Inhalable targeted gold nanoparticles for accelerating lung delivery and treating lung inflammation

Abstract

The invention provides a method for preparing inhalable targeted gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation. The method comprises the following steps: preparing plasmids; carrying out plasmid transformation; carrying out protein expression induction; carrying out protein purification; and the gold nanoparticles (Au (at) PEG-RBD NP) combined with the spikes RBD are prepared. Nanoparticles (NP) in air accumulate more readily in lungs with lipopolysaccharide-induced acute respiratory distress syndrome (ARDS) than in healthy lungs and enter alveolar epithelial cells that express angiotensin converting enzyme (ACE) 2 and L-SIGN (both are Spike receptors activated in ARDS hamsters). The nanoparticles (NP) treat tissue damage, oxidative stress, and inflammation more effectively than corticosteroids, and do not remain gold or create toxicity in the major organs one year after inhalation. The gold core can be used for inhibiting p38 alpha mitogen activated protein kinase and polo-like kinase 3. Similar therapeutic effects have been observed in hamsters suffering from hydrochloric acid induced ARDS. This self-therapeutic nanoparticle (NP), when combined with biomimetic and non-invasive delivery, provides a safe, efficient, and targeted treatment for lung inflammation.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 719519, filed November 12, 2024, the entire contents of which, including any figures, tables or graphs, are incorporated herein by reference.

[0003] Background Technology

[0004] Acute respiratory distress syndrome (ARDS) is a leading cause of death, affecting approximately 10% of intensive care unit patients, with a global mortality rate of 40%. [1] Rupture of the air-blood barrier (ABB) is a direct pathophysiological cause of ARDS. [2] This can cause biological fluids to leak into lung tissue and impair oxygen diffusion into the blood. Severe inflammatory symptoms may appear within 12 hours of exposure to bacteria or chemical irritants. [3] Therefore, timely management will improve treatment outcomes. [4] However, conventional treatments take 5-7 days to achieve a clinical response and do not target the underlying cause of the disease. [5,6] For example, invasive mechanical ventilation and anti-inflammatory drugs with side effects. [7] .

[0005] Nanoparticles (NPs) are being researched for ARDS management. [8] Due to the highly vascularized nature of the lungs, past lung nanomedicines have mostly been delivered intravenously to the lung endothelium (see Tables 1 and 2). However, when carriers, drugs, and targeting ligands are all included, they are often large and easily cleared by the liver, which can lead to systemic toxicity. Lung-specific delivery is possible, but requires specialized lipid engineering. [9] Importantly, intravenous delivery does not guarantee delivery to the lung epithelium, which is ABB's protective layer for the lungs.

[10] Regulating ARDS-related immune responses

[11] and regulate tissue repair

[12] Key structural components. Other nanoparticles (NPs) are delivered locally to the lung epithelium using intratracheal instillation (Table 2). However, infusion may increase airway pressure and lead to cardiac arrest.

[13] Infusion of NPs only accumulates in a narrow confined tissue region. Interestingly, inhalation offers rapid, non-invasive lung delivery and broad tissue distribution

[14] , but has only recently seen rare application.

[15] (Table 1). It is hypothesized that inhaled NPs targeting the lung epithelium of ARDS can support rapid, safe and effective treatment.

[0006] Currently, there are four main types of treatment for ARDS: mechanical ventilation, corticosteroids, small molecule anti-inflammatory drugs, and inhaled NPs. Mechanical ventilation is invasive to the trachea and only relieves symptoms. Furthermore, surgical opening of the trachea can lead to local infection, while inhaled NPs are non-invasive. Corticosteroids have strong anti-inflammatory effects, although they have serious systemic side effects and are not specifically for ARDS. Small molecule anti-inflammatory drugs (such as statins and ACE inhibitors) have been extensively tested in human trials for ARDS (but are not yet approved), but statins offer no protection against ARDS, and ACE inhibitors produce only mild efficacy. NP-based therapies are relatively safer than the methods mentioned above. Several commonly used NPs are used to deliver ARDS drugs to the lungs, such as liposomes, polymeric NPs, and exosome-derived NPs. However, liposomes require complex manufacturing processes, polymeric NPs have low drug loading efficiency, and exosome-derived NPs are more difficult to manufacture and have low yields.

