A facile injectable carbon dot/oxidative polysaccharide hydrogel with potent self-healing and high antibacterial activity
A B S T R A C T
Bacterial infection is one of the most formidable problems in wound healing, which inflicts severe pain on pa- tients while causing wound ulceration. Here, we prepared an injectable self-healing carbon dot hydrogel with outstanding antibacterial activity only using ε-poly(L-lysine) carbon dot (PL-CD) and oxidized dextran (ODA). The particle size of PL-CD prepared by pyrolysis of poly-L-lysine was about 3 nm. Moreover, PL-CD with abundant -NH2 on its surface could not only act as nodes to connect ODA through Schiff base to construct PL-CD@ODA hydrogel network, but also offer excellent antibacterial properties. As the contacting and releasing antibacte- rial action of the PL-CD@ODA hydrogel, nearly 100 % of the 107 CFU/mL of S. aureus was killed after 10 min of contacting. In addition, PL-CD@ODA hydrogel showed flexible injectability and extremely strong self-healing properties after being severely damaged. When 1000 % shear stress applied to the hydrogel, complete healing could be achieved within a few seconds.
1.Introduction
Bacterial infection has always been the biggest obstacle to wound healing (Alwaili, 2004; Qian et al., 2019). The breeding of bacteria not only infects and festers wound, but also can result in bacterial biofilms, which have a protective effect on pelagic bacteria and accelerate the reproduction of bacteria (Flemming, Neu, & Wozniak, 2007). Hydrogels are widely used in medical wound dressings due to their advantages of flexibility, porosity as well as water absorption and retention (Qu et al., 2019; Rezvanian, Ahmad, Amin, & Ng, 2017; Zepon et al., 2019). Furthermore, hydrogels with antibacterial properties can effectively prevent wound infection and relieve the patient’s pain (Ghavaminejad et al., 2015; Jiang et al., 2016; Kim et al., 2014). However, traditional hydrogels often do not possess antibacterial properties, and it is neces- sary to introduce exogenous antibacterial constituents to fulfill bacte- ricidal requirements. In general, the antibacterial properties could be introduced to hydrogels in two ways. One is to select precursors with antibacterial properties to prepare hydrogels with intrinsic antibacterial effects to give contacting antibacterial result (Salick, Kretsinger, Pochan, & Schneider, 2007). The other is to load the antibacterial agents into the hydrogel network to achieve the releasing antibacterial effect (Chen et al., 2017; Qu et al., 2018). However, for antibacterial hydrogels, it is best to have both contacting and releasing antibacterial capabilities to achieve lasting and durable effects (Liu et al., 2020). But most of these hydrogel systems are too cumbersome (Hoque, Bhattacharjee, Prakash, Paramanandham, & Haldar, 2017; Juby et al., 2012; Mohan, Lee, Pre- mkumar, & Geckeler, 2007).
Therefore, it is of great significance to design a simple gel system with both contacting and releasing bacteri- cidal action. Carbon dots (CDs) are surface-functionalized small carbon nano- particles (Cao et al., 2012), usually 2—10 nm in diameter, and are easy to prepare and controllable in structure. The stable fluorescence properties, good water stability and dispersibility as well as biocompatibility of CDs have always attracted attention of researchers (Han et al., 2012). In recent years, a large number of documents have reported the application of CDs as antibacterial agents in the field of biomedicine (Li, Han, Cao et al., 2020, 2020; Yang et al., 2016). CDs can not only exhibit good antibacterial activities against pelagic bacteria, but also effectively destroy bacterial biofilms (Li, Liu, Zhang et al., 2020; Ran et al., 2019). Moreover, CDs have multiple antibacterial mechanisms, which is conducive to avoid the emergence of bacterial resistance, showing a good antibacterial prospect (Liu et al., 2020, 2020; Bing, Sun, Yan, Ren, & Qu, 2016). The reports of loading CDs in the hydrogel network to improve the gel performance often appear (Bhattacharya, Nandi, & Jelinek, 2017; De, Kuila, Kim, & Lee, 2017). However, to best of our knowledge there are no previously published reports using only carbon dots and a polymer to form hydrogel with antibacterial property.Another concern for hydrogels is the durability of material and ease of use.
