Abstract

   Current pharmacotherapy remains futile in acute alveolar inflammation
   induced by Gram-negative bacteria (GNB), eliciting consequent
   respiratory failure. The release of lipid polysaccharides after
   antibiotic treatment and subsequent progress of proinflammatory cascade
   highlights the necessity to apply effective inflammation management
   simultaneously. This work describes modular self-assembling peptides
   for rapid anti-inflammatory programming (SPRAY) to form nanoparticles
   targeting macrophage specifically, having anti-inflammation and
   bactericidal functions synchronously. SPRAY nanoparticles accelerate
   the self-delivery process in macrophages via lysosomal membrane
   permeabilization, maintaining anti-inflammatory programming in
   macrophages with efficacy close to T helper 2 cytokines. By pulmonary
   deposition, SPRAY nanoparticles effectively suppress inflammatory
   infiltration and promote alveoli regeneration in murine aseptic acute
   lung injury. Moreover, SPRAY nanoparticles efficiently eradicate
   multidrug-resistant GNB in alveoli by disrupting bacterial membrane.
   The universal molecular design of SPRAY nanoparticles provides a robust
   and clinically unseen local strategy in reverse acute inflammation
   featured by a high accumulation of proinflammatory cellularity and
   drug-resistant bacteria.
     __________________________________________________________________

   Modular peptide assemblies enable inhaled nanoparticles to exert
   immunomodulation and rescue bacteria-induced lung inflammation.

INTRODUCTION

   As the final stage of unmanaged alveolar inflammation, acute lung
   injury (ALI), featuring hypoxemia and bilateral pulmonary infiltrates,
   results in respiratory failure with 38.5% mortality ([48]1, [49]2).
   Gram-negative bacteria (GNB) infection is one of ALI’s most common and
   notorious risk factors. Its outer membrane component,
   lipopolysaccharide (LPS), rapidly activates the Toll-like receptor 4
   (TLR4)/CD14 signaling cascade in alveolar macrophage (AlvM) and
   polarizes it to the M1 phenotype. Subsequently, the expansion of
   proinflammatory cellularity in lung edema further leads to epithelial
   and endothelial damage ([50]3), causing edema accumulation and
   impairment of gas exchange ([51]4). The swift progress of ALI
   necessitates concerted therapy, including oxygenation and ventilatory
   ([52]5), plus most essential antibacterial therapy and certain
   inflammation constriction ([53]6). Clinically, the long-term systemic
   dosing of antibiotics is applied to combat lethal infection caused by
   GNB but results in poor pulmonary drug accumulation ([54]7), of which
   the growing number of drug-resistant strains may further compromise the
   efficacy ([55]8). In addition, the action of antibiotics, e.g.,
   ampicillin, cotrimoxazole, and azithromycin, increases the LPS release
   from GNB to deteriorate lung injury or cause severe side effects such
   as endotoxin shock ([56]9), emphasizing the anti-inflammatory therapy
   in GNB-induced ALI. However, clinically available
   immunosuppressive-based pharmacotherapy is frequently feeble because of
   (i) uncleared therapeutic outcomes (e.g., glucocorticoids) ([57]10),
   (ii) incompetence in constructing a regenerative environment ([58]11),
   and (iii) the reduction of host defense to existing infection ([59]12).
   For example, the popular anticytokine therapies (e.g., tocilizumab)
   only neutralize proinflammatory cytokines or related receptors but may
   render a more susceptible respiratory tract to invading or colonized
   pathogens ([60]13).

   To overcome these predicaments, an inhalable nanoparticle orchestrated
   anti-inflammatory, antibacterial, and regenerative functions to secure
   the recovery process in pulmonary lesions while favoring the local
   delivery to maximize therapy effectiveness. Notably, microenvironment
   predominated by anti-inflammatory macrophages (M2) is necessary for the
   recovery of acute inflammation by inaugurating downstream protective
   and regenerative processes ([61]14), thus acting as a universal
   anti-inflammatory target. However, the direct AlvM targeting to induce
   M2 activation is currently very limited in GNB-infected lung ([62]15).
   Because regenerative medicine modulates macrophages by the delivery of
   drugs ([63]16, [64]17), nucleic acid ([65]18) or stem cells ([66]19)
   were still in its preclinical stage. The self-functioned biomaterials
   to modulate macrophage are specifically designed for topical use on
   epidermal ([67]20, [68]21), cardiovascular ([69]22), and tumor ([70]23)
   rather than respiratory tract, and thus may not be suitable for the
   harsh alveolar environment. Recent studies indicate that the M2
   macrophage polarization and bacteria eradication could be
   simultaneously achieved by cationic peptides ([71]24), including human
   host defense peptides (e.g., HD6 defensin) ([72]25), naturally
   occurring antimicrobial peptides (e.g., melittin), or other synthetic
   peptides ([73]26). However, the immunomodulation of most cationic
   peptides is too diverse to satisfy the clinical requirements ([74]27).

   This work reports a category of nanoparticles composed of modular
   self-assembly peptides for rapid anti-inflammatory programming (SPRAY)
   that compulsively reverse acute proinflammatory response by reshaping
   macrophages. We constructed SPRAY by conjugating a membrane-permeable
   motif (MPM) with a cationic peptide sequence to trigger
   anti-inflammatory functions. MPM comprised a biphenyl group (Bip) and
   two phenylalanine residues (FF). MPM assists the self-assembly process
   by dexterity to enhance π-π stacking (fig. S1) ([75]28, [76]29).
   Further, it interacts with the lipid bilayer, potentiating the membrane
   disruption under local accumulation ([77]30–[78]32). The cationic
   sequence is truncated from existing peptides or designed
   sophisticatedly ([79]Fig. 1A). To prove our conception, a short library
   of candidates was engineered and screened to generate the optimal
   SPRAY, called BLKR, by selecting a truncated sequence from
   antimicrobial peptide SAAP-148 ([80]33), termed LKR (table S1). Our
   result suggested that MPM conjugation helps with the formation of BLKR
   nanoparticles (SPRAY-BLKR-NPs) and enhances cytoplasmic delivery of
   BLKR for anti-inflammatory M2 polarization and inhibition of M1-related
   signaling. Moreover, MPM renders SPRAY-BLKR-NPs to efficiently
   eradicate multidrug-resistant Escherichia coli (MRE) in mice through
   membrane disruption in injured airspace and reverses ALI within a week.
   Together, we envisioned SPRAY nanoparticles as the express medication
   for treating GNB-induced ALI and hold enormous translational potential.

Fig. 1. The molecular design and the discovery of a biocompatible SPRAY with
dual M2 macrophage polarization and antibacterial activity.

   [81]Fig. 1.
   [82]Open in a new tab

   (A) BLKR was screened out as the optimal SPRAY that is comprised of an
   MPM and anti-inflammatory heptapeptide, exerting two prominent
   therapeutic roles: (i) SPRAY nanoparticles achieve anti-inflammatory
   immune programming in AlvMs through enhanced cytoplasmic delivery and
   amplifies the M2-related signaling transduction. (ii) SPRAY
   nanoparticles disrupt the membrane integrity of GNB and switch the
   inflammatory state when treating GNB-induced ALI. (B) The schematic
   illustration for the screening processes of optimal SPRAY. (C) The
   percentage of CD206^+ M2 macrophage after peptide treatment at the
   concentration of 16 μM. The dotted line in blue noted the percent of
   CD206^+ cells treated by blank culture medium. The dotted line in red
   noted the percentage of CD206^+ cells treated by IL-4 and IL-10. The
   peptides ranked in the top four are shown in red. n = 3, mean ± SD. (D
   and E) The cytocompatibility of anti-inflammatory SPRAY candidates to
   lung epithelial cells [Beas-2B; (D)] and macrophage [THP-1; (E)]. (F)
   The heatmap plotting of the hemolysis rate of anti-inflammatory SPRAY
   candidates. The up-limitation of the acceptable hemolysis rate was set
   as 5% (white). Data are presented as mean value, n = 9. (G) The MIC
   value against MRE of anti-inflammatory SPRAY candidates, n = 3
   biologically replicates. The MIC value (in micrograms per milliliter)
   in the third column was calculated from the value in the second column
   (in micromolar).

RESULTS

The optimization of SPRAY to complete M2 macrophage polarization

   To screen the optimal SPRAY, we applied the initial turn of screening
   by analysis of CD206 expression level in peptide-treated macrophage
   because CD206 represents the M2 phenotype polarization ([83]Fig. 1B).
   Through the quantification of the percentage of CD206^+ cells in total
   CD11b^+F4/80^+ bone marrow–derived macrophages (BMDMs), BKII, BLKR,
   BRLI, and BKRA ranked the top four anti-inflammatory potencies, with
   the overall M2 macrophage percentage at near 28.84, 28.67, 24.32, and
   16.83% ([84]Fig. 1C and fig. S2), respectively.

   Subsequently, the second turn of screening shall identify general
   cytotoxicity of candidates against various cells that might be exposed
   to SPRAY during in vivo absorption and distribution process, including
   the epithelial cells, immune cells, and red blood cells (RBCs).
   Hemolysis is the primary concern during the translation of cationic
   antimicrobial peptides. Thus, we next performed an imperative
   examination of the hemolytic effect led by peptide candidates ([85]Fig.
   1F). BKII and BLKR exhibited no detectable hemolytic activity even at
   256 μM [16-fold of minimal inhibitory concentration (MIC)]. BRLI is
   eliminated from the candidate pool because of its 7.49% hemolysis rate
   at 128 μM. Moreover, epithelial cells and macrophages are the most
   ubiquitous two classes of pulmonary cells to uptake incoming peptides
   or nanoparticles through the respiratory tract ([86]34, [87]35). After
   demonstrating BLKR and BKII have no notable cytotoxicity toward human
   bronchial epithelial cells (BEAS-2B) ([88]Fig. 1D), we also showed that
   only BLKR renders the eventual viability larger than 80% in the human
   monocytes (THP-1) ([89]Fig. 1E), the results of which are extremely
   encouraging because macrophages are more susceptible. Moreover, BLKR
   showed no significant cytotoxicity when treating another two kinds of
   epithelial cells [human embryonic kidney (HEK) 293T and HS-5 (human
   marrow stromal cells)] and antigen-presenting cells (RAW264.7, DC2.4),
   further demonstrating the cytocompatibility of BLKR (fig. S3).

   Last, considering a large portion of ALI was raised following GNB
   infection, the optimal SPRAY candidate shall exert vigorous
   antibacterial ability as a necessary simultaneous therapy. E. coli, the
   common cause of GNB-induced nosocomial pneumonia, was selected as the
   model pathogen ([90]36, [91]37). The MIC value of BLKR against MRE is
   16 μM. Admittedly, BKII failed to show antibacterial activity even at
   256 μM and therefore was not chosen despite its strong
   anti-inflammatory ability ([92]Fig. 1G).