[0007] Furthermore, current NP delivery routes have limited efficacy, particularly (1) intravenous (iv) or intraperitoneal (ip) and (2) intratracheal (it). Intravenous or intraperitoneal injection provides systemic delivery. To maximize delivery efficiency to the lungs, targeted ligands are typically used, resulting in most injected NPs accumulating in the liver and spleen. Intratracheal injection is invasive and risky, potentially increasing airway pressure and leading to cardiac arrest. In addition, infused NPs (in fluid) often accumulate in small tissue areas due to inertial impaction. Summary of the Invention

[0008] There is still a need in this field for improved designs and technologies for nanoparticle-based drug delivery methods targeting the lungs.

[0009] According to one embodiment of the present invention, a method for preparing inhalable targeted gold nanoparticles for accelerating lung delivery and treating lung inflammation is provided. The method includes preparing a plasmid; performing plasmid transformation; inducing protein expression; purifying the protein; and preparing spike RBD-conjugated gold nanoparticles (Au@PEG-RBD NP). Plasmid preparation involved mixing DH5α competent cells with the plasmid; incubating the mixture on ice; heat-shocking the mixture; incubating the mixture on ice; adding preheated lysogeny broth to the mixture; and incubating the cells in the mixture with orbital shaking. The transformed cells were then spread onto LB agar plates containing ampicillin and incubated. A colony of cells was selected and grown in LB / ampicillin with shaking. Cells were collected by centrifugation and the plasmid was purified to obtain the plasmid encoding the His-labeled RBD (pET11a-RBD-8xHis). Furthermore, plasmid transformation included thawing previously incubated at -80°C on ice. The process involves: storing competent cells; adding plasmids to competent cells to obtain a mixture; keeping the cells in the mixture on ice; heat-shocking the cells in the mixture in a water bath and re-incubating the cells on ice; adding fresh LB medium and shaking the mixture; centrifuging the cells in the mixture and discarding the supernatant; resuspending the remaining pelleted cells; seeding the bacteria onto LB agar plates with antibiotics and incubating the bacteria. Protein expression induction includes transforming Origami B cells pre-transformed with the molecular chaperone plasmid pG-KJE8 using the RBD expression plasmid pET11a-RBD; culturing the results on LB agar plates; selecting one single colony from the results; adding the colony to fresh LB medium with antibiotics; adding overnight cultures to fresh LB medium; and culturing the results until OD. 600 The target concentration was approximately 0.5; induction was performed by adding an induction buffer containing isopropyl β-D-1-thiogalactopyranoside and L-arabinose; cells were collected by centrifugation for purification or at -80°C. The cells were then stored. Protein purification involved resuspending Origami B cells in protein purification buffer and lysing them using an ultrasonic processor; centrifuging the lysate; collecting the supernatant of the lysate; filtering the supernatant using a syringe filter; incubating the lysate with Ni-NTA resin; transferring the supernatant and resin together to a blank gravity column; discarding the liquid flowing through the lysate-resin mixture; adding washing buffer to elute non-specifically bound proteins; eluting the RBD protein product by adding elution buffer and dialyzing against storage buffer; and determining the purity and concentration of the protein product by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the Bradford assay, respectively. The preparation of spike RBD-bound gold nanoparticles (Au@PEG-RBD NP) includes: synthesizing citrate-capped gold nanoparticles (cit-AuNP) with a diameter of approximately 20 nm; boiling HAuCl4, adding sodium citrate under vigorous stirring and maintaining the mixture at boiling; cooling the result to obtain cit-AuNP; and adding freshly dissolved thiol (HS)-PEG at a 1:1 molar ratio. 20k -Methoxy or HS-PEG 20k nitrilotriacetic acid (NTA) (Biochempeg) at per nm 2 The total concentration of 5 PEG molecules on the surface of the nanoparticles was added to a cit-AuNP solution; the mixture was stirred, NiCl2 was added, and the mixture was stirred again to obtain Au@PEG-NTA-Ni. 2+ NP; His-labeled RBD protein was added to Au@PEG-NTA-Ni under stirring. 2+ NP solution was prepared to obtain Au@PEG-RBD NP; and the obtained NP was dialyzed against Nanopure water by centrifugation and filtration. Attached Figure Description

