syringe (Scheme 1b). PAA-2APBA/PVA + laponite gelation was assessed using a vial inversion test (Fig. 1a). Solutions of either PVA or PAA-2APBA + laponite did not gel, while gelation occurred quickly (about 1 minute) after mixing of PAA-2APBA + laponite and PVA solutions, con rming that boronate-PVA cross-linking occurred. When the volume ratio of laponite solution increased to 40% v/v (PAA-2APBA precursor solution 60% v/v), the transparency of the hydrogel decreased signi cantly ( Figure S2). In addition, the high grafting rate of precursor1 (see Supplementary Information) performed high crosslinking density in the hydrogel. Therefore, subsequent experiments are performed based on laponite 20% v/v (NCgel1) unless otherwise speci ed.

Characterization of nano-composite hydrogel
Wide pH adaptability of hydrogels Due to the unique structure of 2APBA, it can break through the constraints between the environmental pH and the pKa of PBA, and act as a "bridge" in a wider pH range (

Scanning electron microscopy
The hydrogels formed at room temperature were then freeze-dried to obtain a SEM specimen. Figure 2 showed the morphology of the hydrogels and nano-composite hydrogels, namely gel5, NCgel5, gel2.5, NCgel2.5, and gel1, NCgel1 respectively (see Supporting Information for the details of naming). The threedimensional network structure of the NCgel5 appears loose, and the diameter of its pores is also large.
Compared with NCgel5, the network structure of NCgel2.5 is denser, and the hole diameter is relatively small. The network structure of NCgel1 is much denser, and the hole diameter is the smallest than the rst two nano hydrogels. With same laponite content, the network structure of the hydrogel became denser and the internal pore size was smaller with the increase of the 2APBA grafting rate (Fig. 2b, 2d, 2f). More 2APBA side chains appeared in the PAA polymer, more borate ester bond would form between PAA and PVA polymer chains, so the crosslinking density was greater. In addition, the hydrogels containing laponite had a better network structure compactness (Fig. 2), which indicated that laponite might act as a cross-linking point and promote the intertwining of polymer networks(Xiang, Peng et al.

Self-healing and injectable properties
We used gel1 and NCgel1 to investigate their self-healing characteristics. After healing for three minutes without external pressure, the healed gel could be handled with a tweezer to indicate its mechanical integrity (Fig. 3a). The healed gel could bear its own weight, and the macroscopic size can be restored to the initial state. As for NCgel1, the broken hydrogels could also heal without any external stimulus, restore size, and bear its own weight (Fig. 3b). As mentioned above, the reversible covalent bonds between 2APBA and the cis-diols in PVA is a dynamic equilibrium. The broken hydrogel will generate a key rearrangement at the fracture interface by B-O, and spontaneously repair into a whole structure.
Mechanical testing demonstrated the shear storage modulus (G') is higher than shear loss modulus (G"), which meant gel1 and NCgel1 behaving as gels ( Figure S3). The rheological results showed the selfhealing properties of both gel1 and NCgel1. As shown in Fig. 3c, G' is higher than G" from 0s to 120s at a low oscillation displacement (0.02 rad,1.468Hz), which indicated that the materials behaved as gels.
From 120s to 240s, a higher oscillation displacement (0.5 rad, 1.468Hz) was exerted to damage the structure of hydrogels (G' decreased from ~ 600 to 50 Pa). At this time, the materials tended to be uid rather than gels (G">G'). After the destroyed hydrogels were allowed to heal for 180s, the gel showed mechanical recovery at a low oscillation displacement (0.02 rad, 1.468Hz).
NCgel1 also showed gel behavior and reversibility of self-healing. The G' of NCgel1 was about 1500 Pa, as compared with 600 Pa of gel1, indicating that laponite could signi cantly improve the mechanical properties of the hydrogel (Fig. 3d). After the hydrogel structure was destroyed, NCgel1 can still recover tõ 90% of the initial state within 3 minutes, ensuring that the mechanical properties of the hydrogel could be recovered.
In addition, we tried to inject the already-formed NCgel1 through the syringe, and allowed the squeezedout gel fractures to form integral gel again. In order to facilitate the distinction, the gels were stained with a red dye (left, sulforhodamine B solution) and blue (right, methylene blue solution) (Fig. 3e). The NCgel1 could be easily extruded through a 0.5 mm diameter needle (Supplementary Video 1). Such nanocomposite hydrogels were used to wrote 'NPU' through the syringe, and the different line segments of the 'NPU' could heal together into a whole structure through the self-healing performance (Fig. 3f).