   On the basis of above results, BLKR is distinguished for its
   combinational embracing of M2 polarization ability, biocompatibility,
   and bacterial eradication and would be comprehensively investigated in
   the following experiments. We further demonstrated that the
   anti-inflammatory property of this optimal SPRAY is highly sequence
   specific, because two scrambled sequences of BLKR, BWLR and BVRR,
   failed to significantly improve CD206 expression in BMDMs (fig. S6, A
   to C), even though their uptake quantity in BMDMs is on a similar scale
   as that of BLKR (fig. S6, D to F), respectively.

The optimal SPRAY induces anti-inflammatory M2 macrophage in vitro

   We used BMDMs to examine the influence of BLKR on the M2 polarization
   and the corresponding mechanism. The flow cytometry results ([93]Fig.
   2A and fig. S38) revealed that LKR failed to activate the desired M2
   phenotype. However, nearly 27% of BMDMs in the treatment of BLKR
   transformed into CD206^+ M2 macrophage, suggesting MPM is an essential
   framework for anti-inflammatory programming. Notably, interleukin-4
   (IL-4) and IL-10 generate 33% M2 macrophages. Meanwhile, LPS rather
   than BLKR or LKR eventually rendered more than 80% of CD86^+ macrophage
   in the total population ([94]Fig. 2B and fig. S4). F-actin ([95]Fig.
   2C) staining indicated that BMDMs presented round-shaped morphology at
   the M0 state. Upon the treatment of BLKR, it transformed into a long
   fusiform structure, which is consistent with the observation by the
   stimulation of the IL-4 and IL-10. The immunofluorescence staining
   revealed that CD206 protein is highly expressed on the cell membrane
   and partially in the cytoplasm under the treatment of BLKR, suggesting
   that BLKR shares similar M2 polarization ability as T helper 2 (T[H]2)
   cytokines do. Real-time quantitative polymerase chain reaction (qPCR)
   analysis indicates that BLKR significantly heightened the mRNA levels
   of several emblematic anti-inflammatory markers, including Arginase-1
   (Arg-1), CD206, YM-1, and Fizz-1 ([96]38) ([97]Fig. 2D). Because the
   T[H]2 cytokines secreted by M2 macrophage are regulatory to numerous
   tissue-repair responses during the inflammation ([98]39), we further
   tested the levels of T[H]1/T[H]2 cytokines in BMDM culture supernatant
   by the enzyme-linked immunosorbent assay (ELISA). Compared to untreated
   cells, BLKR up-regulated IL-4, IL-10, and IL-5 levels by 7.9-, 4.3-,
   and 2.0-fold ([99]Fig. 2E, and fig. S5), respectively. The treatment of
   BLKR does not affect the secretion of interferon-γ (IFN-γ) and
   down-regulated the production of tumor necrosis factor–α (TNF-α) by
   1.9-fold (fig. S5). These results indicate that BLKR might play a role
   in tissue repair during inflammation.

Fig. 2. The N-terminal modification enables optimal SPRAY to potentiate the
M2-type macrophage polarization.

   [100]Fig. 2.
   [101]Open in a new tab

   (A) The flow cytometry analyzing the CD206 and CD86 expression levels
   demonstrates that BLKR rather than LKR induces M2 macrophage. (B) The
   quantification of M2 macrophage percentage (CD11b^+F4/80^+CD206^+
   cells) in (A), n = 3, mean ± SD. (C) Representative CLSM images of
   BMDMs treated by indicated peptide (16 μM) for 24 hours (green, CD206;
   red, F-actin; blue, DAPI). Scale bar, 20 μm. (D) Real-time qPCR
   analysis of mRNA expression of M2-related marker (Arg-1, CD206, YM-1,
   and Fizz-1), n = 3, mean ± SD. (E) The ELISA test of the T[H]2
   cytokines levels (IL-10, left; IL-4, right) in the BMDM supernatant, n
   = 3, mean ± SD. (F) The GO enrichment analysis in BLKR group compared
   with LKR group. (G) KEGG pathway enrichment analysis for BLKR versus
   LKR group. (H) Heatmap plotting for clustering 33 differentially
   expressed genes under LKR and BLKR treatments in IL-17, MAPK, and TLR
   pathways. n = 3. (I) Schematic representation of the IL-17, MAPK, and
   TLR signaling cascade regulated by BLKR to trigger M2 macrophage
   polarization and inhibit M1 phenotype polarization. IRF-7, interferon
   regulatory factor-7; FOS, Fos proto-oncogene; CD14, cluster of
   differentiation 14 for LPS binding; p38, p38 mitogen-activated protein
   kinase, CCL17, C-C motif chemokine ligand 17; CCL2, C-C motif chemokine
   ligand 2; CD25, interleukin 2 receptor subunit alpha; CCL5, C-C motif
   chemokine ligand 5. Statistical analysis in (B), (D), and (E) was
   conducted using one-way analysis of variance (ANOVA).

SPRAY-induced transcriptome reprogramming to control M2 polarization

   To unravel the possible biological signal transduction uniquely
   manipulated by BLKR. We conducted the transcriptomics analysis on the
   LKR- or BLKR-treated BMDMs. Among all detected genes, BLKR and LKR
   shared 11,754 genes, and the rest are singly distributed in certain
   groups (fig. S7). The principal components analysis and M-versus-A (MA)
   plotting demonstrated the essential transcriptome reprogramming in
   response to BLKR treatment, as the samples treated with either LKR or
   BLKR are well splitted with markedly different regulations on gene
   pattern (figs. S8 and S9). To further explore the function affected by
   BLKR treatment, we performed Gene Ontology (GO) enrichment analysis. In
   detail, up to 196 differentially expressed genes were eminently
   T[H]1/T[H]2 cytokines biosynthesis process, namely, the production of
   IL-4, IL-5, IL-12, and IFN-γ ([102]Fig. 2F). Then, we concluded the top
   12 significant signaling pathways affected by the BLKR according to
   Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis
   (BLKR versus LKR) ([103]Fig. 2G). Seeking the regulatory functions of
   essential genes, we subsequently plotted the clustering of up to 33
   differentially expressed genes to comprehensively reveal that the key
   immunoreactions are mainly enriched in IL-17, mitogen-activated protein
   kinase (MAPK), and TLR pathways. In general, the genes related to T[H]2
   responses are up-regulated to varying degrees, while the expression of
   most genes in TLR signaling is inhibited ([104]Fig. 2H). Herein, we
   normalized the expression level of related genes in IL-17, MAPK, and
   TLR signaling (fig. S10). Specifically, the lower expression of IL-17Ra
   further caused the down-regulation of FOS through the MAPK pathway,
   then triggered downstream expression of CD25 and CCL17 to potentiate
   T[H]2 cell responses ([105]40, [106]41), and suppress T[H]17 cell
   responses through IL-17 signaling pathway. Moreover, we found BLKR
   stimulated the downstream expression of CCL2, demonstrating M2
   phenotype may be enabled through the increasing level of
   immunosuppressive CCL2 expression ([107]42, [108]43) ([109]Fig. 2I).

   Besides promoting M2 macrophage-related T[H]2 immunoreactions, BLKR
   also blocked the proinflammatory signal transduction. CD14 is a
   well-documented LPS-binding protein to activate TLR4. The mRNA
   expression level of CD14 under BLKR treatment is lowered by 1.8-fold,
   suggesting that the cellular sensitivity to LPS is down-regulated (fig.
   S10). To verify this, we find the impotence of LPS for further M1
   repolarization in BMDMs after the preincubation of BLKR at 16 or 128
   μM. Notably, such inhibition cannot be completed by using IL-4 and
   IL-10 (fig. S11).

Characterization of the self-assembly property of SPRAY

   The critical micelle concentration (CMC) values of BLKR and LKR is 89
   and 2560 μM ([110]Fig. 3A), respectively, suggesting that the MPM
   enables original LKR to acquire an enhanced self-assembly ability by
   precisely increasing the hydrophobicity at the N terminus. The circular
   dichroism (CD) indicates that MPM conjugation alters the secondary
   structure of peptide to potentiate the formation of nanoparticles. The
   original negative peak at 203 nm transformed into a positive peak at
   188 nm together with a negative peak at 209 nm.

Fig. 3. The N-terminal modification renders self-assembly to induce
nanoparticle formation and cytoplasmic release of functional
anti-inflammatory sequence.

   [111]Fig. 3.
   [112]Open in a new tab

   (A) The CMC measurement for BLKR and LKR. (B) The CD spectra of BLKR
   peptide. (C) The representative TEM images of the self-assembly
   structures of LKR and BLKR (128 μM). (D) The representative CLSM images
   demonstrating the uptake behavior of BLKR in BMDMs. Blue, DAPI-stained
   nuclei; red, lysotracker; green, FITC-labeled BLKR. Scale bar, 100 μm.
   The peptide trapped in the endosome was noted with green arrow. The
   peptide that escaped from the endosome was indicated with white arrow.
   (E) The representative Bio-EM images of BMDMs treated by BLKR for 15
   min, 1 hour, 2 hours, and 4 hours. The endocytosis process was noted
   with black arrow. The peptide distributed in the endosome was indicated
   with blue arrow. The integral endosomal membrane was noted with white
   arrow. The disruption of the endosomal membrane was marked with red
   arrow. (F) The histogram of FITC fluorescence intensity of BMDMs
   treated by BLKR or LKR for 1 hour. (G) The quantification of mean
   fluorescence intensity of FITC-labeled peptide in (F). Data are
   presented as mean ± SD, n = 3 biologically replicates. (H) The
   histogram plotting the FITC signal of BMDMs treated by FITC-labeled
   BLKR (16 μM) for 15 min in absence (Control) or presence of endocytosis
   inhibitor MβCD (5 mM), Amiloride (50 μM), EIPA
   (ethyl-isopropyl-amiloride; 50 μM), CPZ (chlorpromazine; 40 μM), and
   Filipin III (5 μg ml^−1). (I) The scheme of SPRAY’s cellular uptake
   processes and cytoplasmic release. Statistical analysis in (G) was
   conducted using one-way ANOVA.