Self-healing hydrogels will provide great convenience for pa- tients, without frequent replacement caused by exercise and other ruptures, which greatly reduces the possibility of secondary injuries (Chen et al., 2019; Liu et al., 2015; Wang et al., 2018). For a long time, Schiff-base based hydrogels have attracted interest since long due to its easily available raw materials, inject ability, simple preparation and self-healing. Hydrogels constructed by Schiff-base can achieve self-repair by imine bond. (Du et al., 2019; Huang et al., 2018; Vicente et al., 2017). Moreover, Self-healing hydrogels formed by covalent bonds are more stable, have stronger repair ability and better tolerance, which prompts Schiff-base based hydrogels to be favored by the public. Besides, the shape of wound is usually irregular. Conventional hydrogel dressings cannot meet the application requirements, but injectable hydrogels are adaptable to the wound of any shape, which perfectly overcome this obstacle (Hoque, Prakash, Paramanandham, Shome, & Haldar, 2017; Xiang et al., 2019; Zhao et al., 2017). The injectable hydrogels, completed gelation in a syringe and then punched out through a needle, are particularly appealing because of their attractive features including homogeneous matrix within any defect size and shape, as well as the less invasive delivery procedure (Tseng et al., 2015). In this study, we designed a hydrogel system using only ε-poly(L-lysine) carbon dot (PL-CD) and oxidized dextran (ODA). ε-poly(L-lysine) (PL) was selected to prepare PL-CD through a simple one-step pyrolysis. The large specific surface area of PL-CD may efficiently favor the enrichment of -NH2 (Natarajan et al., 2019), which is not only helpful for PL-CD to play the role of nodes to construct gel network with ODA, but also can exert antibacterial activity as the retention of -NH2 (Li, Liu, Zhang et al., 2020). Furthermore, the PL-CD@ODA hydrogel based on Schiff base may not only have intrinsic antibacterial property but also can release PL-CD through the reversible imine bond to fulfill bacteri- cidal action. The PL-CD@ODA hydrogel constructed by Schiff base are expected to show good self-healing, injectability and remarkable anti- bacterial properties.
2.Experimental Section
ε-poly(L-lysine) (Mw ~ 4000), Dextran (Mw ~ 70,000) and NaIO4 were purchased from Shanghai Dipper Biotechnology Co., LTD, Nanjing Lattice Chemical Technology Co., LTD and Nanjing Juyou ScientificEquipment Co., LTD, respectively. Dialysis membrane (MWCO = 5000 Da) and dialysis membrane (MWCO = 14000 Da) were purchased from Nanjing Middle East Chemical Glass Instrument Co., Ltd. Syringe waspurchased from Jiangyin Medical Device Co., Ltd. Tryptone and other Luria–Bertani (LB) nutrient broth related reagents were purchased from Nanjing Wanqing Glass Instrument Co., Ltd. All reagents were used directly without further purification.1 g of ε-poly(L-lysine) was taken in a crucible and placed in an oven. The temperature was increased to 240 ◦C per minute at 4 ◦C and maintained for 3 h. Then, cooled naturally to room temperature. Theresidue was ground to powder in a mortar, then dissolved in 20 mL of deionized water. After 10 min sonication, the mixture was centrifuged (10000 rad/min) for 10 min. The supernatant was dialyzed (MWCO =5000 Da) for 48 h and lyophilized. A light yellow flocculent product was obtained with a yield of 89.7 %.4 g of dextran and 3.4 g of NaIO4 were dissolved in 50 mL PBS so- lution and stirred for 24 h at room temperature in dark to obtain an orange solution. 1 g of ethylene glycol was added to terminate the re- action for 2 h. Then the products were dialyzed (MWCO = 14000 Da) for48 h and lyophilized. The degree of oxidation of dextran determined bythe hydroxylamine hydrochloride method was 54.7 %.15 % (w/v) of PL-CD in PBS solution and 10 % (w/v) of ODA in PBS solution were simply mixed in a 5 mL cartridge in different volume ra- tios respectively. Then they were mixed well by an oscillator and poured into the mold. The gel formed in situ at room temperature.The high resolution transmission electron microscopy (HRTEM) image of PL-CD was obtained at 200 kV using a transmission electron microscope (JEM-2100, JEOL Ltd, Japan).