[0010] Figure 1A-1H This study shows spike receptor-binding domain (RBD) coated polyethylene glycol (PEG) stabilized gold nanoparticles (Au@PEG–RBD NP). Figure 1A and 1BThe expression levels of ACE2 and L-SIGN receptors in the lungs of Syrian hamsters with LPS-induced ARDS are shown. Left: Lung tissue stained with L-SIGN or ACE2 (brown) at different time points after ARDS induction. Representative images of three tissue sections from each hamster, n=3 hamsters / group. Right: Time-dependent expression levels of ACE2 and L-SIGN in ARDS lungs detected by ELISA. n=3 in each of the three experiments. Figure 1C The synthetic route of Au@PEG–RBD NP is shown. ~20 nm, citrate-terminated gold NP reacts with thiolated -PEG-NTA, supported on Ni. 2+ The ions conjugated the His-labeled spike RBD protein. Au@PEG-methoxyNP served as an untargeted control. Figure 1D In this study, healthy hamsters showed no significant immunogenicity after inhaling Au@PEG–RBD NP (red) for 28 days. Intranasal injection of mammalian RBD protein served as a control (blue). BALF IgA and serum IgG levels represent humoral immune and mucosal immune responses, respectively. In each of the three experiments, n=3. Figure 1E The association of Au@PEG-methoxy and Au@PEG–RBD NPs with HEK293 cells in vitro is shown. After 1 hour of incubation, cells exhibited normal or overexpressed levels of ACE2 or L-SIGN receptors. n=3 per group in 3 experiments. Statistical significance was determined by Student... t Verification calculation. Figure 1F Representative confocal images of HEK293 cells after 1 hour of incubation with Cy5.5-labeled Au@PEG–methoxy and Au@PEG–RBD NP (red). Au@PEG–RBD NP preferentially binds to HEK293 cells overexpressing L–SIGN or ACE2 (green). Blue = nucleus. Figure 1G The in vitro binding of Au@PEG-methoxy and Au@PEG-RBD NP to healthy hamster lung tissue is shown. In three experiments, n=3 per group. Statistical significance was determined by Student... t Verification calculation. Figure 1HConfocal images of lung tissue from healthy hamsters 0.5 hours after intratracheal injection of Au@PEG–methoxy and Au@PEG–RBD NP (green) are shown. Au@PEG–RBD NP binds to ACE2 on cells in vitro (brown). Representative images of three tissue sections from each hamster are shown, with n=3 hamsters per group. Figure 1B , 1D In 1E and 1G, data are presented as mean ± SEM. No significant difference was observed within ns; P > 0.05. P<0.05; P<0.01.

[0011] Figures 2A-2F The in vivo lung distribution of Au@PEG–RBD NPs inhaled by LPS-induced ARDS hamsters is shown. Figure 2A The experiment showed ARDS hamsters being exposed to NP air for 2 hours, then returned to their holding space to await another inhalation 10 hours later or being euthanized immediately. Figure 2B The relationship between the content of Au@PEG-methoxy (blue) and Au@PEG–RBD (red) NPs in the lungs of healthy and ARDS hamsters and the number of inhalations is shown. Data are expressed as mean ± SEM. There were 3 experiments, with n=3 in each group. Statistical significance of the two NP types was compared using Student... t Verification calculations. Figure 2C The tissue-level distribution of NPs in BALF supernatant (green), BALF cells (red), and lavage lung tissue (blue) is shown in healthy hamsters (left) and ARDS hamsters (right). Data are expressed as mean ± SEM. In three experiments, n=3 per group. Figure 2D In the confocal reflectance images of ARDS hamster lung tissue, Au@PEG–RBD NPs (green; silver enhancement) are widely distributed in epithelial cells (brown: E-cadherin) in both the lobes (low power field) and alveoli (high power field). The black numbers represent the Pearson correlation coefficient (PCC) between gold NPs (green) and cell types (brown). Representative images are from three tissue sections from each hamster, with n=3 hamsters per group. Figure 2E In the confocal reflectance images of ARDS hamster lung tissue, Au@PEG–RBD NPs (green; silver enhancement) show strong association with cells expressing L–SIGN and ACE2 (brown). Black numbers represent PCCs between gold NPs (green) and receptor types (brown). Representative images are from three tissue sections from each hamster, with n=3 hamsters per group. Figure 2FThis study demonstrates a reduction in lung accumulation of inhaled Au@PEG–RBD NP (purple) (instead of Au@PEG–methoxy NP (green)) following intranasal instillation of anti-L-SIGN or anti-ACE2 antibodies into ARDS hamsters. Data are presented as mean ± SEM. n=3 per group in one experiment. Figure 2B and Figure 2F In Chinese: ns indicates no significant difference; P>0.05; P<0.05; P<0.01.