   As shown by transmission electronic microscope (TEM; [113]Fig. 3C),
   BLKR forms nanoparticles, SPRAY-BLKR-NPs, with a diameter around 20 nm
   in physiological conditions at 128 μM, which could be favored for
   intrapulmonary delivery to reach the lower respiratory tract. However,
   the addition of LKR in the same condition results in an undetectable
   nanostructure, further demonstrating the importance of MPM in the
   formation of nanoparticles. We further demonstrated that SPRAY-BLKR-NPs
   retained desired particle properties in injured alveolar environment
   for up to 1.5 hours, which promisingly ensures the efficient uptake
   process during the real therapy (fig. S12). Moreover, the accelerated
   storage experiment proved that BLKR retained strong self-assembly
   property after being exposed to refrigeration, high temperature, and
   light exposure, evidenced by corresponding nonsignificantly changed
   reconstituted particle sizes (fig. S13).

SPRAY renders the enhanced cytoplasmic delivery

   Confocal laser scanning microscopy (CLSM; [114]Fig. 3D) shows that the
   green fluorescence from fluorescein isothiocyanate (FITC)–BLKR
   colocalized with the red fluorescence from Lyso-Tracker, implying that
   BLKR is uptaken by BMDMs through endocytosis and trapped in the
   endosomes at the first 1 hour. After incubating for 4 hours, more green
   fluorescence appeared in the cytoplasm, suggesting that the previously
   trapped BLKR started to escape from the endosome. Twelve hours after
   the coincubation, more peptides entered into the cytoplasm and
   distributed in the cytoplasm in a dotted pattern. No noticeable green
   fluorescence of FITC-LKR appeared in cells at all tested time points
   (fig. S14). The quantified mean fluorescent intensity measured by flow
   cytometry of BLKR is about 30- and 195-fold larger than the LKR group
   and control group, respectively ([115]Fig. 3, F and G). These results
   suggested that the nanoparticles facilitate the cytoplasmic delivery of
   functional anti-inflammatory peptide sequence by enhancing the cellular
   endocytosis and accelerating endosomal escape through latent membrane
   destabilization.

   We next used well-established endocytic inhibitors to investigate the
   endocytosis mode involved in the BLKR uptake by BMDMs. The addition of
   Filipin III reduces the percentage of FITC-positive cells from 95.28 to
   73.68% ([116]Fig. 3H), while leading to a 43.01% loss of BLKR uptake
   quantified by FITC intensity, suggesting that caveolae-mediated
   endocytosis contributes to the cellular uptake of BLKR (fig. S15). In
   addition, ethyl-isopropyl-amiloride (EIPA) and chlorpromazine (CPZ),
   inhibitors for macropinocytosis/clathrin-mediated endocytosis, also led
   to the decrease of cellular uptake of BLKR by 22.14 and 24.47%,
   respectively. We further demonstrated that such multiple endocytic
   pathways of BLKR in BMDMs by the combination of different inhibitors.
   Notably, when Filipin III, CPZ, and EIPA were applied simultaneously,
   only 33.47% of cells had positive BLKR signals. The absence of Filipin
   III significantly lowered the inhibition rate as the combination use of
   CPZ and EIPA reached 70.58% SPRAY-positive cells (fig. S16). These
   results together illustrate the cellular uptake of BLKR undergoes the
   aforementioned different endocytic pathways. Bio-EM provides direct
   evidence for the intracellular fate of BLKR ([117]Fig. 3E). Within the
   commencing 15 min, BLKR entered the macrophage, which was verified by
   the observation that peptide assemblies accumulated on the cell surface
   and were incorporated into the emerging vesicles. Forty-five minutes
   later, the BLKR assembles were sparsely distributed in endosomes, then
   further condensed in the endosomes at 2 hours, reflected by the pattern
   within the endosome gradually darkened. Notably, the structure of the
   endosomal membrane destructed at 4 hours to render the cytoplasmic
   release ([118]Fig. 3I).

SPRAY nanoparticles accumulated in injured lung through targeting AlvMs

   On the basis of the above encouraging results, we evaluated the
   capacity of SPRAY-BLKR-NPs to reverse the pulmonary aseptic
   inflammation after establishing the ALI mice model by the intratracheal
   injection of LPS (10 mg kg^−1) in mice on day 0 ([119]Fig. 4A). The
   local administration of our nanoparticle was selected for its maximized
   lung retention, meanwhile simulating the actual medication process
   based on the inhalation of anti-inflammatory agents in patients with
   ALI. Therefore, the pulmonary biodistribution of SPRAY was checked
   first. Our CLSM results confirmed that SPRAY-BLKR-NPs were not
   distributed in airway epithelial cells or endothelial cells but
   predominantly in the immune cells that infiltrated in alveoli. (fig.
   S17). Thus, we next comprehensively investigate the uptake status of
   BLKR in various types of pulmonary immune cells in injured lung. The
   uptake of SPRAY-BLKR-NPs mainly occurred in myeloid cells, in which
   nearly 51.01% of AlvMs uptook SPRAY particles within 1 hour. Less than
   10% of neutrophils, dendritic cells, and eosinophils show positive BLKR
   signals. In contrast, lymphoid cells, including T cells and B cells,
   show negligible BLKR uptake ([120]Fig. 4G and fig. S39). Encouragingly,
   we further directly visualized the colocalization of SPRAY-BLKR-NPs
   with CD68^+ pulmonary macrophages in the alveolar space at 1 hour after
   administration ([121]Fig. 4F). In comparison, LKR exhibited extremely
   little accumulation in injured lung tissue, witnessed by its virtually
   undetectable fluorescence in both healthy and injured lung.

Fig. 4. SPRAY nanoparticles are promising to reverse the inflammation in
LPS-induced ALI via M2 macrophage polarization.

   [122]Fig. 4.
   [123]Open in a new tab

   (A) Scheme illustrating the medication schedule for ALI mice. (B) Body
   weight monitoring. (C to E) Hematology monitoring of plasma white blood
   cell (WBC) counts (C), percentage of NEU in plasma (D) and BALF (E), n
   = 3. WBC, white blood cells; NEU, neutrophils. (F) Biodistribution of
   BLKR in injured lung at 1 hour after administration. Scale bar, 100 μm.
   Red, CD68; green, FITC-BLKR; blue, DAPI. (G) Uptake distribution of
   SPRAY-BLKR-NPs in various lung immune cell types, n = 3. AlvMs,
   alveolar macrophages; DCs, dendritic cells; Eos, eosinophils. (H)
   Representative images of lung H&E staining on day 6 (wide view, scale
   bar, 2.5 mm; enlarged region, scale bar, 50 μm). (I) Quantification of
   inflammatory cells in alveoli, min to max; +, mean value. (J)
   Representative immunofluorescence staining of pulmonary macrophages on
   day 6. Wide view, scale bar, 400 μm; enlarged region, scale bar, 10 μm
   (red, CD86; green, CD206; blue, DAPI). (K and L) Percentage of CD86^+
   (K) and CD206^+ macrophages (L) in total pulmonary cells. (M)
   Representative immunohistochemistry staining of SFTPC (20× field) on
   day 6. Scale bar, 100 μm. SFTPC, surfactant protein C. (N) Percentage
   of SFTPC^+ cells in total pulmonary cells, n = 3. (O and P) The IL-6
   (O) and IL-4 (P) concentration in BALF on day 6, n = 5. Data in (B) to
   (E), (G), (I), (K), (L), and (N) to (P) are presented as means ± SD.
   Statistical analysis was conducted using one-way ANOVA.

   We next measured the overall pulmonary retention of peptide molecules
   in injured lung on the single dosage of SPRAY-BLKR-NPs (fig. S18).
   Herein, more than 83.54% of dosed BLKR remained in the lung tissue at
   15 min after administration. Moreover, the pulmonary area under the
   curve of BLKR is more than 47.90 and 154.13 times larger than that in
   liver or kidney within 24 hours. These results indicated that BLKR is
   predominantly accumulated and metabolized in lung tissue. Near 10% BLKR
   was left in entire respiratory tract at 12 hours after administration,
   suggesting that a twice-administration (every 12 hours) schedule is
   needed to maintain pulmonary peptide content.

   Besides, a small amount of BLKR also accumulated in liver at the first
   1 hour and then gradually decreased, suggesting that the liver could
   also metabolize BLKR that leaked through the air-blood barrier in
   alveoli. Our further measurement indicated BLKR is metabolizable by
   liver microsome in human, monkey, dog, rat, and mouse, with related
   half-life ranging from 4.04 to 37.94 min (table S5).

SPRAY nanoparticles inhibit the proinflammatory status in aseptic ALI

   On the basis of our screening of effective dosage range, SPRAY-BLKR-NPs
   was dosed at 0.38 mg kg^−1 (128 μM, 50 μl) twice a day (every 12 hours)
   on day 1 for treating aseptic ALI (fig. S19 and [124]Fig. 4A). In
   subsequent daily clinical inspection, with the loss of 14% body weight
   by day 2, the weight turning points for mice that received
   SPRAY-BLKR-NPs occurred on day 3, as the weight maintained then
   elevated on day 5. The mice that received LKR lost nearly 18% body
   weight on day 3 and failed to show recovery later ([125]Fig. 4B).

   The composition of leukocytes in bronchoalveolar lavage fluid (BALF)
   and plasma exhibits the fundamental status of the pulmonary migration
   of inflammatory cells ([126]Fig. 4, C to E, and fig. S20). On day 2, we
   found that plasma leukocytes in mice from all groups elevated to nearly
   6 K μl^−1, while the down-regulation occurred on day 6 ([127]Fig. 4C).
   The plasma neutrophils only count for 51.36% of total leukocytes,
   1.6-fold smaller than Saline group ([128]Fig. 4D). This result
   indicated that neutrophil mobilization might be partially inhibited by
   SPRAY-BLKR-NP treatment. Mitigation also occurred in mice received
   saline and LKR. However, the plasma neutrophil percentage in the
   SPRAY-BLKR-NP group was still significantly lower than the level in
   other two groups on day 6, respectively. There was a distinct delay in
   the mitigation of pulmonary neutrophil migration. Neutrophils
   predominantly accumulated in the lung lumen during the acute phase of
   ALI, of which the percentages in all groups were higher than 70% until
   day 4. On day 6, we lastly found that only the BALF neutrophil
   percentage in the SPRAY-BLKR-NP group dropped to 45.8%, suggesting that
   the proinflammatory immune status was principally inhibited ([129]Fig.
   4E).

   The histological analysis revealed that alveolar inflammatory cell
   infiltration occurred intensively on day 2 as the critical feature for
   the onset of ALI. The mice received SPRAY-BLKR-NPs showed the most
   considerable diminution of alveolar inflammatory cell count from day 2
   to day 6 ([130]Fig. 4H and figs. S21 and S22). By quantifying the
   counts of inflammatory cells in the alveoli, the infiltration condition
   in SPRAY-BLKR-NP group started to mitigate at day 4, as the
   SPRAY-BLKR-NP treatment lowered the cell counts to nearly 1.4-fold more
   diminutive than Saline group. Such mitigation became more significant
   on day 6 because SPRAY-BLKR-NPs decreased the inflammatory cell counts
   in alveoli to about 1.9-fold compared to LKR group ([131]Fig. 4I).