The fluorescence spectros- copy of PL-CD was performed with an F-2700 fluorescence (FL) spec- trophotometer (Hitachi, Japan) and the emission spectra were recorded in the wavelength range of 300–460 nm. Fourier transformed infrared (FTIR) spectra of PL-CD, ODA and PL-CD@ODA hydrogels were recor-ded by FTIR spectrometer (Nicolet iS 10, Thermo Fisher Scientific, USA) by collecting 32 accumulative scan in 525 cm—1 to 4000 cm—1 region against a blank KBr pellet background. The Ultraviolet-Visible spectrawere obtained by Thermo Scientific Evolution 220 spectrophotometer. The morphology of the hydrogels was observed after lyophilizing at – 50◦C and spraying gold using a scanning electron microscope (SEM)(Quanta 250FEG, FEI, USA). XPS measurements were performed by an X-ray photoelectron spectrometer ESCALAB 250 ultrahigh vacuum (1 × 10-9 bar) apparatus with an Al Kα X-ray source and a monochromator.The hydrogels were anchored on silicon wafers. The X-ray beam size was 500 μm, and survey spectra were recorded with pass energy (PE) 150 eV and high-energy resolution spectra were recorded with PE 20 eV. Pro- cessing of the XPS results was carried out using AVANTGE program.Rheological measurements of PL-CD@ODA hydrogels were per- formed by Anton Paar MCR 300. A series of PL-CD@ODA hydrogels with different ratios were prepared in the shape of a disc with a diameter of25 mm and a thickness of 2 mm. During the test, the temperature of the sample stage was set to be constant at 37 ◦C. The angular frequency sweep range was from 0.1 % rad/s to 100 % rad/s, and the amplitude γwas 0.5 %. Strain sweep range γ from 0.01%–1000%, the angular fre- quency was fixed at 1 Hz. RHEOPLUS software recorded the changes in storage modulus G’ and loss modulus G”.The gelation time of PL-CD@ODA hydrogels was determined by the vial inversion method.
Simply speaking, a certain amount of 10 % (w/v) of ODA and 15 % (w/v) of PL-CD in PBS solution were added into a small glass bottle. Once the two solutions were in contact, the timing started. Then shaking the glass bottle by the vortex gently to mix the solutions evenly, and continuously changing the tilt angle of the glass bottle. When inverting the glass bottle, the liquid in the bottle solidified and stopped flowing, the timer was terminated, which indicated that the PL- CD@ODA hydrogel had been formed at this point.First, prepare PL-CD@ODA hydrogel. After completion of gelation, destroy the morphology of the gel (crushed, cut, or scratched), then leave the damaged gel in the humidor at 37 ◦C for a period of time andrecord the self-healing property of PL-CD@ODA hydrogel.The rheometer was used to quantitatively evaluate the self-healing properties of PL-CD@ODA hydrogels. All hydrogel samples were pre- pared as a disc with a diameter of 25 mm and a thickness of 2 mm. The value of the critical strain region was recorded using the strain ampli- tude sweep method (γ from 0.1%–1000%). Then, using other hydrogel discs to test the self-healing performance by alternating strain sweeptests at a fixed angular frequency (1 Hz). Switch the amplitude oscilla- tion strain from small strain (γ = 0.1 %, 240 s for each interval) to large strain (γ = 1000 %, 600 s for each interval), and perform 2 cycles.The injectability of PL-CD@ODA hydrogels were performed with a disposable sterile syringe with a 22-gauge needle. 15 % (w/v) of PL-CD in PBS solution and 10 % (w/v) of ODA in PBS solution were vortex mixed for 12 s to make homogenous mixture and put them into a sy- ringe. After gelation, the hydrogel was extruded through the needle to test its injectability.Gram-positive Staphylococcus aureus (S. aureus) ATCC 25923 pur- chased from Nanjing Bizhi Biotechnology Co., Ltd. was chosen as the model pathogen for antibacterial activity experiments.
