Polymeric DNase-I nanozymes targeting neutrophil extracellular traps for the treatment of bowel inflammation

Abstract

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a family of chronic disorders along the gastrointestinal tract. Because of its idiopathic nature, IBD does not have a fundamental cure; current available therapies for IBD are limited to prolonged doses of immunomodulatory agents. While these treatments may reduce inflammation, limited therapeutic efficacy, inconsistency across patients, and adverse side effects from aggressive medications remain as major drawbacks. Recently, excessive production and accumulation of neutrophil extracellular traps (NETs) also known as NETosis have been identified to exacerbate inflammatory responses and induce further tissue damage in IBD. Such discovery invited many researchers to investigate NETs as a potential therapeutic target. DNase-I is a natural agent that can effectively destroy NETs and, therefore, potentially reduce NETs-induced inflammations even without the use of aggressive drugs. However, low stability and rapid clearance of DNase-I remain as major limitations for further therapeutic applications. In this research, polymeric nanozymes were fabricated to increase the delivery and therapeutic efficacy of DNase-I. DNase-I was immobilized on the surface of polymeric nanoparticles to maintain its enzymatic properties while extending its activity in the colon. Delivery of DNase-I using this platform allowed enhanced stability and prolonged activity of DNase-I with minimal toxicity. When administered to animal models of IBD, DNase-I nanozymes successfully alleviated various pathophysiological symptoms of IBD. More importantly, DNase-I nanozyme administration successfully attenuated neutrophil infiltration and NETosis in the colon compared to free DNase-I or mesalamine.

Keywords: Colitis; DNase-I; Inflammatory bowel disease; Nanoparticle; Neutrophil extracellular trap.

Scheme 1

Schematic illustration of the fabrication of DNase-NZ and their application in the treatment of colitis. a DNase-NZ was fabricated using the synthetic polymer PLGA as the core. Polydopamine was used as a bio-adhesive coating for the conjugation of DNase-I. Nanozymes were PEGylated to improve their stability in vivo. b The negative surface charge of DNase-NZ allows passive recruitment to the damaged colon epithelium. Infiltrated particles attenuate NET-associated inflammations by inhibiting NET accumulation and interrupting the immune cascade. The inset demonstrates the advantage of DNase-NZ. With improved stability and enzyme activity, DNase-NZ can successfully degrade NET structures at inflamed sites while free DNase-I is rapidly cleared from the host environment. Scheme created using BioRender.com. PLGA, poly(lactic-co-glycolic acid); DSS, dextran sodium sulfate; NET, neutrophil extracellular trap

Fig. 1

Changes in various neutrophil-related biomarkers during acute colitis in C57BL/6 mice. a-m Biomarker levels were analyzed from the colon of normal and colitis mice (n = 3 per group). a GM-CSF, b G-CSF, c IL-17, d MIP-2, e KC, f MIP-1, g MIP-1, h IP-10, i MIG, j IL-10, k IL-6, l IL-1β, and m IFN-γ levels were simultaneously analyzed using the Luminex® assay. Biomarkers were categorized as either a–e those involved in neutrophil recruitment, f–i those released from activated neutrophils, j anti-inflammatory, or k–m pro-inflammatory. n Changes in plasma DNase-I levels during the development of acute colitis (n = 10). DNase-I levels in normal mice were averaged and used as the baseline (100%) to calculate relative changes in colitis mice. All analyses were performed in duplicates. Data are presented as mean ± SEM a-m or min./median/max. n Statistical significance was assessed using a one-tailed Student’s t-test. *p < 0.0332, **p < 0.0021, ***p < 0.0002

Fig. 2

Characterization of DNase-NZ. a–d Size distribution, average diameter, and polydispersity index of a PLGA NPs, b Dopa@PLGA NPs, c PEG@D-PLGA NPs, and d DNase-NZ. a–d Insets represent a scanning electron micrograph of each particle imaged at 10,000× magnification. Scale bar: 200 nm. e Surface charges of PLGA NPs, Dopa@PLGA NPs, PEG@D-PLGA NPs, and DNase-NZ. f The enzymatic activity of DNase-NZ was confirmed using gel electrophoresis. g The enzymatic activities of DNase-NZ were successfully preserved after prolonged incubations (1, 3, 6, 12, 24, and 48 h) at 37 ℃. h In vitro cytotoxicity of DNase-NZ on L929 cells at various concentrations (0.5, 1, 2, 5, 10, 100, 500, and 1000 μg/mL) after 24 h incubation. i NET-degrading ability of DNase-NZ was confirmed after incubation with NETosis-induced bone marrow derived neutrophils (BMDNs). Data represented as mean ± SD. e, h, i Statistical significance was assessed using a two-tailed Student’s t-test. ***p < 0.0002, ****p < 0.0001

Fig. 3

Therapeutic efficacy of DNase-NZ against DSS-induced colitis in mice. a Scheme illustrating the experimental groups and treatment schedule. C57BL/6 mice were provided with 2.5% DSS dissolved in drinking water for five days to induce acute colitis. PBS, mesalamine (100 mg/kg), free DNase-I (500 U), or DNase-NZ (500 U) was intra-rectally administrated daily for 7 days (Day 0–6). All mice were killed for analysis on day 10. b Changes in body weight recorded daily (n = 5). Statistical significance was assessed against G2. c, d Image and quantitative measurement of colon lengths on day 10 (n = 5). e Disease activity index (DAI) scored daily based on weight loss, stool consistency, and rectal bleeding (n = 5). Statistical significance assessed against G2 f, g Quantitative analysis of pro-inflammatory cytokine levels including f IL-1β and g IL-6 using commercial ELISA kits (n = 5). h Quantitative analysis of colon MPO activity on day 10 (n = 5). i Representative H&E and AB-PAS staining images of colon tissues on day 10. Arrows in AB-PAS stained images indicates presence of mucin. Scale bars: 250 µm. j Histological scores based on microscopic appearance of H&E stained images (n = 5). Data are presented as mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-test. *p < 0.0332, **p < 0.0021, ***p < 0.0002

Fig. 4

Changes in immune cell population and NETosis-related genes after treatment with DNase-NZ. a Representative flow cytometry plots (left) of leukocytes as identified by CD45⁺ cells in the lamina propria of mice (gated on live cells). Numbers within the plot indicate the average percentage of cells in the corresponding subset. The bar graph (right) represents the percentage of cells positive for CD45. (n = 5) b Representative FACS plots (left) of the neutrophils as identified by Ly6G and CD11c in the lamina propria of mice (gated on CD45 + leukocytes). Numbers within the plot indicate the average percentage of cells in the corresponding subset. The bar graph (right) represents the percentage of cells positive for Ly6G and CD11c. c–e Relative mRNA expression levels of genes, including c Cxcl5, d Elane, and e Padi4, were determined using quantitative PCR assay (n = 5). Gapdh was used as an internal reference and relative expression levels were calculated against control mice. All assays were performed in at least duplicates. Data are presented as mean ± SEM. Statistical significance was assessed using two-tailed Student’s t-test. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001