Pulmonary M2 macrophage polarization by SPRAY-BLKR-NPs expedited tissue
regeneration

   To directly verify the efficient inflammatory mitigation that was
   observed mainly contributed to the M2 macrophage polarization inducing
   through SPRAY-BLKR-NP treatment, we used immunofluorescent staining of
   lung tissues to differentiate CD86^+ M1 macrophage and CD206^+ M2
   macrophage ([132]Fig. 4J). The mice received ineffectual
   anti-inflammatory therapy, including saline and LKR, to maintain the
   substantial accumulation of M1 macrophage in the lung tissue. In
   contrast, the SPRAY-BLKR-NPs reduced the M1 macrophage percentage from
   23% to nearly 10%, indicating that the progression of the inflammatory
   state is largely inhibited ([133]Fig. 4K). Moreover, SPRAY-BLKR-NPs
   rendered the M2 macrophage to dominate the entire macrophage population
   as its percentage was boosted from 9 to 18% ([134]Fig. 4L). Such a
   switch in dominated macrophage phenotype proves that SPRAY-BLKR-NPs is
   promising to reverse the proinflammatory state in ALI to a regenerative
   anti-inflammatory state. Furthermore, this reduction of the M1
   macrophage population resulted in the descending pulmonary IL-6 level.
   All three groups show similar BALF IL-6 levels larger than 400 pg ml^−1
   on day 2, then gradually decreased, in which the SPRAY-BLKR-NPs most
   timely accomplished local IL-6 level below 65 pg ml^−1 on day 4
   ([135]Fig. 4O). Notably, IL-6 is mainly secreted by the macrophages or
   neutrophils in pulmonary tissue and then enters blood circulation. The
   systemic cytokine fluctuation might be less sensitive than the local
   site. Our tracking of systemic IL-6 levels also clued to such
   mitigation of inflammation by witnessing the significant reduction in
   serum IL-6 level only at day 4 (fig. S23A).

   The differentiation of M2 macrophage would secrete IL-4, a key T[H]2
   cytokine ([136]44), to trigger a regenerative microenvironment for
   alveolar epithelial stem cell proliferation ([137]45) and type II
   pneumocyte differentiation ([138]46). We subsequently confirmed that
   the establishment of such pulmonary regenerative environment as BALF
   IL-4 level in SPRAY-BLKR-NP group is near 222 pg ml^−1, about two times
   higher than mice received LKR. These distinctions were further
   expanded, as evidenced by the finding that BALF IL-4 elevated to 426 pg
   ml^−1 at day 6, which is threefold larger than the LKR group ([139]Fig.
   4P). We also observed a steady plasma IL-4 level lower than 100 pg
   ml^−1 at tested time points (fig. S23B), suggesting that the switch of
   inflammation status mainly occurred locally in lung tissue without a
   systematic manner. During recovery, M2 macrophages facilitate the
   proliferation and differentiation of type II pneumocytes to secrete
   lung surfactant protein C (SFTPC) as a protective agent to accelerate
   tissue regeneration ([140]47, [141]48). As shown in [142]Fig. 4M,
   SPRAY-BLKR-NPs induced the most obvious proliferation of type II
   pneumocytes on day 6. In detail, type II pneumocytes accounted for
   approximately 28% of total pulmonary cells in SPRAY-BLKR-NP group,
   which was at least 1.5-fold larger than the level in the Saline or LKR
   group. Notably, less than 5% of cells are demonstrated to be SFTPC
   positive in healthy mice lungs ([143]Fig. 4N).

Pulmonary monocyte infiltration regulated by optimal SPRAY in healthy lung

   To study the potential immunomodulatory effect on monocytes led by BLKR
   in healthy mice lungs. The healthy mice are administrated with BLKR at
   different dosages twice and then analyzed 24 hours later for acute
   immunomodulation inspection ([144]Fig. 5A). First, we find no elevation
   of monocyte percentage in peripheral blood ([145]Fig. 5F) at all the
   tested concentrations. Next, the potential monocyte infiltration caused
   by BLKR in the lung parenchyma is investigated. CD64 and
   tyrosine-protein kinase Mer (MERTK) were selected to gate the pulmonary
   monocyte from CD45^+ pulmonary leukocytes. Then, CD11b was applied to
   differentiate AlvMs and interstitial macrophages (IMs) (fig. S40). The
   positive control, LPS, significantly lifted the IM percentage to nearly
   70% in total CD45^+CD64^+MERTK^+ monocytes ([146]Fig. 5C), suggesting
   pronounced monocyte infiltration. In contrast, the mice lung treated
   with BLKR (128 or 256 μM) showed a similar level of CD45^+CD64^+MERTK^+
   monocyte as Saline group. Besides, the percentage of IMs and AlvMs
   maintained the same after the treatment with BLKR in the total lung
   CD45^+ leukocytes ([147]Fig. 5D).

Fig. 5. Potential pulmonary macrophage infiltration and adverse effects of
optimal SPRAY in healthy mice.

   [148]Fig. 5.
   [149]Open in a new tab

   (A) The experiment designed to analyze macrophage recruitment in
   healthy mice. (B) The representative images of H&E staining of lung
   tissue of healthy mice received BLKR at indicated dosage.
   Representative areas were shown in the first row (scale bar, 100 μm).
   The enlargement of focused area (black frame) was shown in the second
   row (scale bar, 50 μm). The infiltrated immune cells were noted using
   black arrow. (C) The flow cytometry analysis of IMs and tissue-resident
   AlvMs infiltration in pulmonary tissue induced by peptide treatment.
   The pulmonary macrophage was gated by CD64a and MERTK from CD45^+
   cells. (Upper row). The IMs and AlvMs were then gated using CD11b from
   CD45^+CD64^+MERTK^+ cells (lower row). (D and E) The quantification of
   AlvMs (D) and IMs (E) in CD45^+ leukocytes. n = 3 mice per group, mean
   ± SD. (F) The percentage of monocyte (MON) in total leukocytes in
   peripheral blood. n = 3 mice per group, mean ± SD. (G) Blood parameters
   at 24 hours after the BLKR administration. n = 5 mice, violin plot,
   quartiles are shown in dotted lines, mean values are shown in solid
   lines. WBC, white blood cell; HGB, hemoglobin; MCV, mean corpuscular
   volume; PLT, platelets. Statistical analysis was conducted using
   one-way ANOVA.

   The histological analysis got a similar conclusion because no monocyte
   infiltration was observed in the alveolar site after BLKR treatment
   (128 or 256 μM). Notably, inflammatory cell infiltration occurred in
   lung when treated with higher concentrations (512 or 1204 μM). These
   results together suggested that BLKR is safe to use locally and without
   altering monocyte infiltration at concentrations up to 256 μM
   ([150]Fig. 5B).

Biosafety evaluation of SPRAY nanoparticles

   The results also confirmed that such short-term (1 day, two doses)
   exposure to SPRAY-BLKR-NPs would not lead to hemolysis, anemia, or
   systemic inflammation as similar plasma levels of RBC counts,
   hemoglobin, mean corpuscular volume, and platelets were found in the
   mice receiving different dosages of SPRAY-BLKR-NPs. Besides,
   SPRAY-BLKR-NPs did not alter the plasma leukocyte level ([151]Fig. 5G)
   or cause acute injury in the main organs, suggesting the excellent
   biocompatibility of our system (fig. S24).

   We also performed the safety profile of SPRAY-BLKR-NPs under long-term
   (14-day) exposure to respiratory tract (fig. S25A). No significant
   abnormality in blood parameters was detected during or after the
   consecutive 14-day dosing period (fig. S25, B to F). Liver and kidney
   function remained unaffected throughout the evaluating period, which
   might be due to the fact that BLKR is mainly distributed in the lungs
   rather than the liver and kidneys (fig. S25, G to K). Further
   histological inspection confirmed the absence of any sign of acute
   injury in the main organs at 2 weeks after discontinuation of
   nanoparticle exposure (fig. S25L).

   Potential damage to the respiratory system under long-term exposure was
   particularly focused (fig. S26A). We applied three times lung function
   tests on days 6, 15, and 27, respectively, to reveal similar levels of
   inspiratory capacity, forced expiratory volume (FEV0.2), resistance,
   static compliance, forced vital capacity and elastance in
   nanoparticle-treated mice as healthy mice did (fig. S26, B to G).
   Further BALF examination of pro/anti-inflammatory cytokines levels
   (fig. S26, H and I) and lung hematoxylin and eosin (H&E) staining (fig.
   S26J) suggest the nonoccurrence of T[H]1/T[H]2-related inflammation or
   fibrosis.

SPRAY nanoparticles exert promising bactericidal activity

   Besides the rapid activation of TL4 signaling cascade in immune cells,
   GNB could also directly injure the integrity of alveolar endothelium to
   increased permeability that allows liquid to migrate from the lung
   endothelium to the interstitial alveolar lumen ([152]3). During the
   progress of GNB colonization, type II pneumocytes are weakened to
   absorb the excessive apical fluid ([153]4). Moreover, GNB and toxins
   could leak into blood circulation because of increased endothelial
   permeability and eventually lead to devasting sepsis. Thus, we next
   comprehensively evaluate the antibacterial activity of SPRAY-BLKR-NPs.

   In vitro experiments show that SPRAY-BLKR-NPs killed 99.961% MRE and
   99.997% methicillin-resistant Staphylococcus aureus (MRSA) at 128 μM
   ([154]Fig. 6A and fig. S27). Then, we investigated whether
   SPRAY-BLKR-NPs is qualified for specialized pulmonary bacterial
   eradication. In injured alveoli, accumulated phospholipids and proteins
   provide nonspecific adhesion and compromise the efficacy of cationic
   agents. Thus, competent antibacterial agents shall especially tolerate
   the harsh environment of the lungs. We mimic the healthy and injured
   alveoli microenvironment using the BALF extracted from untreated mice
   and mice with aseptic ALI, in which SPRAY-BLKR-NPs reached anti-MRE
   efficiency at 98.83% in healthy BALF and 96.41% in ALI BALF ([155]Fig.
   6B). Moreover, during pulmonary infection, the changed airway
   microenvironment would enhance the bacteria attachment on epithelium to
   lastly form biofilms, which essentially prevents the normal
   phagocytosis by macrophages, facilitates the growth of drug resistance,
   and severely deteriorates the injury condition. The results suggested
   that SPRAY-BLKR-NPs eradicated both the MRE and MRSA biofilm as
   evidenced by the live/dead staining using SYTO9/Propidium Iodide (PI)
   ([156]Fig. 6C and fig. S28). The crystal violet staining further showed
   that BLKR lowered relative biomass by 2.06-fold in MRE ([157]Fig. 6D)
   and 1.62-fold in MRSA (fig. S29). These results proved the feasibility
   of using SPRAY-BLKR-NPs to combat local pulmonary bacteria eradication.