Before the ex-periments, S. aureus was cultivated in Luria–Bertani (LB) nutrient broth at 37 ◦C for 24 h and determined its concentration.The antibacterial activities of PL and PL-CD were investigated against S. aureus using bacterial colony counting method. 500 μL of 10 μg/mL sample suspension was mixed with 500 μL of 107CFU/mL freshS. aureus suspension and start the timer. 100 μL of mixed suspension was coated uniformly on the LB plates at 0, 10, 30, 60 and 120 min, respectively. After incubation at 37 ◦C for 24 h, the viable colony number was counted for antibacterial activity evaluation. Each experi- ment was repeated three times.The PL-CD@ODA hydrogels were prepared as a disc with a diameter of 1 cm and a thickness of 3 mm and transferred to a LB agar plate covered with 100 μL of 106 CFU/mL fresh S. aureus suspension. After incubating at 37 ◦C overnight, measure the bacterial inhibition area around the gel disc. Each experiment was repeated three times.2 mL of PL-CD@ODA hydrogels were prepared with different ratios in 12-well plates, respectively. When the gelation completed, add 20 μL of 107 CFU/mL fresh S. aureus suspension to each hydrogel surface. After incubating at 37 ◦C for 10 min in a relative humidified atmosphere, add 1 mL of sterile PBS to each well to resuspend the surviving bacteria with a moderate sonication (f =50 Hz). 100 μL of suspension was coveredwith a LB agar plate. The results were observed after incubation for 24 hat 37 ◦C. 20 μL of 107 CFU/mL S. aureus suspension dropped into the well without hydrogels was treated as a control. Count the number of col-onies on the agar plates and calculate the killing ratios of PL-CD@ODA hydrogels against S. aureus. Each experiment was repeated three times.All results were analyzed by ANOVA at P < 0.05 significance level. Standard deviation is used as an estimate of the standard uncertainty associated with measurement. 3.Results and discussions The PL-CD@ODA hydrogel was prepared by Schiff base between the amino from the PL-CD and the aldehyde from the ODA (Fig. 1c). The PL- CD prepared by one-step pyrolysis was mainly distributed between2.8—3 nm in particle size, with a yield of up to 89.7 % (Figs. 1a and 2 a).Fig. 2 b showed that the PL-CD expressed stable and excitation wavelength-dependent fluorescence. The minimum inhibitory concen- tration of PL-CD against S. aureus was only 0.5 μg/mL (Table S1), lower than that of PL. In addition, it is worth mentioning that PL-CD could achieve 100 % sterilization after 10 min of contact with S. aureus, which got the better of PL (Fig. S1). Next, dextran was oxidized to provide aldehyde (Figs. 1b and S2), and the oxidation degree of ODA measured by the hydroxylamine hydrochloride method was 54.7 %.The gelation information was shown in Fig. S3a. It was found that gels could be successfully formed under the ratios of 1:5, 1:4, 1:3, 1:2,1:1, 2:1, 3:1, 4:1 and 5:1, while the 1:6 and 6:1 were not. Moreover, 15 % (w/v) of PL in PBS and 10 % (w/v) of ODA in PBS could not form gels at any ratio. This result may be related to the PL-CD with abundant -NH2 on its surface, which provided a node for the cross-linking with the ODA polymer chains. In the gelation process, primary amino from PL-CD and aldehyde in ODA interacted to construct a PL-CD@ODA gel network through Schiff base. Next, we measured the gelation time of PL- CD@ODA hydrogels with different ratios by the vial inversion method (Fig. S3b). The gelation time of the 1:1 was the shortest and could be formed in about 20 s at room temperature. Comparing the gelation time of the gels of 1:5, 1:4, 1:3, 1:2, and 1:1, it was found that the higher the percentage of PL-CD in the gel, the shorter the gelation time. As the content of PL-CD increased, it supplied more amino required for Schiff base with aldehyde, which promoted the occurrence of cross-linking reactions. However, for 1:1, 2:1, 3:1, 4:1 and 5:1, the opposite was true. The continuous increase of PL-CD ratio resulted in the decrease of ODA, while the deficiency of aldehyde concentration was not conducive to gel formation. Moreover, it indicated that the gelation time of the 2:1 was