[0012] Figures 3A-3F The efficacy of Au@PEG-RBD NP in LPS-induced ARDS in hamsters was demonstrated. Figure 3A In the study, ELISA showed that Au@PEG–RBD NP (red) was more effective than Au@PEG–methoxy NP (blue) and hydrocortisone (brown) in reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines in BALF supernatant. Figure 3B In the study, Western blot analysis showed that Au@PEG–RBD NP was more effective than Au@PEG–methoxy NP and hydrocortisone in reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines in lavaged lung tissue. Figure 3C The results show that Au@PEG-RBD NP (red) was more effective than Au@PEG methoxy NP (blue) and hydrocortisone (brown) in reducing ROS levels in BALF cells. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc test for multiple comparisons. Figure 3D In the study, histological images of lung tissue damage scores showed that Au@PEG-RBD NP was more effective than Au@PEG-methoxy NP and hydrocortisone in reducing tissue damage. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc test. Representative images were obtained from three tissue sections per hamster, n=9 hamsters / group. Figure 3E This study demonstrates how blocking with anti-L-SIGN antibody reversed the efficacy of Au@PEG–RBD NP to levels similar to Au@PEG–methoxy NP. Data are presented as mean ± SEM. There were 6 n=6 groups in each of the 3 trials. No significant difference was observed within ns; P>0.05. P<0.01; P<0.001; P < 0.0001. Figure 3F In the study, plethysmography showed that after three inhalations of Au@PEG-RBD NP, key lung function parameters returned to healthy levels. Figure 3A and 3C In this study, data are expressed as mean ± SEM. In each of the three experiments, n=9. There were no significant differences within ns; P>0.05. P<0.05; P<0.01; P<0.001; P<0.0001.

[0013] Figure 4A-4I The mechanism by which gold NP exerts its anti-ARDS effect in LPS-induced ARDS hamsters is illustrated. Figure 4A In, through Z'-LYTE TM Analysis of 250 kinases showed that 0.2 nM Au@PEG–methoxyNP inhibited only 5 kinases by >70%, and only MAPK14 (p38α) and PLK3 were reported to be associated with ARDS. Figure 4B The 10-point titration curves and IC50 values ​​of Au@PEG-methoxy NP against MAPK14 (p38α) and PLK3 are shown. Figure 4C In the middle, (left) Western blot analysis showed that Au@PEG–RBD NP, and a lesser degree of Au@PEG–methoxy NP, inhibited p-p38α and PLK3 in lavaged lung tissue (top) and BALF cells (bottom) of ARDS hamsters compared to untreated ARDS hamsters. n=3 in one experiment. (Right) Quantification of Western blot data. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc test for multiple comparisons. Figure 4D In the study of Western blot analysis of lavage lung tissue, Figure 4E In a BALF supernatant ELISA study, the anti-inflammatory effect of inhaled Au@PEG-methoxy NP was ablated by the p38α activator, anisoxin. Data are presented as mean ± SEM. There were 3 experiments, with n=9 in each group. Statistical significance was assessed using one-way ANOVA. Figure 4FIn the volcano plot, the distribution of differentially expressed proteins (DEPs) obtained from pairwise comparisons between the "Au@PEG–methoxyNP group" and the "untreated group" is shown. n=3 (absolute fold change > 2; p < 0.05). Of the 2487 proteins tested, only 4 DEPs were upregulated (red), and 22 DEPs were downregulated (blue). Figure 4G The top 5 DEPs and their expression differences and p-values ​​are shown. Figure 4H In LPS-induced Western blot analysis of BEAS-2B cells, Au@PEG-methoxyNP reduced total Elavl1 expression, but the p38α inhibitor VX-702 only affected the nucleus-to-cytosol translocation of Elavl1 in vitro. Au@PEG-methoxyNP can inhibit Elavl1 independently of p38α. Figure 4I It shows Figure 4H Quantitative analysis of Western blot data was performed. Data are expressed as mean ± SEM. There were 3 experiments, n=3 per group. Statistical significance was assessed using one-way ANOVA. No significant difference was observed within ns; P>0.05. P<0.05; P<0.001; P<0.0001.