Fig. 6. SPRAY-BLKR-NPs exert bacteriacidal activity through membrane
disruption and rescue mice from MRE-induced ALI.

   [158]Fig. 6.
   [159]Open in a new tab

   (A) In vitro viability of MRE after peptide (128 μM) treatment. (B) Ex
   vivo assay evaluating the anti-MRE activity of peptide (256 μM) in
   alveolar environments. (C) Representative CLSM imaging of MRE biofilm
   treated by indicated peptide (green, SYTO9; red, PI). Scale bar, 160
   μm. (D) Crystal violet assay of MRE biofilm, mean ± SD, n = 3. (E)
   Representative SEM images of peptide-treated MRE. The membrane
   disruption was indicated by white arrow; the debris of MRE is indicated
   by black arrow. Scale bar, 400 nm. (F) The NPN fluorescence test
   demonstrating the BLKR treatment influenced the membrane integrity of
   MRE. n = 3, violin plot. (G) Membrane depolarization of MRE measured by
   DiSC[3](5) probe. n = 3, mean ± SD. (H) Representative Bio-EM images of
   MRE (white arrow, membrane disruption). Scale bar, 500 nm. (I) Scheme
   illustrating peptide treatment for MRE-induced ALI. (J) The plotting of
   pulmonary MRE burden after indicated administration, n = 6, mean ± SD.
   (K) NEU counts in plasma. Each dot represents data from one mouse, n =
   5. P value was calculated by comparing BLKR group with Saline group at
   each time point. (L) Representative H&E staining images of lung (0.5×
   field for wide view, scale bar, 2.5 mm; 20× field for enlarged region,
   scale bar, 100 μm). (M) The survival rate. n = 15. (N and O) The IL-6
   (N) and IL-4 (O) concentration in BALF on day 6. n = 6, mean ± SD.
   Statistical analysis was conducted using one-way ANOVA.

The membrane disruption mechanism ensures bacteria eradication by SPRAY
nanoparticles

   SEM imaging indicated that the healthy E. coli has long cylindrical
   morphology with slight wrinkles on its integral cell membrane. In
   contrast, most bacterial cell membranes become disintegrated (white
   arrow) after SPRAY-BLKR-NP treatment, while some bacteria directly
   lysed into cell debris (black arrow), suggesting SPRAY-BLKR-NPs induce
   membrane disruption to eradicate MRE ([160]Fig. 6E). Such disruption
   was also observed in MRSA (fig. S30). To prove that such membrane
   disruption is the predominant mechanism, we used
   N-phenylnaphthalen-1-amine (NPN) assay to perform membrane integrity
   analysis. The results show a marked boost of normalized NPN
   fluorescence in SPRAY-MRE treated by SPRAY-BLKR-NPs compared to
   untreated MRE ([161]Fig. 6F).

   The membrane potential examination using a voltage-sensitive dye
   DiSC[3](5) indicated a sharp uplift of DiSC[3](5) fluorescence
   intensity within the first 5 min after the addition of SPRAY-BLKR-NPs
   ([162]Fig. 6G), clearly affirming the close interaction between BLKR
   with the components of bacterial membrane. LNPs composed of
   1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PE),
   1,2-dioleoyl-sn-glycero-3-phosphoglycerol (PG), and cardiolipin (DPG)
   (79:17:4) were extruded to investigate the detailed interaction mode
   between GNB membrane with BLKR. CD showed the appearance of standard β
   sheet secondary structure (positive peak at 196 nm; negative peak at
   214 nm) once BLKR binds to the artificial GNB membrane (fig. S31),
   indicating that BLKR undergoes reassembling process and conformational
   change to form ordered nanostructure during bacteria eradication. After
   verifying membrane disruption, we further explored the potential
   intracellular disorder the peptide leads. According to the Bio-EM
   images of the bacterial section, the appearance of membrane disruption
   is confirmed in MRE or MRSA ([163]Fig. 6H and fig. S32) in
   SPRAY-BLKR-NP group. Cytoplasmic morphology changes are absent,
   suggesting that membrane disruption is the main contributor to overall
   antibacterial efficacy. In addition, such physical antibacterial
   mechanism expanded the application of SPRAY to kill other GNB strains,
   such as Klebsiella pneumoniae and Pseudomonas aeruginosa (tables S3 and
   S4) and may help to delay the development of drug resistance ([164]49).

Nanoparticles of BLKR rescue mice by in vivo MRE eradication

   Etiological studies revealed that bacterial infection is one of the
   most prevalent causes of lung injury. GNB could cause lobar pneumonia
   ([165]50), in which the invading bacteria multiply in the alveoli site
   and release toxins to provoke ALI ([166]51). After establishing
   bacteria-induced ALI by local infection with a lethal dose of MRE in
   pulmonary tissue, we first screen the therapeutic dosage range by
   administrating the mice with SPRAY-BLKR-NPs every 12 hours during the
   first 2 days, in which the dosage at 0.76 mg kg^−1 (256 μM, 50 μl) per
   administration ensured the primary MRE eradication adequacy and the
   following recovery process to rescue the overall survival condition
   (fig. S33, A to E). According to the plotting of pulmonary MRE burden,
   the first dose of SPRAY-BLKR-NPs reached 96.03% MRE eradication
   efficiency compared to Saline group, whose second dose of
   SPRAY-BLKR-NPs further eliminated nearly 78.39% of residual MRE to
   approach the 98.76% anti-MRE efficiency ([167]Fig. 6J). Thus, the first
   two doses of BLKR rather than LKR are competent for diminishing
   pulmonary MRE burden. There is no significant difference in the
   pulmonary MRE quantity between the first and second doses of saline,
   demonstrating that the self-immunity could not eradicate resident MRE
   on day 1.

   Furthermore, the clinical available antibiotic, ceftazidime required at
   least three consecutive days of parenteral systemic administrations
   (six doses) to reach the similar pulmonary MRE eradication rate as
   SPRAY-BLKR-NP group reached on day 1 (fig. S37, A and B). Thus, the
   promising pulmonary MRE eradication allowed SPRAY-BLKR-NPs to reach
   nearly 80% survival rate in the first 2 days ([168]Fig. 6M). In
   contrast, the 2-day survival rate of mice received LKR and ceftazidime
   only reached 20 and 58.3%, respectively.

SPRAY-BLKR-NPs inhibit the progression of pulmonary injury by switching the
inflammation status

   The exclusive anti-MRE therapy usually fails to rescue the mice with
   pulmonary MRE infection. Once the peptide lysed the MRE, the remains of
   bacterial cell walls containing LPS or other toxins would successively
   raise the secretion of proinflammatory cytokines, resulting in
   exacerbating lung injury. Thus, an add-on therapeutic effect,
   inflammation reversal, is waiting to be valid for trial on
   SPRAY-BLKR-NPs.

   We primarily focused on the fluctuation of neutrophil levels to track
   the mitigation of overall inflammatory conditions. The hematology
   results indicated that SPRAY-BLKR-NPs successfully forestalled the
   neutrophil mobilization as merely 2.28 K μl^−1 plasma neutrophils, at
   least twofold less than the level in mice received LKR. On day 6, the
   surviving mice treated with SPRAY-BLKR-NPs showed similar plasma
   neutrophil counts as day 1, while neutrophil concentration slightly
   decreased in the LKR group to 4.24 K μl^−1 ([169]Fig. 6K). Similar
   mitigation can be found in the plasma leukocyte counts (fig. S34). Such
   mitigation in the proinflammatory status is also proven by the decrease
   in BALF IL-6 level ([170]Fig. 6N). The number of infiltrated cells is
   almost identical in the alveoli of all groups on day 2 (fig. S35) but
   much less in the alveoli of nanoparticle group on day 6 ([171]Fig. 6L).
   In comparison, the six doses of ceftazidime show less capacity than our
   nanoparticle in inhibiting inflammation progress, testified by the
   significantly slower drop of BALF IL-6 and plasma neutrophil counts
   during therapy (fig. S37, C and D).

   To investigate whether SPRAY-BLKR-NPs accelerate the establishment of
   anti-inflammatory microenvironment, we briefly measured the IL-4 level
   in BALF. The IL-4 in the SPRAY-BLKR-NP group is nearly twofold larger
   than the level in the LKR group by reaching 127 pg ml^−1 ([172]Fig.
   6O), suggesting that the immune surroundings favored tissue
   regeneration. Hence, the edema and infiltrated cell is nearly
   diminished only in the alveoli of mice received SPRAY-BLKR-NPs on day
   10 (fig. S36). Notably, such accelerated construction anti-inflammatory
   environment was not seen in Ceftazidime group (fig. S37, E to G). Our
   survival rate monitoring could also support this conclusion.
   SPRAY-BLKR-NPs reached the final 73.3% 10-day survival rate, suggesting
   that less than 7% of mice later died because of the following
   toxin-induced lung injury, while only 20 and 13.7% of mice survived in
   LKR group and Saline group on day 10, respectively ([173]Fig. 6M).

DISCUSSION

   The ineffectiveness of antimicrobial and anti-inflammatory
   pharmacotherapy in GNB-induced ALI messages the urgency to develop a
   single-molecule–based therapeutic for suppressing proinflammatory
   reactions, combating bacterial infection and constructing a
   regenerative environment. This work conceived a previously unidentified
   category of an anti-inflammatory peptide molecule, SPRAY, and screened
   out BLKR as the optimal candidate for immuno-programming in
   macrophages. In molecular engineering, we conjugate essential MPM motif
   to a minimalistic anti-inflammatory sequence, LKR, which constitutes
   crucial membrane disruption and self-assembly ability to form
   nanoparticles. In undifferentiated macrophage, MPM enhances cellular
   uptake and cytoplasmic release of anti-inflammatory sequence, amplifies
   the anti-inflammatory signaling transduction, and diminishes the
   cellular sensitivity to the proinflammatory toxin to potentiate the M2
   polarization. In GNB, the MPM participates in the lysis of cells by
   disrupting bacterial membrane.