shorter than that of the 1:2. This may be the result of steric hin- drance effect. With the increase of ODA concentration, the movement of ODA polymer chains were limited. However, with a further increase in ODA concentration, the gelation time of 3:1, 4:1, and 5:1 was longer than that of 1:3, 1:4, and 1:5, respectively, which was attributed to the decrease of the aldehyde groups.In order to illuminate the conversion of specific groups of PL-CD andODA during the formation of hydrogel, we conducted a Fourier trans- form infrared spectroscopy experiment (Fig. 3a). For PL-CD, 3451 cm—1and 3288 cm—1 were attributed to υNH2 and υOH, respectively and the obvious characteristic absorption peak at 1654 cm—1 belonged to υCO– from amide (Li, Liu, Cao et al., 2020). For ODA, 3352 cm—1 belonged to υOH, and a weak peak at 1728 cm—1 was assigned to υCO–. There werenot obvious signals of aldehyde in FTIR. It was probable that the carbonyl groups were extensively acetalized and the oxidized dextran aldehyde may exist in the form of hemiacetal (Liu et al., 2009; Maia, Carvalho, Coelho, Simoes, & Gil, 2011). The FTIR spectrum of thePL-CD@ODA gel showed peaks of υNH2, υOH, υC = O, respectively at 3346 cm—1, 3243 cm—1, 1665 cm-1 and the obvious υC = N at 1673 cm—1, indicating that PL-CD@ODA gel was successfully synthesized by imine bond (Du et al., 2019). Besides, the peak positions of the activegroups of -NH2, -CHO and –COOH in PL-CD@ODA showed slight devi- ation compared with those in the precursors, which indicated that the chemical environment of these active groups had changed.X-ray photoelectron spectroscopy (XPS) results revealed the state of different atoms in PL-CD and PL-CD@ODA hydrogel. Deconvolution of the C 1s XPS spectrum of PL-CD showed three kinds of carbon bonds,corresponding to sp3 (C–C) at 282.2 eV, CO at 285.7 eV, and CN–– at283.5 eV (Fig. 3b) (Bhattacharya et al., 2019; Xu et al., 2019). AfterPL-CD interacting with ODA, an additional peak at 283.4 eV appeared in C 1s of PL-CD@ODA (Fig. 3c), which was attributed to the C–N of iminebond. This was one of the evidences of Schiff base reaction occurring to form a gel (Bhattacharya et al., 2019). Consistent with the C1 s, C–N(398.8 eV) could also be found in the N1 s XPS spectrum ofPL-CD@ODA, but not in the N1 s of PL-CD (Fig. 3d and e). Furthermore, N1 s XPS spectrum of PL-CD confirmed the presence of primary amines on the surface of PL-CD, and some of the primary amines were still preserved after interaction with ODA, which would contribute to the contacting antibacterial ability of PL-CD@ODA hydrogels. Comparingthe XPS spectra before and after the reaction between PL-CD and ODA, it was found that the binding energy of C–O and N–H2 shifted slightly,which were derived from the formation of imine bond (Xu et al., 2019). Additionally, the relative percentages of carbon, nitrogen and oxygen in the PL-CD and the gel, based upon XPS, were given in Table S2.The UV–vis absorption spectra of PL-CD and PL-CD@ODA hydrogel in Fig. 3f was consistent with the FTIR, providing further evidence to explain the assembly of PL-CD@ODA gel. The absorption peak of solublePL-CD at 270 nm corresponded to the π-π* transition of the aromatic sp2 domains (C–C and CC–), while the shoulder peak at 300—400 nm was attributed to the n-π* transition of the C–O, CO and CN–– bonds (Jianet al., 2017; Li et al., 2018). After the reaction of PL-CD and ODA, the π-π* transition peak widened and a new peak appeared at 400—470 nm, which reflected the formation of imine bond. The apparent broadening and redshift of the π-π* peak was due to the immobilization of PL-CD in the hydrogels (Bhattacharya et al., 2019).Four gels with continuous changes in PL-CD content of 1:3, 1:2, 1:1 and 2:1 were selected for SEM (Fig. 4). It was found that the PL- CD@ODA hydrogels had an appearance of loofah flesh. With the in- crease of the percentage of PL-CD in the gel, the porosity of PL-CD@ODA gradually increased, and when it reached 2:1, all pores disappeared. By comparing the three gels of 1:3, 1:2 and 1:1, as the increase of PL-CD content, the pores of PL-CD@ODA hydrogel gradually increased and