Acute lung injury (ALI) is a severe diffuse pulmonary disease, wherein endothelia–epithelial barrier disruption leads to lung edema and acute hypoxemic respiratory failure [1,2]. Inflammation is a hallmark event closely associated with the onset and progression of ALI, which involves a cascade of reactions including the production of pro-inflammatory cytokines, infiltration and activation of neutrophils, and generation of cytotoxic reactive oxygen species [3,4]. As such, anti-inflammatory therapy is one of the most effective modalities for the management of ALI in the clinical setting [5,6]. While anti-inflammatory small-molecular drugs, such as corticosteroids and nonsteroidal drugs, possess potent efficiency in improving the disease status, they often cause cumulative toxicity in non-target organs and trigger systemic immune disorders due to lack of cellular or molecular specificity [7].

Antibodies-mediated suppression of anti-inflammatory cytokines/chemokines serves as a potential alternative for anti-ALI therapy, mainly due to their efficiency and selectivity [8]. Among numerous cytokines, tumor necrosis factor-α (TNF-α) serves as a critical mediator in the inflammation process [9], [10], [11], and its level in the alveolar lavage fluid is positively correlated with the severity of ALI [12]. Infliximab, a TNF-α monoclonal antibody, has been demonstrated to effectively reduce intra-alveolar effusion and lung tissue destruction by neutralizing the excessive TNF-α [13]. However, the high cost, immunogenicity, and resistance of antibodies have prompted the development of alternative therapeutic strategies with desired efficacy, better biosafety, and low cost [14].

RNA interference (RNAi) is a site-specific mRNA degradation mechanism mediated by small interfering RNA (siRNA) [15], [16], [17], and it holds great potentials for anti-inflammatory therapy [18], [19], [20]. Effective delivery of TNF-α siRNA (siTNF-α) to alveolar macrophages, the major source of TNF-α production [21,22], could serve to reduce TNF-α production toward ALI treatment. Compared with systemic injection, pulmonary administration affords various advantages such as noninvasiveness, immediate availability, local targeting, and decreased systemic side effects [23,24]. Additionally, the low nuclease activity of pulmonary alveoli prevents fast degradation of siRNA [25]. However, the success of pulmonary RNAi is greatly challenged by the lack of siRNA delivery systems that can cooperatively overcome the epithelial barrier of lung tissues and the cellular/endolysosomal membrane barrier of alveolar macrophages [23].

To allow siRNA condensation and intracellular siRNA delivery, cationic polymers (polycations) with membrane-penetrating capabilities are often employed [26,27]. Among them, cell penetrating peptides (CPPs) with potent membrane activities are widely utilized materials for the intracellular delivery of siRNA cargoes [28], [29], [30]. CPPs normally contain cationic arginine residues in their primary structure, and they often adopt helical secondary structure [31,32]. Mechanistic studies have demonstrated that the arginine motif as well as the rigid helical structure is dominantly related with their membrane penetration potency [33,34]. However, CPPs are often short (< 25 residues); their membrane-penetrating domain may be shielded and the overall positive charges could be neutralized after complexation with the siRNA molecules, thus reducing their affinities with cell membranes [35]. Additionally, higher membrane activity is often accompanied with higher toxicity, and CPPs at high concentrations would often cause irreversible damage to target cells. Therefore, a membrane-penetrating polypeptide with strong siRNA-binding affinity yet low cytotoxicity is highly necessitated to realize efficient intracellular siRNA delivery.

Apart from the membrane barrier, the mucus layer in the lung tissues poses another intrinsic barrier against polycation-mediated pulmonary siRNA delivery [36,37]. The positively charged polyplexes, assembled from the polycation and siRNA via electrostatic attraction, are easily entrapped by the mucus layer covering the respiratory epithelia, mainly because of the strong electrostatic attraction with negatively charged mucin glycoproteins [38,39]. As such, inhaled polyplexes experience fast mucociliary clearance, and minimal amount could traverse the mucus layer to reach the alveolar macrophages underneath [40,41]. While dense coating of polyplexes surface with PEG could facilitate the mucus penetration by diminishing adhesive interaction with mucus, it will at the meantime reduce the affinity with cell membranes to prevent effective cellular internalization and gene knockdown [42,43]. As such, it is of great demand to synchronously penetrate both mucus and cell membrane to achieve efficient trans-mucosal siRNA delivery into alveolar macrophages.

To address the above critical challenges, a mucus/cell membrane dual-penetrating nanosystem was developed to enable pulmonary delivery of siTNF-α toward ALI treatment. A membrane-penetrating helical polypeptide containing both guanidine and zinc dipicolylamine (Zn-DPA) as charged motifs on side chains was first developed. The obtained polypeptide, P-G@Zn, afforded potent membrane activities and siRNA condensation capacities due to its stable helical structure, sufficient backbone length, and abundant cationic groups. Excessive guanidine groups often lead to higher cytotoxicity due to the cationic charge density and strong membrane activity, and thus we herein partially replaced guanidine groups with Zn-DPA, the organometallic complexes of Zn coordination. Because Zn-DPA features low toxicity and selective affinity with phosphate-containing siRNA molecules and cell membranes [44,45], we reason that partial replacement of guanidine groups with Zn-DPA would diminish the cytotoxicity of helical polypeptides without compromising their siRNA binding and membrane penetration capabilities. To enable mucus penetration, carboxylated mannan (Man-COOH) as an anionic polysaccharide was further designed to coat the P-G@Zn/siRNA polyplexes, which would shield the cationic charges of the polyplexes to reduce the entrapment by the mucus layer. Because Man-COOH could bind the mannose receptors over-expressed on macrophages [46,47], we further hypothesize that the Man-COOH coating should increase rather than decrease the macrophage internalization level of polyplexes via mannose receptor (CD206)-mediated endocytosis, thus imparting higher TNF-α knockdown efficiency toward the treatment of ALI (Scheme 1).