A pH-responsive hydrogel with potent antibacterial activity against both aerobic and anaerobic pathogens

Jingjing Hu a, Zhao Zheng a, Cenxi Liu a, Qianyu Hu a, Xiaopan Cai b, Jianru Xiao b and Yiyun Cheng *ac
aShanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P.R. China. E-mail: yycheng@mail.ustc.edu.cn
bDepartment of Orthopedic Oncology, Changzheng Hospital, The Second Military Medical University, Shanghai, 200003, PR China
cSouth China Advanced Institute for Soft Matter Science and Technology, South China University of Technology, Guangzhou 510640, China

Received 28th September 2018 , Accepted 7th November 2018

First published on 9th November 2018

Aminoglycosides are a family of antibiotics with a wide-spread antibacterial spectrum and high antibacterial activity. However, they are less effective against anaerobic pathogens due to the requirement of aerobic respiration to exert their antibacterial functions. Here, we prepared a pH-responsive hydrogel with potent antibacterial activities against both aerobic and anaerobic pathogens. The hydrogel was prepared by reacting oxidized dextran with aminoglycoside and an ornidazole analogue via an acid-labile Schiff base linkage. The prepared hydrogel was injectable, self-healing, biocompatible, and showed a pH-responsive drug release behavior. More importantly, the gel was efficient in killing both aerobic and anaerobic pathogens. This study provides a facile strategy to expand the antibacterial spectrum of aminoglycoside hydrogels.

Bacterial infection usually occurs in burns, wounds, and lots of surgical operations.1,2 It may cause chronic infection, muscular death, sepsis, and even threaten life. There is an urgent need to develop efficient antibiotics or antibacterial materials to treat antibiotic-resistant bacteria.3–10 Aminoglycosides are a family of antibiotics which possess a wide-spread antibacterial spectrum and highly potent antibacterial activity.11–13 Due to possible biofilm formation at low doses and adverse effects such as ototoxicity and nephrotoxicity at high doses, on-demand local delivery of aminoglycosides has been urgently required to obtain a sustained and suitable drug concentration at the infected sites.14,15 Hydrogels, a class of three-dimensional crosslinked networks, have been prevalent as antibacterial agents for local infection treatment nowadays,16–19 as antibacterial hydrogels possess unique advantages including high water retention capacity, good tissue adaptability, sustained local drug delivery, and excellent biocompatibility.20–24 In a recent study, we reported a class of aminoglycoside hydrogels by crosslinking oxidized polysaccharides with amine-abundant aminoglycosides via acid-labile Schiff base linkages.25 The gel stiffness, degradation rate and drug release kinetics could be precisely controlled by modulating the aminoglycoside dose during gel preparation. Although the aminoglycoside hydrogels showed high antimicrobial activities against both Gram-negative and Gram-positive pathogens in vitro and in vivo, they still have a limitation that they are less effective against anaerobic bacteria.26 Aminoglycosides exert their antibacterial functions by transporting across the cytoplasma membrane and binding to the ribosome of bacteria to disrupt protein synthesis. This process requires metabolic energies from the electron transport system in an oxygen-dependent manner.27 However, the infections in clinical practice usually involve complicated infections, as the aerobic bacteria growth may decrease the blood-supply and oxidation–reduction potential of infected tissues, which accelerate the proliferation of anaerobic bacteria. The anaerobic bacteria may induce serious infections such as brain abscesses, myocardial necrosis, osteomyelitis, and peritonitis.11 Therefore, there is a need to develop an antibacterial hydrogel that can efficiently kill both aerobic and anaerobic pathogens.

Ornidazole is a typical 5-nitroimidazole antibiotic drug widely used in the treatment of anaerobic infections, such as bacterial vaginitis, endometritis, and periodontitis.28 It exerts an antibiotic function under hypoxic conditions by reducing the nitro group on the drug to a reactive amine group which binds with microbial DNA to interfere with nucleic acid synthesis.29 Here, ornidazole was conjugated on a generation 1 (G1) amine-terminated poly(amidoamine) (PAMAM) dendrimer to yield a prodrug (G1-orni). The dendrimer was used as a scaffold here due to its well-defined molecular structure, monodispersity, and high density of amine groups on the surface.30–33 An oxidized dextran was then crosslinked by a mixture of G1-orni and an aminoglycoside drug tobramycin via acid-labile Schiff base linkages. The hydrogel was expected to possess high antibiotic activity against both aerobic and anaerobic pathogens.