[0014] Figures 5A-5C The long-term toxicity of Au@PEG–RBD NP after three inhalations in LPS-induced ARDS hamsters was demonstrated. Figure 5A The organ-level distribution of Au@PEG–RBD (red) and Au@PEG-methoxy (blue) NPs is shown. One year after inhalation, gold was not detected in various major visceral organs, indicating that the gold NPs had been cleared. Data are from one experiment with n=3. Figure 5B (lungs) and Figure 5C Histological images of other major internal organs showed no abnormal tissue morphology compared to healthy controls one year after inhalation of Au@PEG-RBD NP. Representative images are from one tissue section of each hamster, n=3 hamsters / group.

[0015] Figures 6A-6E The efficacy of Au@PEG–RBD NP in HCl-induced ARDS hamsters was demonstrated. Figure 6AThe expression of ACE2 and L-SIGN receptors in the lungs of Syrian hamsters with HCl-induced ARDS is shown. Left: Western blot of lung tissue blotted with L-SIGN or ACE2 at different time points after ARDS induction. n=3 hamsters / group. Middle: Time-dependent expression of ACE2 and L-SIGN in ARDS lungs by ELISA. n=3 per group in 3 experiments. Right: Lung tissue immunostained with L-SIGN or ACE2 (brown) at different time points after ARDS induction. Representative images are from 3 tissue sections from each hamster, n=3 hamsters / group. Figure 6B In the study, ELISA showed that Au@PEG–RBD NP (red) was more effective than Au@PEG-methoxy NP (blue) and hydrocortisone (brown) in reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines in BALF supernatant. Figure 6C In the study, Western blotting showed that Au@PEG–RBD NP most effectively reduced pro-inflammatory cytokines in lavaged lung tissue. Figure 6D The results show that Au@PEG-RBD NP (red) was more effective than Au@PEG-methoxy NP (blue) and hydrocortisone (brown) in reducing ROS levels in BALF cells. Statistical significance was assessed using one-way ANOVA and Tukey's post-hoc test for multiple comparisons. Figure 6B and Figure 6D In this study, data are expressed as mean ± SEM. In each of the three experiments, n=9. Figure 6E This study showed that blocking with anti-L-SIGN antibody reversed the efficacy of Au@PEG–RBD NP to a level similar to that of Au@PEG–methoxy NP. Data are presented as mean ± SEM. In the three experiments, n=6 per group. There were no significant differences in efficacy within ns; P>0.05. P<0.05; P<0.01; P<0.001; P<0.0001.

[0016] Figure 7 The expression levels of ACE2 (brown) in the lungs of ARDS hamsters at different time points after LPS induction are shown. ACE2 signaling was strongest at 36 hours post-induction. Representative images are from 7 tissue sections from each hamster, n=3 hamsters / group.

[0017] Figure 8 The expression levels of L-SIGN (CD299; brown) in the lungs of ARDS hamsters at different time points after LPS induction are shown. L-SIGN signaling was strongest at 12 hours post-induction. Representative images are from 7 tissue sections from each hamster, n=3 hamsters / group.

[0018] Figure 9 The expression levels of mannose receptor (CD206; brown) in the lungs of ARDS hamsters at different time points after LPS induction are shown. mannose receptor signaling was low within 72 hours post-induction. Representative images are from 7 tissue sections from each hamster, n=3 hamsters / group.

[0019] Figure 10 The expression levels of DC-SIGN (CD209; brown) in the lungs of ARDS hamsters at different time points following LPS induction are shown. DC-SIGN signaling was lower within 72 hours post-induction. Representative images were obtained from 7 tissue sections from each hamster, with n=3 hamsters per group.