the morphology gradually became regular. The pores in PL-CD@ODA gels may be caused by the super-hydrophilicity of PL-CD (Kato, Sakai, & Shibata, 2003). In the presence of aqueous solution, with the increase of the content of PL-CD, the water content of PL-CD@ODA gel enlarged. After being lyophilized, the porous structures with different pore sizes were obtained. A few holes were found in the hydrogel of 2:1 and many wrinkles were observed on its surface. It may be due to the large amount of PL-CD, which made the cross-linking between ODA polymer chains more sufficiently dense. It was reported that the porous structures of hydrogels played an important role in its properties and applications in biomedical engineering (Ma et al., 2003; Zawko & Schmidt, 2010).Angular frequency sweep was used to study the stability of the hydrogels under cyclic shear forces. As shown in Fig. 5a, the storagemodulus G' of each PL-CD@ODA hydrogel was significantly higher than the loss modulus G'' by more than an order of magnitude, showing that PL-CD@ODA adopted a gel organization (Li et al., 2018). The storagemodulus G' of the 1:1 hydrogel was the highest as well as its loss modulus G'', which was related to its regular gel network structure. The 1:2 and the 1:3 were slightly different in the storage modulus G', and both were lower than that of 2:1, which also was decided by their un-even gel structure. And the loss modulus G” of these three hydrogels had the similar performance. In addition, the storage modulus G’ and the loss modulus G” were basically stable within the angular frequency of 30 rad/s, indicating the PL-CD@ODA hydrogels had relatively stable structure.Amplitude sweep (Fig. 5b) showed the dependence of the storage modulus G' and the loss modulus G'' of the PL-CD@ODA hydrogels on the applied strain (γ%). It was worth noting that, before the gels collapsed (G” > G’), all PL-CD@ODA hydrogels had elasticity up to 400% strain at a fixed angular frequency of 1 Hz. The extraordinary me- chanical strength of the gel reflected the contribution of the imine bond network within the PL-CD@ODA gel (Bhattacharya et al., 2019).
The self-healing properties of hydrogels gave them the ability to withstand damage and extended the usage time, greatly reducing the risk and cost of secondary infection. The photographs and fluorescent images in Fig. 6a–c provided a vivid demonstration of the self-healing properties of the PL-CD@ODA gels. After being crushed, the hydrogel chippings restored to a small disc without any external intervention in a mold within 1 h at 37 ◦C (Fig. 6a), showing extraordinary self-healing property, which was realized by imine bonds (Bhattacharya et al.,2019). Fig. 6b showed the fluorescence image (exc. 365 nm) of the PL-CD@ODA gel cut in the middle and then recovered from self-healing. After being cut into two pieces, PL-CD@ODA hydrogel could be self-repaired and fused into one piece within 1 min. Fig. 6c emphasized the self-healing effect of PL-CD@ODA gel when local deformation (scratch) was applied (Fig. 6c, i). Specifically, after 20 min of scratch, the cracks on the gel surface gradually disappeared (Fig. 6c, ii), and after 30 min, the morphology of PL-CD@ODA gel recovered to that beforescratch (Fig. 6c, iii). As shown in Fig. 6d, the higher the PL-CD content, the shorter the self-healing time. The 2:1 gel only took 32 ± 6 s to achieve self-healing. The abundant –NH2 on the surface of PL-CDgreatly ensured the possibility of Schiff base, which powerfully pro- moted the self-repair process of PL-CD@ODA hydrogel. The injectable properties of PL-CD@ODA gels were demonstrated in Fig. 6e. PL-CD@ODA hydrogels could be extruded through a 22-gauge needle without blocking, indicating their good injectability due to their shear dilution (Wu, Chen, Huang, Yan, & Zeng, 2020). The injectability enabled PL-CD@ODA hydrogels to be adaptable to the wound of any shape.We further quantitatively evaluated the self-healing properties of PL- CD@ODA hydrogels through rheological testing (Fig. 7). For 1:1 PL- CD@ODA hydrogel, at first the gel was placed under 0.1 % strain for 4 min at 37 ◦C for which G’ > G”.