Ornidazole was modified on the G1 PAMAM dendrimer through a facile reaction, and the average number of conjugated ornidazole molecules on each dendrimer was calculated to be 1.0 through the ratio of integrated areas for H1 to Ha in the NMR spectrum (Fig. S1). The sharp peak at 1614.1 in the mass spectrum (MS) further proved that most G1 dendrimers were conjugated with an ornidazole ligand, and the low peak intensity at 1797.2 suggested that a small fraction of the G1 dendrimer was modified with two ornidazole moieties (Fig. 1a). The minimum inhibition concentration (MIC) of G1-orni was found to be 9.1 nM and 36.3 nM against Clostridium sporogenes (C. sporogenes) and Bacaeroides fragilis (B. fragilis), respectively, which was slightly higher than 2.3 nM and 9.2 nM for free ornidazole molecules, respectively (Table S1), suggesting that the conjugation of ornidazole on the G1 dendrimer maintains the antibiotic activity of ornidazole against anaerobic bacteria such as C. sporogenes and B. fragilis (Fig. 1b). Tobramycin was chosen as the model drug of aminoglycosides, and the drug is inefficient in killing anaerobic bacteria. An oxidized natural polysaccharide dextran was then mixed with tobramycin and G1-orni to prepare the proposed hydrogel (Fig. 1c). The gel was formed within several minutes, and exhibited a prominent shear shining property (Fig. 2a). The gel viscosity dropped from 216 Pa s to 1.09 Pa s when the shear rate was increased from 0.015 S−1 to 10.0 S−1. The hydrogel was also capable of being injected through a syringe. It can be seen that the viscosity of the gel decreased under the extrusion, while it returned back to the gel state after passing through the needle. Besides, the gel viscosity was investigated at continuous alternative shear rates. As shown in Fig. 2b, the hydrogel was collapsed to the liquid state at a shear rate of 20 S−1, while the modulus returned to its initial value when the shear rate recovered to 1 S−1, indicating the recovery of the gel state.

image file: c8bm01211c-f1.tif
Fig. 1 (a) Synthesis and mass spectrum of the synthesized G1-orni. (Observed m/z + 1 = 1614.1 Da, theoretical m/z = 1613.1 Da). (b) Bactericidal mechanism of G1-orni against anaerobic bacteria. (c) Preparation of antibiotic hydrogels consisting of both tobramycin and G1-orni for the treatment of both aerobic and anaerobic pathogens.

image file: c8bm01211c-f2.tif
Fig. 2 Mechanical properties of the prepared hydrogel. (a) The shear shinning property of the gel, and the inset is an image of the hydrogel injected through a syringe. (b) Thixotropic property of the gel determined by a continuous viscosity measurement, performed by breaking and recovery of the gel using an alternative shear rate of 20 and 1 S−1, respectively. (c) Thixotropic property of the gel determined by a continuous step strain between 200% and 1%, respectively. (d) Self-healing property of the gel. The middle hole in a ring-shaped gel was healed (upper) and different shaped gels were constructed by assembly of small gel blocks (lower).

Moreover, the gel showed a good thixotropic property (Fig. 2c). We also found that the gel is self-healing as it was formed through a dynamic Schiff base linkage. As shown in Fig. 2d, the hole in the middle of a ring-shaped gel was filled automatically after 3 hours incubation, and the small gel blocks attached to each other could join together as a uniform bulk, which can be fabricated into different shapes, such as a clover and a chain.

It is well known that bacterial growth is associated with decreased pH in the microenvironment.34–36 As shown in Fig. 3a and S3, the colour of the bacterial suspension added to a pH-sensitive dye bromothymol blue turned from blue to yellow during the incubation, suggesting the increased acidity of the bacterial suspension caused by bacterial growth. The generated acidic environment may cleave the Schiff base linkage in the hydrogel to trigger the release of antibiotics. 92% G1-orni molecules were released in a pH 5.0 buffer which mimics the bacterial suspension, while only 20% of the drug was released in a pH 7.4 buffer (Fig. 3b). Similarly, tobramycin showed an acid-responsive release profile, and 94% of the drug was released in pH 5.0 versus 26% in pH 7.4 (Fig. 3c). The hydrogel also exhibited an acid-triggered degradation behaviour. The storage modulus of the hydrogel was 229 Pa after immersion in a pH 7.4 buffer for 0.5 h, while the value was 144 Pa in a pH 5.0 buffer (Fig. 3d). These results suggest that the Schiff base linkages in the gel degrade much faster in the acidic microenvironments. We further evaluated the biocompatibility of the gels towards NIH 3T3 cells. The extract of the gel showed minimal toxicity on the treated cells at concentrations up to 5.0 mg mL−1. In addition, no obvious haemolytic activity of the gels can be observed at a gel concentration of 20 mg mL−1.