[0020] Figure 11 The expression levels of MGL (CD301; brown) in the lungs of ARDS hamsters at different time points after LPS induction are shown. MGL signal was low within 72 hours post-induction. Representative images are from 7 tissue sections from each hamster, with n=3 hamsters per group.

[0021] Figure 12 The expression levels of TMPRSS2 (brown) in the lungs of ARDS hamsters at different time points following LPS induction are shown. TMPRSS2 signaling was lower within 72 hours post-induction. Representative images were obtained from 7 tissue sections from each hamster, with n=3 hamsters per group.

[0022] Figures 13A-13E The expression and purification of the spike RBD protein (with an 8x His tag at the C-terminus) are shown. Figure 13A As shown, among the three types of Escherichia coli tested ( E. Coli In the strain, Western blot analysis showed that the expression of soluble spike RBD protein was highest in Origami B (right), which was evidenced by thick bands in the soluble fraction (supernatant of lysate), which was due to immunostaining of spike RBD and its His tag. Figure 13B and 13CThe results showed that among the five folding chaperone plasmids tested, the spike RBD plasmid (pET11a-RBD-8xHis) co-expressed with pG-KJE8 in Origami B cells resulted in the highest RBD expression and the strongest binding [or lowest dissociation constant] to the ACE2 and L-SIGN receptors. Kd )). Figure 13D The stepwise protein detection during the purification process is shown. All elute fractions were collected, centrifuged and filtered three times at 4°C (MWCO 30 kDa) for later use. Figure 13E The use of a 30 kDa MWCO membrane to remove other impurity bands is shown for further purification of the spike RBD protein [300 and 500 mM imidazole elution buffers ( Figure 13D We collected two elute fractions of the RBD protein. The expected molecular weight of the spike RBD protein is 25 kDa.

[0023] Figure 14 This demonstrates the validation of the conjugation of the spike RBD protein on Au@PEG NP. Initially, the spike RBD protein (with a His tag) binds to Au@PEG–NTA–Ni. 2+ The NTA group of the NP is conjugated, forming Au@PEG–RBD NP. Unreacted spike RBD protein was removed by centrifugation and the NP was washed with Tris buffer. Imidazole was then added to the purified Au@PEG–RBD NP to release the bound spike RBD protein. Finally, Western blot analysis confirmed the release of spike RBD protein from the Au@PEG NP, as evidenced by the protein bands after separate immunoblotting of the spike RBD and its His tag. The addition of free imidazole disrupts the interaction between the nickel-nitrotriacetic acid (Ni-NTA) on the PEG chain and the histidine tag of the spike protein. The released spike RBD protein was detected by ELISA (fraction 3), and the number of RBD proteins on the gold NP was ~306.

[0024] Figure 15 The batch consistency of the synthesized Au@PEG-methoxy NP and Au@PEG-RBD NP is shown, confirmed by displaying a hydrodynamic size distribution histogram of three representative batches. Each batch was synthesized at intervals of 2–3 months.

[0025] Figure 16 The stability of Au@PEG–RBD NPs varies with spike RBD loading. Au@PEG–RBD NPs with 100% PEG chain spike RBD loading aggregated after 24 hours of incubation in BALF (likely due to protein-protein interactions), but this aggregation was easily reversed by sonication. Conversely, Au@PEG–RBD NPs with only 50% PEG chain spike RBD loading remained stable in BALF for 3 days. For our subsequent studies, we selected 50% RBD as the default loading for constructing Au@PEG–RBD NPs.

[0026] Figure 17 Representative TEM images of citrate-capped gold NPs, Au@PEG-methoxy NPs, and Au@PEG-RBD NPs with negative staining via EMStainer are shown. The PEG shell appears as a halo around the gold core due to its low electron scattering ability relative to the gold core and the surrounding heavy-metal background stain. The physical diameter of the NPs (measured manually by TEM counting, 500 NPs per group) is smaller than the hydrodynamic diameter (measured by DLS) because the TEM images were captured under dry conditions.

