Bioinspired nanogels as cell-free DNA trapping and scavenging organelles for rheumatoid arthritis treatment

Abstract

Excessive cell-free DNA (cfDNA) in the serum and synovium is considered a causative factor of rheumatoid arthritis (RA). Thus, cfDNA scavenging by using cationic polymers has been an effective therapeutic avenue, while these stratagems still suffer from systemic toxicity and unstable capture of cfDNA. Here, inspired by the biological charge-trapping effects and active degradation function of enzyme-containing organelles in vivo, we proposed a cationic peptide dendrimer nanogel with deoxyribonuclease I (DNase I) conjugation for the treatment of RA. Benefitting from their naturally derived peptide components, the resultant nanogels were highly biocompatible. More attractively, by tailoring them with a larger size and higher surface charge density, these cationic nanogels could achieve the fastest targeting capability, highest accumulation amounts, longer persistence time, and superior DNA scavenging capacity in inflamed joints. Based on these features, we have demonstrated that the organelle mimicking cationic nanogels could significantly down-regulate toll-like receptor (TLR)-9 signaling pathways and attenuate RA symptoms in collagen-induced arthritis mice. These results make the bioinspired DNase I conjugated cationic nanogels an ideal candidate for treating RA and other immune dysregulation diseases.

Keywords: bioinspired; cell-free DNA; nanogel; peptide dendrimer; rheumatoid arthritis.

Fig. 1.

Organelle-inspired cationic nanogels with DNase I conjugation inhibit RA development by locally binding and eradicating cfDNA. (A) Biomimetic construction of organelle-inspired cationic nanogels with DNase I decoration. (B) The cationic nanogels with cfDNA-driven targeting ability to inflamed joints effectively inhibit the TLR9 activation by i) intercellular and ii) intracellular neutralization of pathogenetic cfDNA.

Fig. 2.

The characterization of G3KBPY dendrimer and cationic nanogels. (A) Chemical structure of the G3KBPY dendrimer. (B) FITR spectra and (C) Ultraviolet (UV) spectra of G3K and G3KBPY dendrimers. (D–G) Hydrodynamic size distribution and TEM images of G3K2, G3K5, G3K10, and DG3K10 nanogels. Average particle size and zeta potential (ZP) of DG3K10 after (H) dilution with PBS and (I) reservation for 1 wk. Data are expressed as mean ± SD.

Fig. 3.

Cationic nanogels inhibit cell inflammatory response due to a strong binding efficiency with extracellular and intracellular cfDNA. (A) EtBr competitive binding assay to measure the DNA binding efficiency of nanogels in PBS and in 10% FBS, respectively. The nanogels and DNA were mixed at a mass ratio of 1:1. (B) Biological activity of chemically anchored DNase I at pH 5 and 7.4, respectively. Cationic nanogels suppress NA-mediated activation of TLR9 in (C) Ramos BlueTM cells and (D) RAW264.7 cells. (E) MyD88, TRAF6, IL-β, and TNF-α enhanced expressions with CpG are down-regulated by cationic nanogels in Ramos BlueTM cells. (F) Intracellular trafficking of Quasar 670–labeled CpG and FITC-labeled cationic DG3K10 nanogels in RAW264.7 cells at different time points (Scale bar: 10 μm.) White spots marked by the white arrows indicate the colocalization of CpG and cationic nanogels. D: DAPI, L: LysoTracker, F: FITC-labeled DG3K10, and Q: Quasar 670-labeled CpG. (G) Cationic nanogels inhibit TNF-α and IL-6 expression of RA-FLS. RA-FLS was incubated with 1 μM of CpG 2006 for 4 h. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group, using one-way ANOVA followed by a post hoc test.

Fig. 4.

The mechanistic studies of the favorable therapeutic performance of DG3K10 in treating RA. (A) Dynamic in vivo NIRF images of DG3K10 from 0 to 2 h. (B) The biodistribution of nanogels in extra-articular organs and joints at 2 h and 24 h postinjection by ex vivo NIRF imaging. Quantification of mean NIRF intensity of cationic nanogels in ex vivo biodistribution study of CIA mice at (C) 2 h and (D) 24 h after i.v. injection. (E) A). liver; B). spleen; C). heart; D). lung; E/F). kidneys; G/H). forelimbs; I/J). hindlimbs. Data are expressed as mean ± SD.

Fig. 5.

DG3K10 nanogels improve the arthritic condition of SD rats induced by CpG. (A) Schematic representation of the establishment and treatment of the CpG-induced rat RA model. (B) Photos and thermographic (TG) images of right hind paws after administration of 7 d. The circled region indicated the swollen popliteal lymph node. (C) Quantification of paw temperatures at day 8. (D) The RA repairment of ankle joints was monitored by micro-CT images. Yellow arrows indicated the bone erosion. (E) The BMD of ankle joints were calculated from the micro-CT reconstruction. (F) Serum cfDNA concentration in RA rats after the treatment of nanogels and MTX for 7 d. Data are expressed as mean ± SD. ***P < 0.001 compared to the control group, using one-way ANOVA followed by a post hoc test.

Fig. 6.

In vivo therapeutic effects of cationic nanogels in the CIA model. (A) Schematic representation of the establishment and treatment of the CIA mice model. (B) Hind paw swelling, clinical scoring of (C) hind paws of CIA mice in the process of establishment and treatment by cationic nanogels. (D) Serum cfDNA concentration in CIA mice with the treatment of nanogels at different time points. (E) The RA progression and repairment of knee joints were monitored by micro-CT images. (F) Illustration of RA joint destruction and regeneration. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, using one-way ANOVA followed by a post hoc test.

Fig. 7.

In vivo histology outcomes with the treatment of cationic nanogels in the CIA model. (A) H&E, toluidine blue, safranin O, and TRAP staining, and (B) NLRP-3, TNF-α, and IL-6 immunofluorescent staining of the knee joint of CIA mice at day 70 after immunization. Coexpression and colocalization of TNF-α and IL-6 were confirmed by line profile analysis. (Scale bar, 200 μm.)

Fig. 8.

Therapeutic mechanisms of DG3K10 nanogels in the CIA model. (A) Volcano plot of differentially expressed genes between DG3K10-treated and model groups. (B) GO, (C) KEGG, and (D) Reactome enrichment analyses of differentially expressed genes in the DG3K10-treated and model group. Enrichment analyses were performed using Database for Annotation Visualization and Integrated Discovery (DAVID) Bioinformatics. BP, biological processes; CC, cellular components; MF, molecular functions. (E) Heat map of the immune regulation-related genes. (F) Heat map of the TLR signaling transduction-related genes.