image file: c8bm01211c-f3.tif
Fig. 3 (a) pH conditions of the medium during bacterial growth. The inset is the image of the bacterial suspension taken at 4, 5, 6, 7, and 8 h, respectively. Bromothymol blue was used as a pH indicator. Release profiles of (b) G1-orni and (c) tobramycin from the hydrogel in PBS buffers of pH 7.4 and pH 5.0, respectively. (d) Storage modulus of the hydrogel after immersion in PBS buffers of pH 7.4 and pH 5.0 for 0.5 h, respectively. (e) Cytotoxicity of the hydrogel extract on NIH 3T3 cells at different concentrations. (f) Hemolysis of the hydrogel. Triton X-100 (0.5%) and PBS were used as the positive and negative controls, respectively. The insets are the photographs of blood cell suspensions treated with Triton X-100, hydrogel (20 mg mL−1) and PBS, respectively.

The in vitro antibacterial activity of the hydrogel against both aerobic and anaerobic pathogens was further investigated. As shown in Fig. 4, the tobramycin gel showed high antibacterial activity against the aerobic pathogens including Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), but was less effective against anaerobic ones such as C. sporogenes and B. fragilis. However, the G1-orni gel showed an opposite result, which efficiently killed C. sporogenes and B. fragilis, but was less effective against S. aureus and P. aeruginosa. For comparison, the complex gel containing equal amounts of active antibiotics to the G1-orni gel and tobramycin gel, respectively, was used. A live-dead staining assay was further used to illustrate the antibacterial effects of hydrogels. As revealed in Fig. 4e and f, most of the G1-orni gel treated P. aeruginosa and tobramycin gel treated B. fragilis were stained green (live bacteria), suggesting the weak antibacterial activities of G1-orni against P. aeruginosa and tobramycin against B. fragilis, respectively. However, the complex gel treated P. aeruginosa and B. fragilis were stained red (dead bacteria) during the same period (Fig. 4g and h). These results suggest that the tobramycin/G1-orni hydrogel can efficiently inhibit the growth of both aerobic and anaerobic pathogens. Additionally, the morphology of bacteria treated with gels was observed by SEM to further evaluate the antibacterial activity. As shown in the inset images in Fig. 4e and f, the membranes of P. aeruginosa and B. fragilis were still intact and smooth after incubation with the G1-orni gel and tobramycin gel, respectively, while seriously damaged membranes were observed for both P. aeruginosa and B. fragilis incubated with the complex gel (inset images in Fig. 4g and h).

image file: c8bm01211c-f4.tif
Fig. 4 In vitro antibacterial activity of the hydrogels against (a) S. aureus, (b) P. aeruginosa, (c) C. sporogenes, and (d) B. fragilis. The contents of tobramycin and G1-orni in the complex gel were equal to those in the tobramycin gel and G1-orni gel, respectively. Fluorescence images of the bacteria by live-dead staining: (e) P. aeruginosa treated by the G1-orni gel, (f) B. fragilis treated by the tobramycin gel, (g) P. aeruginosa and (h) B. fragilis treated by the complex gel. Scale bar in the staining image, 200 μm. Scale bar in the SEM image, 1 μm. *p < 0.05, N.S.p > 0.05.

In conclusion, we prepared a hydrogel with high antibacterial activity against both aerobic and anaerobic pathogens by crosslinking oxidized dextran with tobramycin and G1-orni. The injectable hydrogel exhibited thixotropic and self-healing properties and was acid-responsive due to the contribution of acid-labile Schiff base linkage during gel formation. This study provides a facile strategy to expand the antibacterial spectrum of aminoglycoside hydrogels and may have potential applications in the treatment of complicated infections in clinical practice.

Conflicts of interest

There are no conflicts to declare.


This work was financially supported by the National Natural Science Foundation of China (21725402) and the Shanghai Municipal Science and Technology Commission (17XD1401600).

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8bm01211c

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