Inflammation, a crucial process in the host immune response, is inherently protective when tightly regulated. However, when prolonged or dysregulated, it can lead to chronic inflammatory disorders, contributing significantly to the onset and progression of various chronic diseases. Emerging studies underscore the implication of cell-free DNA (cfDNA) in initiating the development of organ-specific or systemic autoimmune diseases, including but not limited to systemic lupus erythematosus [1], rheumatoid arthritis [2], cardiovascular diseases [3], preeclampsia [4] and the development of toxic shock [5]. Recognition of cfDNA by Toll-like receptor 9 (TLR9) on the endosomes of immune cells triggers intracellular signaling cascades, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), leading to pro-inflammatory cytokine production [6]. Consequently, inhibiting the TLR9 pathway is hypothesized as a novel treatment target [7]. While TLR antagonists may induce systemic immune suppression, in time and accurate scavenging of cfDNA offers a promising approach to prevent TLR activation [8].

For this purpose, multiple cationic materials have been developed in recent years, showing therapeutic effects on many inflammatory diseases, such as rheumatoid arthritis [9], psoriasis [10], periodontitis and diabetic chronic wound [11]. Despite the development of cationic materials for scavenging cfDNA, challenges persist, including the high toxicity and short blood circulation of cationic nanoparticles (cNPs). Positively charged nanoparticles could be easily cleared by reticuloendothelial system, reducing the scavenging affinity [12]. Efforts to address these issues, such as the introduction of hydroxyl shells or poly(ethylene glycol) segments to cNPs' shells, have shown progress [13,14]. Furthermore, current cNPs cfDNA scavengers lack specificity and may inadvertently promote cfDNA internalization, counteracting the intended purpose of scavenging [15]. Besides, in the situation of acute injury where abundant cfDNAs are rapidly released, extracellular clearance of the cfDNA before they enter the endosomes could be difficult. Once endocytosed into immune cell endosomes, cfDNA will activate nucleic acid-sensing TLRs that are expressed on endosomal membranes and trigger pro-inflammatory cascades. The current cNPs are powerless against these endocytosed cfDNA.

To address these challenges, we propose pH-sensitive cNPs. Leveraging the pH gradient during endocytosis, pH-sensitive polymers serve as a promising smart delivery vehicle [16]. pH-sensitive cNPs, featuring minimal protonation extracellularly for viability and enhanced protonation intracellularly for better endosomal escape, are introduced. The inflammatory microenvironment's low pH further supports the belief that a proper pH-sensitive cfDNA scavenger can more effectively suppress inappropriate immune cell activation [17]. Notably, there is currently no report on specific intracellular cfDNA scavenging cNPs with reduced indiscriminate extracellular binding. Our hypothesis posits that pH-sensitive cNPs can enhance cfDNA binding efficiency through endosomal interactions, blocking TLR9 recognition intracellularly and ultimately suppressing pro-inflammatory cytokine secretion.

In this study, we synthesized cNPs self-assembled from pentablock polycaprolactone (PCL)/polyethylene glycol (PEG)-based polymers modified with different cationic moieties. The varied cationic segments endow the cNPs with varying pKa values and distinct cfDNA binding features. Through a combination of calf thymus DNA (ctDNA) binding assays, pH titration studies, tumor necrosis factor (TNF)-α secretion testing, and colocalization experiments, we unveiled the mechanism of cellular uptake and the cfDNA-induced pro-inflammatory suppression of cNPs. pH-sensitive cNPs demonstrated reduced extracellular protein adsorption, enhanced DNA binding ability in acid endosomes, and achieved optimal TNF-α suppression (Scheme 1). Finally, the efficacy of pH-sensitive cNPs in suppressing inflammatory cytokines was validated in a traumatic brain injury mice model, marking a significant step forward in our understanding and application of these nanoparticles in inflammation-related pathologies.