Bacterial infection and excessive inflammation following abdominal injury can cause life-threatening complications, leading to multiple organ failure and death. Current strategies for treating abdominal trauma through surgery, fluid resuscitation, and infection control are inadequate for severe cases. New approaches to rapidly reduce inflammation and infection are urgently needed to prevent complications after severe abdominal trauma.

Figure 1: Diagram of the synthesis of hydrogels and their application as wound dressings to the treatment of abdominal trauma

(A) Hydrogels are prepared from oxychondroitin sulfate, cationic polyethylenimide and aminoglycoside antibiotics formed by gel. (B) As a wound dressing for the treatment of abdominal trauma, cationic hydrogels remove anionic pro-inflammatory molecules, such as cell-free DNA(cfDNA), as well as anionic bacteria, preventing infection and reducing inflammation by promoting the transformation of macrophages from a pro-inflammatory phenotype (M1) to a tissue repair phenotype (M2).

Figure 2: Characterization of cationic hydrogels

Laser scanning confocal microscopy (CLSM) images of (A-C) hydrogel. (D-F) Scanning electron microscope (SEM) image. Tob/OCS(A and D), PEI/OCS(B and E), and Tob/PEI/OCS(C and F), Tob/PEI/OCS gels have the densest and tightest networks due to their greater amino abundance and cross-linking density. (G) Energy storage modulus and loss modulus of hydrogel. (H) Viscosity change of hydrogel at shear rate of 1-100 rad s-1. The viscosity of hydrogel decreased sharply with the increase of shear rate. (I)Tob release curves of Tob/OCS and Tob/PEI/OCS hydrogels at pH 5.0 or 7.4. Tob/PEI/OCS hydrogels exhibit PH-dependent degradation and release behavior due to Schiff base fracture and electrostatic interaction. (J) Testing the self-healing ability of a hydrogel by bringing three hydrogel blocks into contact; These blocks form a single hydrogel that remains intact when stretched.

Figure 3: Cationic hydrogels remove pro-inflammatory molecules and bacteria

(A -- D) Hydrogel binding efficiency of (A)TNF-α, (B)CpG, (C)LPS and (D)HMGB1 after 12 h culture. Among the three hydrogels, Tob/PEI/OCS gel has the highest binding efficiency for TNF-α, CpG, LPS and HMGB1. (E -- F) Surface antibacterial activity of hydrogels after culture at 37°C for 2 hours. Cationic hydrogels kill all bacteria. (G) Growth curves of E. coli and (H) Staphylococcus aureus during incubation with hydrogel for 24 hours. Compared with PEI/OCS gels lacking antibiotics, Tob/OCS and Tob/PEI/OCS gels showed stronger antibacterial activity against E. coli and Staphylococcus aureus. (I) 96-hour thermogram of inhibition of bacteria by hydrogel. (J -- L) In the absence or presence of hydrogels, CpG activated HEK-Blue TLR9 reporter cells, LPS activated HEK-Blue TLR4 reporter cells, and CpG activated Raw 264.7 macrophages. All hydrogels reduced CPG-induced TLR9 activation, and the hydrogels also blocked LPS-induced TLR4 activation and reduced TNF-α secretion produced by CpG - and LPS-stimulated RAW 264.7 cells.

Figure 4: Tob/PEI/OCS hydrogels protected mice from complications and death following severe abdominal trauma by removing pro-inflammatory factors from the blood circulation

(A) Schematic of severe abdominal trauma resulting from cecal ligation and puncture (CLP). (B) Mice were monitored for 168 hours (7 days) after CLP (13 mice per group). Seven of the Tob/OCS gel treated mice survived, eight of the PEI/OCS gel treated mice survived, and all 13 of the Tob/PEI/OCS gel treated mice survived. (C) Clinical scores of mice 168 hours (7 days) after CLP. The Tob/PEI/OCS gel treatment group had the lowest score. (D-F) Serum levels of TNF-α(D), IL-6(E), and cfDNA(F) were measured 24 hours after CLP (6 mice per group). Serum TNF-α and IL-6 levels of Tob/PEI/OCS gel treated mice were lower than those of other treatment groups.

Figure 5: Tob/PEI/OCS hydrogels prevent multiple organ failure after abdominal trauma caused by CLP

(A) 24 hours after CLP, the heart, liver, spleen, lungs, kidneys, and colon were taken and stained with HE. Arrows indicate organ damage. (B-D) The corresponding liver (B), kidney (C), and heart (D) injury scores were assessed against established criteria. Hydrogel treatment significantly improved damage to the heart, liver, spleen, lungs, kidneys, and colon, including prevention of necrosis, tissue destruction, and infiltration of white blood cells. (E-G) Serum biochemical indices ALT(E), CRE(F), CK(G) were analyzed. The hydrogel reversed the increase of serum alanine aminotransferase (ALT), creatinine (CRE) and creatine kinase (CK) levels induced by CLP.

Figure 6: After severe abdominal trauma, Tob/PEI/OCS hydrogels polarize peritoneal macrophages from M1 to M2

(A-c) peritoneal levels of (A)cfDNA, (B)TNF-α, and (C)IL-6 in peritoneal lavage fluid 24 hours after CLP. (D-E) peritoneal macrophages were collected 24 hours after CLP and the percentage of (D)M1 and (E)M2 macrophages in peritoneal fluid was assessed by flow cytometry. The gene expression of (F)TNF-α, (G)iNOS and (H)Arg-1 was analyzed by (F-H)RT-PCR. Untreated CLP-induced abdominal trauma was associated with an increase in the M1 polarizing markers TNF-α and iNOS and a decrease in the M2 polarizing marker Arg-1 in peritoneal macrophages. In contrast, Tob/PEI/OCS gel therapy significantly reversed the mRNA expression trend in the model group. These results indicated that Tob/PEI/OCS hydrogels had anti-inflammatory activity by regulating peritoneal macrophages.

Figure 7: Hydrogels exhibit antimicrobial activity after CLP-induced abdominal trauma

(A) Colony images. (B-C) Bacterial CFU in serum and abdominal cavity (PC) of mice in different treatment groups 24 hours after CLP. Clp-stimulated mice had increased bacterial numbers both at the site of infection (the abdominal cavity) and throughout the body (in the blood). Treatment with Tob/OCS and Tob/PEI/OCS gel removed or killed most bacteria, demonstrating the antibacterial effect of TOB-containing OCS hydrogels in vivo.

【 Summary 】

Cationic hydrogels composed of PEI, Tob and OCS were synthesized by Schiff base reaction. This cationic hydrogel clears anionic pro-inflammatory molecules (DAMP and PAMP) and bacteria, and delivers antibiotics, and prevents multiple organ damage and death in mouse models of severe abdominal trauma in cecal ligation and puncture. A single hydrogel administration can achieve a 100% survival rate, and has good biodegradability and biosafety.