Stability and degradation of the hydrogels
The stability and degradation tests of hydrogels were showed in gure S4 and S5. The introduction of laponite signi cantly improved the stability of the hydrogels, and the stability of the hydrogel also increases with the grafting rate of modi ed PAA. In addition, NCgel1 showed glucose-responsiveness.
The NCgel1 was completely dissociated after 40 min immersion in a 2 mg/mL glucose solution, this means that these NC gels have a good application in the biomedical eld ( Figure S4c). According to the experimental data, NCgel1 has the best stability. In the following process of adsorbing pollutants by the hydrogel, it is necessary to ensure that the hydrogel can exist stably to avoid secondary pollution to the environment. So only NCgel1 was used to adsorb cationic organic dyes and metal ions.

Adsorption of organic dyes
The adsorption of methylene blue and malachite green is determined by measuring the absorbance of the solution against time (Fig. 4a, 4b and S6). After putting NCgel1 into the dye solution for 4 h, 63% of methylene blue and 49% of malachite green was quickly removed from the solution phase according to the absorbance value respectively. After 28 h treatment, 71% of methylene blue and 81% of malachite green was removed and trapped into the gel.
Laponite is a silicate with disc-like structure when dispersed in water (Scheme 1b). Typically, more negative charges exist on both bottom surfaces of laponite than positive charges on the side. Therefore, At present, the adsorption of dyes by hydrogels is performed after lyophilization, in which the adsorption process is accompanied by the swelling of the hydrogel (Meng, Peng et al. 2018). The nano-composite hydrogel (NCgel1) does not need to undergo the lyophilization step before adsorption nor a shaking process during the adsorption process. After putting the gel into the solution, about 65% of the dye can be quickly absorbed into the gel at the bottom of the beaker within 4 hours ( Fig. 4a and b). When the samples were kept in the tube for 24 h and centrifuged (1500 rpm, 5 min), the hydrogel sticks to the bottom of the centrifuge tube, and the waste water turned almost clear ( Figure S6a). We also found that the NCgel1 could still adsorb methylene blue at acidic and basic conditions, especially when pH is 9.0, the adsorption e ciency is similar as pH 7.0 ( Figure S6c).

Adsorption of metal ions
Due to the non-degradability of heavy metal ions, the common method for removing heavy metal ions is adsorption (Riederer, Belova et al. 2013, Zhang, Li et al. 2018). This method usually relies on the threedimensional structure of porous materials for adsorption, which is simple and effective.
The heavy metal adsorption of NCgel1 was tested by directly immersing into the Cu 2+ , Cd 2+ , Pb 2+ and Fe 3+ solution. With time, the hydrogel turned blue gradually due to the adsorption of copper ions ( Figure   S7). Freshly prepared NCgel1 showed quick and obvious adsorption to Cu 2+ , and the hydrogel can reach the adsorption equilibrium in about an hour (Fig. 4c) The PAA chain in the hydrogel mainly plays a role in the adsorption of heavy metal ions (Meng, Peng et al. 2018). Due to the large negative charge in the PAA chain, it has a strong adsorption effect on heavy metal cations through electrostatic interactions. At the same time, the introduction of laponite nanoparticles greatly enhances the stability of the hydrogel and at the same time improves the negative charge of the hydrogel, so the hydrogel's ability to adsorb heavy metal ions can be further improved. This hypothesized model is illustrated in Scheme S3. In addition, we evaluated the biocompatibility of the hydrogel, please see the supplementary material for details ( Figure S8, S9, and S10).

Conclusions
In summary, the PAA-2APBA/PVA hydrogel and PAA-2APBA/PVA + laponite nano-composite hydrogel were successfully prepared, which had good self-healing performance and injectability. At the same time, this hydrogel could hold the integrity in a relatively wide range of pH by employing dynamic cross-linking between PVA and 2APBA. In addition, the hydrogel had good biocompatibility, and it can quickly and effectively adsorb cationic organic dyes and heavy metal ions from model waste water. The raw materials of nano-composite hydrogel were cheap and the preparation process was simple. Therefore, this multifunctional nano-composite hydrogel which can adapt to complex pH environments has great application prospects in the elds of waste water treatment, as well as other environmental and biomedical engineering in the future.    (II) cut hydrogel; (III) gel halves placed in contact for three minutes after cutting; (IV) self-adhered hydrogel can suspend its own weight immediately after the two minutes healing time. (c, d) Rheological properties of hydrogel (gel1, c) and nano-composite hydrogel (NCgel1, d) in response to a strain sweep at 1.468Hz initiated at low oscillation displacement (0.02 rad, 120s) followed by high oscillation displacement (0.5 rad, 120s), then hydrogels healed 180 s and returned to low oscillation displacement (0.02 rad, 600s); (e. f) The injectable properties of nano-composite hydrogel (NCgel1, labeled with red or blue dye).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. WangPAA2APBAhydrogelSI.docx