   When treating aseptic ALI, the direct deposition of SPRAY-BLKR-NPs in
   the lower respiratory tract was demonstrated to rapidly mitigate
   neutrophil infiltration and then facilitate the regeneration of
   alveolar epithelium via the proliferation of M2 macrophage. When used
   in bacterial ALI, we also proved its promising efficacy in eradicating
   invading bacteria in lung. Besides, SPRAY-BLKR-NPs do not affect the
   monocyte migration to pulmonary interstitium or cause epithelial
   injury. Thus, it could be regarded as a safe inhalant for potential
   clinical usage. Furthermore, the peptide candidate may be used as a
   dry-powder inhalant or nebulized.

   Overall, this work is a proof of concept that a single-molecule–based
   therapeutic could actualize anti-inflammation, regeneration, and
   antibacteria synchronously to reverse ALI with various etiologies and
   pathogenesis. This clinically unseen molecule may authorize the reverse
   of proinflammatory state in other acute inflammation, such as
   meningitis, arthritis, and endophthalmitis. Moreover, the chemically
   accessible design of SPRAY could be tailored and extended to regulate
   the fate of the other immune cells via MPM-induced cytoplasmic delivery
   of particular functional motifs.

MATERIALS AND METHODS

Materials

   The following materials were used: RPMI 1640 culture medium (no.
   11875093, Gibco), mouse IL-4 recombinant protein (no. BMS338,
   eBioscience), mouse IL-10 recombinant protein (no. 417-ML, R&D
   Systems), mouse macrophage colony-stimulating factor (M-CSF) protein
   (no. 315-02-1MG, PeproTech), LPS (no. L2630, Sigma-Aldrich), and
   avibactam (no. A128068, Aladdin). Peptides were synthesized and
   purified by GL Biochem (Shanghai) Ltd. via the Fmoc solid-phase peptide
   synthesis (SPPS) ([174]52). Biphenyl was conjugated to N terminus using
   4-biphenylacetic acid (no. 196487, Sigma-Aldrich) during the SPPS. MRE
   and MRSA USA300 were a gift from L. Tan, Wuhan Union Hospital, China.
   K. pneumoniae (KP) BNCC358281 was a gift from D. Zhou, Southern Medical
   University, China. P. aeruginosa (PA) PAO1 was the gift from X. Sun,
   Zhejiang University of Technology, China.

The isolation and culture of BMDMs

   The BMDMs were extracted and cultured according to previous literature
   report ([175]53). In short, BMDMs were isolated from the bone marrow of
   6-week-old BALB/c mice (Shanghai SLAC Laboratory Animal Co. Ltd.) and
   further cultured in RPMI 1640/10% fetal bovine serum (FBS)/M-CSF (10 ng
   ml^−1) before the further experiment.

M2 macrophage polarization induced by SPRAY candidates

   To evaluate the M2 macrophage polarization induced by peptide
   candidates, 2 × 10^5 BMDMs per well were seeded into a 12-well plate
   and incubated for 24 hours. The peptide candidates were added to the
   medium to the final concentration of 16 μM and then incubated for 48
   hours. The positive control of the M2 macrophage phenotype was prepared
   by adding IL-4 (20 ng ml^−1) and IL-10 (20 ng ml^−1). The positive
   control of M1 macrophage was prepared by adding LPS (10 ng ml^−1) and
   IFN-γ (20 ng ml^−1). Blank medium was set as the negative control. The
   BMDMs were firstly blocked with anti-CD16/CD32 (no. 553141, BD
   Pharmingen) and stained with Fixable Viability Stain 780 (no. 565388,
   BD Pharmingen), anti–F4/80-BV421 (no. 123132, BioLegend),
   anti–CD11b-FITC (no. 557396, BD Pharmingen), anti–CD206-APC (no.
   17-2061-82, eBioscience), and anti–CD86-PE (no. 553692, BD Pharmingen)
   according to the manufacturer’s instructions. The percentages of CD206-
   and CD86-positive cells were then measured by a flow cytometry analyzer
   (CytoFLEX LX, Beckman Coulter).

Cytocompatibility assay of SPRAY candidates

   Beas-2B (CRL-9609), HEK293T (CRL-3216), THP-1 (TIB-202), and RAW264.7
   (TIB-71) were purchased from the American Type Culture Collection. DC
   2.4 (SCC142) was purchased from Sigma-Aldrich. HS-5 (CC4028) was
   purchased from the Cellbook. All the cells were cultured in a 100-mm
   precoated dish (430167, Corning) according to the manufacturer’s
   instructions with 10% FBS and 1% penicillin-streptomycin (no. 15140122,
   Gibco). To evaluate the cytocompatibility, 1 × 10^4 cells were seeded
   into the 96-well plate per well and incubated for 24 hours. Then, the
   medium was replaced with fresh medium containing each peptide at
   different concentrations. The cell viability was measured using
   3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)
   assay after 24-hour coincubation.

Hemolysis assay of SPRAY candidates

   Peptide candidates were dissolved in phosphate-buffered saline (PBS) to
   512 μM, and then six times twofold dilution was performed. Ten times
   dilution was performed on 2 ml of freshly drawn New Zealand rabbit
   blood using PBS. Then, the RBC pellet was collected by centrifugation
   and washed eight times with PBS before incubating with peptide at
   different concentrations for 3 hours, 37°C. Positive control was set
   using 1% Triton X-100. Negative control was set using PBS. The mixture
   was centrifuged at 10,000 rpm, 10 min, and the absorbance at 570 nm of
   supernatant was measured for further hemolysis rate evaluation.

Material characterization

   Chirascan V100 CD spectrometer (Applied Photophysics) was used to
   measure the CD spectra. LKR and BLKR were dissolved in ultrapure water
   (pH = 7.4) to the concentration of 128 μM and then subjected to CD
   spectrum recording.

   The CMC value of the peptide was revealed by a spectrofluorometer
   (Edinburgh, F55). LKR and BLKR were dissolved in PBS (2.56 mM) and then
   underwent eight twofold dilutions. The pH suspension was adjusted to 7.
   Peptide suspension (100 μl) was mixed with 2 μl of Rhodamine 6G (final
   concentration: 5 μM), then incubated for 24 hours. The wavelength with
   maximum optical density (OD) value (515 to 560 nm) was read out for CMC
   calculation.

   The morphology of peptide assemblies was imaged by Talos L120C G2
   transmission electron microscope (Thermo Fisher Scientific). LKR and
   BLKR were dissolved in PBS (128 μM), then preincubated for 8 hours.
   Suspension (10 μl) was applied on 200-mesh copper grids, followed by
   2-min uranyl acetate staining before TEM imaging.

   To monitor the stability of SPRAY-BLKR-NPs in injured alveolar
   environment, BALF was extracted from ALI mice at 24 hours after LPS (10
   mg kg^−1) administration. The SPRAY-BLKR-NPs were then incubated ALI
   BALF to the final concentration of 256 μM. The diameter of nanoparticle
   at indicated incubation time was measured by a BI-200SM NanoBrook
   ZetaPALS analyzer using number distribution mode.

   To perform the accelerated storage testing for SPRAY, 1.5 mg of BLKR
   powder was placed in 2-ml glass vial and then incubated under low
   temperature (4°C, sealed), light exposure (4500 K, 25°C, sealed), high
   temperature (60°C, unsealed), or high humidity (90% relative humidity,
   37°C, unsealed) for 10 days using medicine Stability Testing Chamber
   (LHH-150GSD, Blue pard, Shanghai). After incubation, the peptide powder
   was checked for appearance, and reconstituted particle size in PBS was
   measured by DLS. The chemical purity was measured by high-performance
   liquid chromatography (Agilent).

The uptake behavior of SPRAY in BMDMs

   The cellular distribution of peptide was analyzed by a confocal laser
   scanning microscope (LSM 800, Zeiss). A total of 2 × 10^5 BMDMs were
   seeded into a 20-mm glass bottom cell culture dish (no. 801001, NEST)
   and then incubated respectively with FITC-labeled LKR and BLKR for
   indicated time points. The nuclei were stained with Hoechst 33342 (no.
   62249, Thermo Fisher Scientific), and the lysosomes were stained with
   LysoTracker Deep Red ([176]L12492, Thermo Fisher Scientific) according
   to the manufacturer’s instructions. Then, the cells were washed with
   PBS three times and cultured in live cell imaging solution (A14291DJ,
   Thermo Fisher Scientific) for further CLSM imaging (Zeiss LSM 980).

   Flow cytometry was used to reveal the cellular uptake behavior of BLKR.
   To evaluate cellular uptake quantity, FITC-labeled LKR and BLKR were
   coincubated with 10^6 BMDMs for 1 hour and then washed with PBS five
   times for further quantification using flow cytometry.

   To quantify the cellular uptake molar level of BLKR, BWLR, and BVRR,
   10^6 BMDMs per well were seeded into a six-well plate and incubated for
   another 24 hours, then incubated with BLKR, BWLR, and BVRR (final
   concentration: 16 μM) in RPMI 1640 culture medium (10% FBS) for 1 hour.
   Ultrapure water (1 ml) was added to each well and incubated for 20 min
   to lyse the cells by hypotonicity. The suspension containing cell
   lysate was lyophilized, followed by adding 400 μl of methanol and
   ultrasonication to dissolve the peptide. The resuspended cell lysate
   was centrifuged at 10,000 rpm for 5 min. The supernatant was filtered
   by a 220-nm filter and then analyzed through the Agilent 1260
   InfinityLab Liquid Chromatography system equipped with InfinityLab
   Poroshell 120 EC-C8 column (4.5 mm by 100 mm, 2.7 μm, no. 695975-906,
   Agilent Technologies).

   The method to evaluate the endocytosis mode was previously described
   ([177]52). Briefly, 10^6 BMDMs were seeded into a 12-well plate and
   then preincubated with single or blended uses of endocytosis inhibitors
   for 30 min, respectively. The medium was discarded, and fresh medium
   containing FITC-labeled BLKR was added to each well to the final
   concentration of 16 μM with the presence of indicated inhibitors,
   followed by 15-min coincubation. The percentage of BLKR^+ cells and
   mean fluorescence intensity were further quantified by flow cytometry.
   In each coincubation, the final concentrations of various indicated
   inhibitors were applied at 5 mM for MβCD, 50 μM for Amiloride, 50 μM
   for EIPA, 40 μM for CPZ and 5 μg ml^−1 for Filipin III.

   Bio-EM (Talos L120C G2 transmission electron microscope) was used to
   provide direct visualization of the peptide’s endocytosis process and
   cytoplasmic releasing. A total of 1 × 10^6 BMDMs were incubated in a
   12-well plate and then treated by BLKR (16 μM, PBS) for 15 min, 1 hour,
   2 hours, and 4 hours. PBS was used as the negative control. Then, the
   medium was discarded, and the cells were gently scraped off with the
   presence of 900 μl of PBS and 100 μl of FBS and centrifuged. The cell
   pellet was fixed with fixation buffer (2.5% glutaraldehyde) and then
   subjected to embedding, staining, and imaging according to the
   instructions from Westlake Electron Microscopy Core Facility.

