Sepsis is a life-threatening organ dysfunction caused by dysregulated host responses to infection [1], primarily driven by macrophage hyperactivation and cytokine storm [2]. Emerging evidences suggest that flap-structure-specific endonuclease 1 (FEN1)-mediated mitochondrial DNA (mtDNA) fragmentation in macrophages plays a critical role in immune dysregulation [[3], [4], [5]]. During sepsis, mtDNA is attacked by reactive oxygen species to form oxidized mtDNA [6], which is cleaved by FEN1 into small fragments and then released from mitochondria into cytosol through the mitochondrial permeability transition pore (mPTP) and voltage-dependent anion channel (VDAC) [3]. Once entering the cytosol, mtDNA is recognized by cytosolic nucleotide-binding oligomerization domain, leucine-rich repeat receptor, and pyrin-domain containing-protein 3 (NLRP3) to trigger inflammasome activation, leading to macrophage pyroptosis and release of pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18 [3,[6], [7], [8]]. Simultaneously, cytosolic mtDNA is recognized by cyclic GMP-AMP synthase (cGAS) DNA sensor to activate stimulator of interferon (IFN) genes (STING) signaling pathway, thereby initiating IFN-based inflammatory responses [[9], [10], [11], [12]].

In addition to activating cytosolic sensors, the cleaved mtDNA can escape from cells through cell death or stress-induced DNA efflux to become cell-free DNA (cfDNA), a key damage-associated molecular pattern capable of triggering robust innate immune responses [[13], [14], [15], [16]]. Elevated level of mtDNA has been detected in the blood of sepsis patients, with its concentration positively correlated with the disease severity and prognosis [17,18]. Once released into the extracellular environment, mtDNA can be taken up by immune cells such as macrophages, and recognized by toll-like receptor 9 (TLR9) in the lysosomes [[19], [20], [21], [22]]. This recognition subsequently activates the nuclear factor κB (NF-κB) signaling pathway, leading to macrophage activation and secretion of various pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-6 [[23], [24], [25]]. More importantly, due to its prokaryotic origin, mtDNA contains a higher proportion of unmethylated and hypomethylated CpG motifs compared to nuclear DNA, making it a robust stimulator of TLR9 that exacerbates systemic inflammation [[26], [27], [28]].

Given the critical pathogenic role of mtDNA in sepsis, FEN1-targeted RNA interference using small interfering RNA (siRNA) holds great promise for protecting mtDNA from fragmentation and inhibiting cytokine storm under septic conditions [3]. Cationic polymers have been widely employed for siRNA delivery, which are capable of condensing siRNA to form cell-ingestible nanocomplexes (NCs) [[29], [30], [31]]. Nevertheless, their positive surface charges lead to poor serum stability and rapid sequestration by the reticuloendothelial system after systemic administration [32,33]. To address this issue, various surface modification strategies, including poly(ethylene glycol) (PEG) modification [34], polyanion shielding [35], and cell membrane camouflaging [36,37], have been explored to neutralize the positive surface charges, thereby prolonging the blood circulation time and enhancing the accumulation at the inflamed tissues. However, these surface modulation strategies will often reduce the affinity of NCs with target cell membranes, hindering effective cellular uptake and gene silencing [32]. Thus, resolving the trade-off between lesion-specific accumulation and efficient uptake by target cells remains a key challenge.

To address this critical issue, biomimetic NCs partially cloaked with macrophage (RAW 264.7) membrane were herein developed to concurrently enhance serum stability and macrophage uptake efficiency for siRNA-mediated sepsis treatment. In particular, a positively charged, guanidine-rich, helical polypeptide (PG) was first utilized to condense FEN1 siRNA (siFEN1) and form the PG/siFEN1 (PsF) NCs, which were further decorated with MM to construct the biomimetic MM/PG/siFEN1 (MPsF) NCs. Optimization of the membrane protein/siFEN1 weight ratios allowed partial MM coating, which improved the serum stability of NCs without compromising their macrophage uptake efficiency. Upon systemic administration in cecal ligation and puncture (CLP)-induced sepsis mice, MPsF NCs exhibited prolonged blood circulation and active accumulation in the inflamed organs, including liver, lung, and kidney, facilitated by MM-mediated immune escape and inflammation homing [32,38]. Subsequently, MPsF NCs were effectively taken up by macrophages and provoked efficient FEN1 silencing, thereby preventing mtDNA cleavage. Consequently, MPsF NCs suppressed cytosolic mtDNA-triggered NLRP3 inflammasome assembly and cGAS-STING signaling as well as cell-free mtDNA-induced TLR9-NF-κB activation, thereby attenuating cytokine storm, inhibiting multiple organ failure, and enhancing resistance to secondary infections (Fig. 1).