Self‐Propelled Proteomotors with Active Cell‐Free mtDNA Clearance for Enhanced Therapy of Sepsis‐Associated Acute Lung Injury

Abstract Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis‐associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell‐free mitochondrial DNA (cf‐mtDNA) in the regulation of alveolar macrophage activation during sepsis‐associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase‐I) and human serum albumin (HSA) via glutaraldehyde‐mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf‐mtDNA clearance. The synthesized DNase‐I/HSA NMs are endowed with self‐propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase‐I/HSA NMs effectively eliminates cf‐mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf‐mtDNA clearance strategy using DNase‐I/HSA NMs is considered to be an attractive approach for sepsis‐associated ALI.


Figure S1 .
Figure S1.Dynamic changes of cf-mtDNA and cf-nDNA BALF levels during sepsisassociated ALI.A, B, Levels of cf-mtDNA (A) and cf-mtDNA (B) in the BALF of septic mice were evaluated by RT-qPCR.n = 8-12.Data represent mean ± SD; differences were compared by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test.*p < 0.05; ns, no significant difference.

Figure S2 .
Figure S2.The activation status of alveolar macrophages following co-incubation with BALF.Alveolar macrophage cells were activated by BALF obtained from septic mice, which was significantly attenuated by DNase-Ⅰ treatment.A-C, mRNA levels of TNF-a (A), IL-1β (B) and IL-6 (C) in alveolar macrophage cells (MH-S) after incubating with PBS or BALF for 6 h were evaluated by RT-qPCR.n = 3. E, F, The expressions of TLR9 (D) and iNOS (E) in alveolar macrophage cells were assessed by fluorescence staining (Scale bar: 50 μm).All cell experiments were performed at least in triplicate.The corresponding groups respectively were: Control (co-incubation with PBS), BALF-PBS (co-incubation with BALF obtained from PBS-challenged mice), BALF-LPS (co-incubation with BALF obtained from LPS-challenged mice), BALF-LPS+DNase-Ⅰ (co-incubation with BALF which obtained from LPS-challenged mice and treated with DNase-Ⅰ for 3 h).Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure S3 .
Figure S3.The mRNA levels of pro-inflammatory cytokines in alveolar macrophages following cf-nDNA and cf-mtDNA challenge.The mRNA levels of pro-inflammatory cytokines were markedly increased in alveolar macrophage cells after treating with cf-mtDNA for 6 h.A-D, mRNA levels of TNF-α (A), IL-1β (B) and IL-6 (C) in alveolar macrophage cells (MH-S) challenged with PBS, cf-nDNA or cf-mtDNA for 6h were evaluated by RT-qPCR.n = 3.The corresponding groups respectively were: Control (treatment with PBS), cf-nDNA (treatment with cf-nDNA), cf-mtDNA (treatment with cf-mtDNA).Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.*p < 0.05, **p < 0.01; ns, no significant difference.

Figure S5 .
Figure S5.The motility performance of DNase-Ⅰ NPs and DNase-I/HSA NMs.The diffusion coefficient of DNase-Ⅰ NPs and DNase-Ⅰ/HSA NMs in the presence of 1.5 μм dsDNA were measured by DLS.n = 6.Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.*p < 0.05, **p < 0.01; ns, no significant difference.

Figure S6 .
Figure S6.Assessment of lung inflammation.The lung inflammation was estimated by measuring the cells infiltration and cytokines secretion in BALF.A, Total number of inflammatory cells in BALF were counted via a haemocytometer (n = 6).B-D, The concentrations of inflammatory cytokines TNF-α (B), IL-1β (C) and IL-6 (D) in BALF were determined by ELISA (n = 8).Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure S8 .
Figure S8.Cytotoxicity of DNase-Ⅰ/HSA NMs.A, Cell viability of alveolar macrophages MH-S treated with various concentrations of DNase-Ⅰ/HSA NMs for 24 h.B, Cell viability of alveolar epithelial cells A549 treated with various concentrations of DNase-Ⅰ/HSA NMs for 24 h.n = 6.Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.ns, no significant difference.

Figure S9 .
Figure S9.The biodistribution of DNase-I/HSA in ALI models.The biodistributions of free DNase-Ⅰ and DNase-Ⅰ/HSA NMs in internal organs (heart, spleen, kidney and liver) 24 h after administration were monitored by IVIS imaging.

Figure S10 .
Figure S10.Blood panel data for in vivo safety evaluation.Blood panel data of normal mice (blank) and mice post either free DNase-Ⅰ or DNase-Ⅰ/HSA NMs administration at 24 h.n = 4 mice/group.Data represent mean ± SD; differences were compared by ANOVA with Tukey's multiple comparisons test.ns, no significant difference.

Figure S12 .
Figure S12.Assessment for the specific amplification of GAPDH plasmid DNA.The specific amplification of GAPDH plasmid DNA (for nDNA), was evaluated by amplification curve (A) and melting curve (B).

Figure S13 .
Figure S13.Assessment for the specific amplification of ND1 plasmid DNA.The specific amplification of ND1 plasmid DNA (for nDNA), was evaluated by amplification curve (A) and melting curve (B).