BMDMs polarization study induced by the optimal SPRAY

   CLSM imaging was used to reveal the CD206 expression and morphological
   change in BMDMs. A total of 2 × 10^5 BMDMs were cultured in glass
   bottom cell culture dish and then incubated with LKR and BLKR (16 μM)
   for 24 hours, respectively. Cells were fixed with 4.2% paraformaldehyde
   for 10 min and penetrated using 0.1% Triton 100-X for 5 min, then
   blocked with the blocking solution containing 1% BSA and 10% goat serum
   for 1 hour. The anti-MMR/CD206 (no. AF 2535, R&D Systems) was applied
   at the final concentration of 0.25 μg for 10^6 cells and incubated
   overnight at 4°C. The AF488-AffiniPure Donkey Anti-Goat IgG (no.
   705-545-147, Jackson ImmunoResearch) was then applied at 2 μg for 10^6
   cells. The cells were then stained with 4′,6-diamidino-2-phenylindole
   (DAPI) and subjected to CLSM imaging.

   To evaluate the inflammatory-related mRNA expression levels of the BMDM
   cells after being stimulated with peptide for 24 hours, the total RNA
   was extracted with TRIzol (no. 15596018, Thermo Fisher Scientific),
   then quantified using NanoDrop (Thermo Fisher Scientific), the mRNA was
   purified, and cDNA was prepared using one NovoScript 1st Strand cDNA
   Synthesis SuperMix (gDNA Purge) Kit (E047-01B, Novoprotein). The
   real-time PCR was then performed subsequently in triplicate using
   NovoStart SYBR qPCR SuperMix Plus Kit (E096-01B, Novoprotein) and a
   Bio-Rad CFX96 real-time PCR system. The relative expression of mRNA was
   normalized to the expression level of GAPDH.

   ELISA was used to measure the cytokines secreted by BMDMs. A total of 1
   × 10^6 BMDMs were seeded in a 6-well plate and then incubated with LKR
   and BLKR (16 μM) for 24 hours with 1 ml of culture media. The
   supernatant was collected for analyzing the concentration of IL-4 (no.
   BMS613, Thermo Fisher Scientific), IL-10 (no. BMS614, Thermo Fisher
   Scientific), IL-5 (no. 431201, BioLegend), TNF-α (no. 430904,
   BioLegend) and IFN-γ (no. 430815, BioLegend), respectively, according
   to manufacturer’s protocol.

   To investigate the effect of peptide pretreatment, BMDMs were incubated
   with LKR and BLKR (16 μM) for 24 hours. Then the medium was not
   discarded. LPS (10 ng ml^−1) and IFN-γ (20 ng ml^−1) were added to each
   well, incubated for another 24 hours, and then subjected to the flow
   cytometry analysis.

Transcriptome sequencing for macrophage polarization

   A total of of 5 × 10^6 BMDMs were incubated with LKR and BLKR (16 μM)
   for 48 hours. RNA extraction was performed using TRIzol Reagent (no.
   15596026, Invitrogen) according to the manufacturer’s instructions.
   Genomic DNA was removed using DNase I (no. 2270B, Takara). Library
   construction and RNA sequencing were completed at Shanghai Majorbio
   Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the
   manufacturer’s instructions (Illumina, San Diego, CA). The expression
   level of each gene was calculated according to the transcripts per
   million reads (TPM). Significantly different expressed genes were
   considered to be slog2 (fold change) | ≥ 1 and P-adjust ≤ 0.05
   (DESeq2). The GO ([178]www.geneontology.org) and KEGG
   ([179]www.genome.jp/kegg/) pathway enrichment analysis was carried out
   using Majorbio Cloud Platform ([180]www.majorbio.com).

Bactericidal activity of SPRAY

   The MIC values of peptide against MRE, PA and KP were measured
   according to previously reported method ([181]54). Ceftazidime (no.
   72558-82-8, Yuanye, Shanghai) and imipenem (GP11075, Glpbio) were used
   as positive control.

   To investigate MRE viability after peptide treatment. MRE (1 × 10^7 CFU
   ml^−1, PBS) in mid-logarithmic growth-phase was incubated with LKR and
   BLKR (128 μM) for 4 hours at 37°C. Then, 10 μl of mixture was
   transferred then quantified to reveal bacterial viability using
   spread-plate method. The method to evaluation the MRSA viability was
   the same as aforementioned steps, but MRSA (1 × 10^8 CFU ml^−1, PBS) in
   mid-logarithmic growth-phase was used. Untreated bacteria were used as
   control group.

   To investigate the anti-MRE activity of BLKR in the healthy and injured
   alveolar environment. Healthy BALF was extracted from untreated mice
   using 1 ml of cold sterile PBS. To extract ALI BALF, 8-week-old BALB/c
   mice received LPS (10 mg kg^−1) followed by BALF extraction at 24 hours
   later using 1 ml of cold PBS. The BALF was sonicated for 5 min to lysed
   living cells before further use. Then, MRE (1 × 10^7 CFU ml^−1) in
   mid-logarithmic growth-phase was resuspended using freshly extracted
   healthy BALF and ALI BALF and incubated with LKR and BLKR for 4 hours
   (128 μM) at 37°C. Then, 10 μl of mixture was transferred then
   quantified to reveal bacterial viability using spread-plate method.

   Living/Dead staining was performed to visualize the biofilm eradication
   induced by peptides. In short, to culture the biofilm, 200 μl of MRE or
   MRSA (5 × 10^6 CFU ml^−1) was preincubated for 48 hours using LB
   medium. Then, the LB medium was replaced by PBS containing. The biofilm
   was then incubated with LKR and BLKR (256 μM, PBS) for 4 hours at 37°C.
   The biofilm was then stained using LIVE/DEAD BackLight bacterial
   viability kit (Invitrogen) and processed with CLSM imaging (Zeiss LSM
   800).

   To visualize the bactericidal phenotype of SPRAY, MRE and MRSA (1 ×
   10^8 CFU ml^−1) were preincubated on a 12-mm round glass slide in a
   12-well plate for 48 hours. Then, the bacteria were washed with PBS
   three times and further incubated with LKR and BLKR (256 μM) for 3
   hours. Next, the bacteria were washed with PBS three times before fixed
   2.5% glutaraldehyde for 24 hours. The imaging process using SEM
   (FE-SEM, GeminiSEM 450) and Bio-EM (Talos L120C G2) was performed at
   Westlake Electron Microscopy Core Facility.

SPRAY-induced bacterial membrane disruption

   NPN uptake assay was performed to prove the bacterial membrane
   integrity altered by peptide treatment. NPN test solution (10 μl; 1 mM)
   was coincubated with MRE or MRSA suspension (1 × 10^8 CFU ml^−1, 5 mM
   glucose, 5 mM Hepes, pH = 7.4) at 0 min. Then, peptide was added to the
   final concentration of 256 μM at 30 min after the NPN addition,
   followed by coincubation for 2 hours. Triton X-100 (1%) was used as the
   positive control. The fluorescence intensity (excitation: 350 nm,
   emission: 420 nm) was then measured by spectrofluorometer (Edinburgh,
   F55).

   Membrane-potential-sensitive dye DiSC[3](5) was used to investigate
   bacterial Membrane depolarization. MRE or MRSA (100 μl; 2 × 10^8 CFU
   ml^−1, 20 mM glucose, 5 mM Hepes, 0.1 mM KCl, pH = 7.4) with DiSC[3](5)
   (20 nM) addition at 0 min was mixed with an equal volume of LKR or BLKR
   (512 μM) at 1 min. The fluorescence intensity (excitation: 622 nm,
   emission: 670 nm) of the mixture was then measured every minute using
   spectrofluorometer (Edinburgh, F55) for the following 29 min starting
   from the time point of peptide addition.

   To evaluate the conformational changes during the peptide-membrane
   interaction, PE, PG, and DPG were mixed at the proportion of 79:17:4 in
   chloroform (final lipid concentration is 5 mM) to mimic the composition
   of GNB membrane ([182]55). The lipid solution was then fired by N[2]
   flow and resuspended using ultrapure water, then extruded through
   0.1-μm pore polycarbonate membrane 15 times using AvantiMini Extruder
   (Alabaster). The resulting LNPs (1 mM) were then incubated with BLKR
   (128 μM) for 30 min before the CD measurement.

Mice studies

   All mice studies were approved by the Institutional Animal Care and Use
   Committee of Westlake University (AP#22-079-WHM). Eight-week-old BALB/c
   mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. Mice
   were anesthetized with the intraperitoneal injection of 2.5% Avertin at
   18 to 20 μl g^−1 before each nonlethal surgery or operation.

Immunomodulatory function and toxicity of SPRAY in healthy mice

   Mice (n = 3) received twice doses of SPRAY-BLKR-NPs (50 μl) through
   intratracheal injection at the concentration of 64, 128, 256, 512, and
   1024 μM, respectively. The time gap between each injection was set as
   12 hours. Then, the mice were euthanized at 24 hours after last BLKR
   administration. Blood was collected through retro-orbital blood
   collection and then analyzed using ProCyte Dx Hematology Analyzer
   (IDEXX). for hematology assay. The main organs were collected for H&E
   staining for the acute toxicity examination.

   To investigate the monocyte infiltration in the lung parenchyma,
   freshly collected lungs that received the treatment above were ground,
   filtered, lysed for 60 min using lung lysis buffer according to
   previous report ([183]56). RBCs were lysed before staining. The
   dispersed pulmonary cells were cultured in RPMI 1640 medium for another
   4 hours and then stained with anti–CD45-BUV395 (no. 564279, BD
   Pharmingen), anti–CD64a-BV650 (no. 740622, BD Pharmingen),
   anti–MERTK-BV786 (no. 747890, BD Pharmingen), CD11b-FITC (no. 561688,
   BD Pharmingen) for further flow cytometry analysis.