When 1000 % strain was applied to thegel for 4 min, G” > G’ and G’ immediately dropped from 96 Pa to 45 Pa,which indicated that the hydrogel network had collapsed. After returning the strain back to 0.1 %, both G’ and G” could immediately return to the original value without any loss. This recovery behavior was reproducible during the cycle test, indicating that PL-CD@ODA hydro- gel of 1:1 had excellent self-healing property. During the cycle test, it was found that the values of G’ had a tendency to decrease slightly, which may be related to the loss of gel moisture during the test instead of losing the self-healing ability. We also performed the same approach to quantitatively assess the self-healing properties for 1:3, 1:2 and 2:1 PL- CD@ODA hydrogels. Similar self-healing properties were recorded in Fig. 7a, b and d.The hydrogels with antibacterial property could effectively avoid the occurrence of bacterial infection, which quickly promoted tissue healing and greatly reduced the pain of patients. We evaluated the antibacterial activity of PL-CD@ODA hydrogels with different structures usingS. aureus. In Fig. 8a, significant inhibitory regions were observed around each sample, which was a manifestation of the releasing bactericidal action of PL-CD@ODA hydrogels. It was known that a large amount of-NH2 on the surface of PL-CD could rapidly interact with the negatively charged bacterial cell membrane through electrostatic interaction and destroy the normal metabolism of bacteria, resulting in killing bacteria (Li, Liu, Cao et al., 2020). Therefore, the releasing antibacterial property of PL-CD@ODA hydrogels was derived from the remarkable antibacte- rial properties of released PL-CD.
The diameters of the inhibition zonesof PL-CD@ODA hydrogels of 1:3, 1:2, 1:1 and 2:1 were 2.05 ± 0.05 cm,2.1 ± 0.05 cm, 2.1 ± 0.05 cm, 2.2 ± 0.05 cm, respectively, indicatingthat the difference in the content of PL-CD would not have a great impact on the releasing antibacterial property of PL-CD@ODA hydrogels. Moreover, it was found that the diameters of the four gel discs changed from the initial 1 cm to 1.3 ± 0.1 cm, 1.3 ± 0.1 cm, 1.5 ± 0.1 cm and1.55 ± 0.1 cm after incubation in Fig. 8a, which showed thatPL-CD@ODA hydrogels had strong water absorption and retention ability and PL-CD had high water solubility. The superior antimicrobial property of PL-CD@ODA hydrogels were further demonstrated by direct contact test in Fig. 8b. After 10 min of contacting with 20 μL of 107 CFU/mL S. aureus at 37 ◦C, the gels killednearly 100 % of the bacteria. All hydrogels showed rapid bacterial killing rates against S. aureus, indicating their excellent contacting antibacterial properties, which was related to the primary amino groups retained in the PL-CD@ODA gel (Fig. 3e). After gelation, the antibac- terial property of PL-CD was endowed to the PL-CD@ODA hydrogels, which indicated that we successfully prepared a carbon dot hydrogel with inherent antibacterial properties. It could be concluded that the PL- CD@ODA hydrogels would have a promising application in wound infection caused by bacteria, especially S. aureus. This facile approach to obtain PL-CD@ODA hydrogels provided a good idea for researchers to develop novel antibacterial hydrogels.
4.Conclusions
In summary, we simply prepared an injectable self-healing hydrogel with dual antibacterial properties using PL-CD and ODA. PL-CD with remarkable antibacterial activity could be used as a node to crosslink the ODA polymer chains, building a three-dimensional gel network. By modulating the ratio of PL-CD and ODA, the purpose of adjusting the porosity and improving the mechanical properties of the gel could be achieved. It was found that PL-CD@ODA hydrogels could be self-healing in a short time after being severely damaged, and this recovery behavior was reproducible. The injectability of PL-CD@ODA hydrogels allowed them to be applicative to the wound of any shape perfectly, which admitted them easier to use. In addition, PL-CD@ODA ε-poly-L-lysine hydrogels had both releasing and contacting antibacterial effect. On the whole, carbon dot/oxidative polysaccharides hydrogel systems may be a meaningful enlightenment on the development of hydrogel, which is worthy of further research and development.