Long-term safety evaluation of the consecutive administrations of SPRAY
nanoparticles

   Mice received twice doses of SPRAY-BLKR-NPs (256 μM, 50 μl, 0.76 mg
   kg^−1) for consecutive 14 days. The time gap between each injection was
   set as 12 hours. Then, the mice were raised for another 13 days. To
   investigate the leukocyte infiltration, hemolysis and anemia, blood
   samples were collected on days 3, 6, 9, 12, 15, 21, and 27 through
   retro-orbital blood collection, and then analyzed Hematology Analyzer
   for WBC and RBC parameters. To evaluate potential liver and kidney side
   effects, blood samples were centrifuged at 4000 rpm for 10 min to
   obtain the serum, then subjected to AST, ALT, ALP, and BUN analysis
   using automatic biochemical analyzer (Catalyst one, IDEXX). To evaluate
   the lung function, trachea was exposed by blunt separation, then 18G
   needle was intubated into trachea and tied by suture. After the trachea
   was connected with ventilator, the lung function test was performed on
   invasive computer-controlled piston ventilator (FlexiVent; SCIREQ Inc.)
   on days 6, 15, and 27. Then, BALF was extracted for IL-4 and IL-6
   measurement using ELISA. The main organs were collected on day 27 for
   H&E staining.

SPRAY nanoparticles reverse LPS-induced ALI in mice

   For modeling of LPS-induced ALI, LPS was administrated through
   intratracheal injection at 10 mg kg^−1. To monitor the biodistribution
   of SPRAY in ALI lung and healthy lung, FITC-labeled BLKR and
   FITC-labeled LKR (128 μM, 50 μl) were administrated locally in mice (n
   = 3) through intratracheal injection at 12 after LPS administration.
   The body weight was monitored daily. Mice were euthanized at indicated
   time point, and lungs were collected for perfusion using PBS and
   embedded in the OCT, then stored at −80°C. Lung was cryosectioned using
   Leica CM3050 S Research Cryostat and stained with ProLong Diamond
   Antifade Mountant with DAPI ([184]P36966, Thermo Fisher Scientific)
   before CLSM imaging. To further visualize the colocalization of SPRAY
   with macrophage, lung sections were further stained with anti-CD68 (no.
   14-0681-82, Thermo Fisher Scientific), AF647 secondary antibody (no.
   A-21247, Thermo Fisher Scientific) and DAPI, then subjected to CLSM
   imaging.

   To investigate the uptake distribution of SPRAY nanoparticles in lung
   immune cells, ALI mice received SPRAY-BLKR-NPs (128 μM, 50 μl) locally.
   At 1 hour after administration, lung was harvested, ground, filtered,
   lysed then stained with viability staining blue (no. [185]L23105,
   Invitrogen), anti–CD45-APC-Cy7 (no. 557659, BD Pharmingen),
   anti–CD3-PE-Cy5.5 (no. 100274, BioLegend), anti–F4/80-BV421 (no.
   123132, BioLegend), anti–Ly6G-APC (no. 560599, BD Pharmingen),
   anti–CD19-BV605 (no. 563148, BD Pharmingen) and anti–CD11c-PE (no.
   12-0114-82, eBioscience) for flow cytometry analysis.

   In the therapy process, LKR and BLKR (128 μM, 50 μl) were administrated
   locally in mice (n = 3) through intratracheal injection at 12 and 24
   hours after LPS injection. BALF was extracted on days 0, 2, 4, and 6
   according to previous literature report ([186]57). The blood was
   harvested through retro-orbital blood collection. The hematology
   parameters and leukocyte differential counting in blood and BALF were
   then measured using ProCyte Dx Hematology Analyzer (IDEXX). ELISA test
   was used to measure the cytokines level in BALF. BALF (n = 5) was
   centrifuged at 500g for 10 min. The concentrations of IL-6 (no.
   MU30044, Bioswamp) and IL-4 (no. MU30385, Bioswamp) in the supernatant
   were analyzed according to the manufacturer’s protocol.

   For the pulmonary histological analysis, mice were euthanized on days
   0, 2, 4, and 6 using CO[2]. The lung was harvested, perfused, and
   embedded in paraffin, then sectioned for H&E staining. The counts of
   inflammatory cells in alveoli were calculated from nine randomly
   selected H&E staining sections; n = 3 mice per group, three sections
   per mouse were selected.

   To screen the therapeutic dosage range of SPRAY for treating aseptic
   ALI, ALI mice were dosed with optimal SPRAY locally every 12 hours two
   times at indicated dosage at day 1. The therapeutic efficacy for tissue
   regeneration was measured by the BALF IL-4 level on day 6.

The immunofluorescence and IHC analysis in ALI mice

   The paraffin sections were firstly dehydrated gradient using
   ethanol-water solution and xylene. Then, the slides were incubated with
   sodium citrate antigen retrieval solution (no. C1032, Solarbio) and 3%
   hydrogen peroxide. The immunostaining for SFTPC-positive type II
   pneumocytes was performed using SFTPC Polyclonal Antibody (Proteintech,
   no. 10774-1-AP) at 1:2000 dilution rate, then followed by the
   previously described methods ([187]58). Slides were scanned using a
   digital pathology imaging system (KFBIO, KF-pro-400). The percentage of
   SFTPC-positive cells was quantified on QuPath-0.2.3 using the positive
   cell detection function (threshold 1: 0.18, threshold 2: 0.3, threshold
   3: 0.4). Twelve randomly selected images (20× field) from three mice
   (four images per mouse) were analyzed in quantification. The
   immunofluorescence staining of CD206-positive and CD86-positive
   macrophages was performed using anti-CD86 (no. MA1-10299, Thermo Fisher
   Scientific) at 1:500. The secondary antibody for CD86 was applied using
   AF647-Goat anti-Rat IgG (no. A-21247, Thermo Fisher Scientific) at
   1:500. Anti-MMR/CD206 (no. AF 2535, R&D Systems) was then applied at
   1:1000. The secondary antibody for CD206 was applied using
   AF488-AffiniPure Donkey Anti-Goat IgG (no. 705-545-147, Jackson
   ImmunoResearch) at 1:500. Nuclei were stained with DAPI and subjected
   to CLSM imaging. To quantify the percentage of CD86-positive cells or
   CD206-positive cells among total pulmonary cell counts, up to 12
   randomly selected images (20× field) and measured using QuPath-0.2.3
   with the fluorescence mode. n = 3 mice per group, at least 5 images per
   mouse were selected.

Pharmacokinetic study of SPRAY-BLKR-NPs

   ALI mice received single dose of SPRAY-BLKR-NPs (256 μM, 50 μl; 18.42
   μg per mouse), then euthanized at predetermined time points. The main
   organs were harvested. Lung tissues were harvested after cardiac
   perfusion using 10 ml of cold PBS. To measure the BLKR content, the
   tissues were lyophilized and crushed into powder with a mortar and
   pestle. The dry weight of tissue was measured. Then, the crushed tissue
   was suspended using the mixture of chloroform and methanol (volume
   ratio = 1:3) followed by freeze grinding at 30 Hz 10 times with the
   assistance of two steel grinding beads (⌀3.2 and ⌀5.2 mm) using a
   bullet blender (JXFSTPRP-II, Shanghai Jinxin). The homogenized tissue
   was centrifuged at 14,000 rpm for 3 min. The supernatant was filtered
   by 220 nm filter, then analyzed through Agilent 1260 InfinityLab Liquid
   Chromatography system using InfinityLab Poroshell 120 EC-C18 column
   (4.5 mm by 50 mm, 2.7 μm, no. 699975-902, Agilent Technologies).

Liver microsomal metabolism of SPRAY

   The metabolic rate of SPRAY in human, dog, monkey, rat, and mouse liver
   microsomes was measured by Shanghai Medicilon Inc. BLKR (1 μM) was
   coincubated with human (bioreclamationIVT), monkey, dog, rat, and mouse
   liver microsomes (Xenotech) (0.75 mg ml^−1), respectively. Mixed
   solution of peptide and microsomes (30 μl) was then mixed with 15 μl of
   NADPH stocking solution (6 mM), and incubation for indicated period.
   Subsequently, 150 μl of stop solution (ACN: MeOH = 1:1) containing
   internal standard (tolbutamide) was added to stop the reaction. The
   suspension was oscillated for 10 min (600 rpm) and centrifuged at 6000
   rpm for 15 min. The supernatant was diluted 2.5-fold with pure water
   for further LC/MS analysis.

SPRAY nanoparticles reverse inflammation in GNB-infected mice

   A total of 2.5 × 10^7 CFU MRE per mouse was locally administrated
   through intratracheal injection at day 0 to model MRE infection-induced
   ALI. Mice received the local administration of LKR and BLKR (256 μM, 50
   μl) at 12, 24, 36, and 48 hours through intratracheal injection,
   respectively. To monitor the survival rate, 45 mice were randomly
   divided into three groups (n = 15) followed by pulmonary MRE infection
   as previously described. The survival rate was monitored daily until
   day 6. Mice were euthanized on days 2, 6, and 10 using CO[2]. The lung
   was harvested, perfused, and embedded, then subjected to H&E staining.
   BALF supernatant was collected using the method above and then
   subjected to an ELISA test for IL-6 and IL-4 concentration. The
   hematology study was analyzed using the blood collected through
   retro-orbital bleeding.

GNB eradication of SPRAY nanoparticles in mice

   To investigate the in vivo anti-MRE efficiency of BLKR. Mice received
   the intratracheal injection of 2.5 × 10^7 CFU MRE at day 0. Peptide was
   administrated through intratracheal injection injection at 12 and 24
   hours after infection. Saline (0.9%) was used as the negative control.
   Then, the entire lung was collected in a sterile 5-ml EP tube at 6
   hours after each dosing. Sterile PBS (2 ml) was added, and the lung was
   homogenized with a handheld homogenizer (Tenlin-A, China) for 30 s.
   Using the spread-plate method, 20 μl of lung homogenate was immediately
   transferred for MRE quantification.

   To screen the therapeutic dosage range of SPRAY for treating
   GNB-induced ALI, MRE-infected mice were dosed with optimal SPRAY
   locally every 12 hours four times at indicated dosage. Pulmonary MRE
   burden was investigated at 12 hours after the final dosing. Lung tissue
   was collected on day 6, then subjected to H&E staining. Survival rate
   was monitored throughout the experiment.

   To compare the therapeutic efficacy of SPRAY-BLKR-NPs with clinically
   available antibiotic, MRE-infected mice (n = 5) received the local
   administration of SPRAY-BLKR-NPs (256 μM, 50 μl, 0.76 mg kg^−1) and
   subcutaneous injection of ceftazidime/avibactam (32/8 mg kg^−1)
   ([188]59, [189]60), respectively, every 12 hours six times. The lung
   was collected at 12 hours after second and sixth administration, then
   homogenized for pulmonary MRE quantification. Blood samples were
   collected on days 1, 5, and 9 for neutrophil quantification using
   Hematology Analyzer. The IL-4 and IL-6 levels in BALF were measured
   using ELISA on days 1 and 5 to investigate the alternation of alveolar
   immuno-microenvironment. Survival rate was monitored throughout the
   experiment. Lung tissue was collected on day 5, then subjected to H&E
   staining.

Acknowledgments