[0027] Figure 18 The batch consistency of aerosolized Au@PEG-methoxy NP and Au@PEG-RBD NP is shown, confirmed by aerodynamic size distribution histograms of three representative batches and aerosolized NP concentrations.

[0028] Figures 19A-19C The stability of Au@PEG–RBD NP in BALF is shown. Figure 19A and 19B The results show that, compared with incubation in water, after 24 hours of incubation in BALF at 37°C, the surface plasmon resonance (SPR) peak of Au@PEG-RBD NP, as measured by UV-Vis spectrophotometry, did not change significantly; the SPR peak remained at 524 nm. Figure 19CIn the study, DLS measurements showed that the hydrodynamic size of both NPs increased by ~20 nm after BALF incubation, but there was no significant difference in the hydrodynamic size between Au@PEG-methoxy NP and Au@PEG–RBD NP. Data are expressed as mean ± SEM. There were 3 experiments, with n=3 per group. Statistical significance was calculated using one-way ANOVA. P<0.05; P<0.01.

[0029] Figures 20A-20C The ligand stability of Au@PEG-RBD NP is shown. Figure 20A As shown, after incubating NPs with fresh BALF extracted from hamsters at 37°C for 48 hours, the supernatant was subjected to Western blotting (lane #1). Next, the NPs were resuspended in 1M imidazole to release the spike RBD protein initially bound to the gold nucleus. The supernatant was then subjected to Western blotting (lane #2) or ELISA. Figure 20B In the study, Western blot analysis showed that almost all RBD proteins remained on the gold nucleus after BALF incubation. Figure 20C In the ELISA results, the total number of RBD proteins on Au@PEG-RBD NPs did not change after BALF incubation. These data indicate the stability of Au@PEG-RBD NPs in BALF, with limited detachment of the target ligand from the NP.

[0030] Figures 21A-21B The binding of Au@PEG-RBD NP to the spike receptor is shown. Figure 21A This demonstrates the ELISA assay method improved in-house.

[63] The binding affinity of Au@PEG–RBD NPs to the ACE2 receptor or L-SIGN receptor was measured. Au@PEG–RBD NPs showed binding affinity at 0.23 and 0.83 nM K, respectively. d Au@PEG–methoxyNP binds to ACE2 (red) and L-SIGN (orange) at the specified values. Au@PEG–methoxyNP does not show significant binding to either ACE2 (dark blue) or L-SIGN (light blue). Data are expressed as mean ± SEM. In 3 experiments, n=6 per group. Figure 21B This paper presents a ligand competition assay of Au@PEG–RBD NP for ACE2 or L-SIGN, measured using our internally modified ELISA method. In each group, the concentration of Au@PEG–RBD NP was kept constant at 3 nM, while the concentration of free RBD was gradually increased. Data are presented as mean ± SEM. There were 3 experiments, with n=6 per group.

[0031] Figure 22 The diagram illustrates nickel ion leakage from Au@PEG–RBD NP. The figure above shows a schematic of the detection process. In this work, to minimize nickel exposure, the Au@PEG–RBD NP solution was dialyzed six times against Nanopure water before aspiration. ICP-MS was used to detect nickel ion concentrations in the supernatant after dialysis and one month of storage. Data are expressed as mean ± SEM. Data were obtained from two experiments (n=3). Statistical significance was determined by student... t Test results showed no significant difference (ns), P>0.05.

[0032] Figure 23 The cytotoxicity of Au@PEG-methoxy NP and Au@PEG–RBD NP is illustrated. A549, BEAS-2B, and HEK293 cells, pre-seeded in 96-well plates, were incubated for 72 hours in medium containing different concentrations of Au@PEG-methoxy NP or Au@PEG–RBD NP. The medium used for A549 and HEK293 cells was complete DMEM, and the medium used for BEAS-2B cells was LHC-8. Cell viability was assessed by measuring absorbance at 570 nm and 600 nm using a Multiskan GO UV absorbance microplate reader. Data are presented as mean ± SD. In two experiments, n=6 per group. We selected 0.2 nM as the incubation concentration for the in vitro cellular uptake study.

[0033] Figure 24 The time-dependent cellular association of Au@PEG-RBD NP with HEK293 cells overexpressing L-SIGN and ACE2 is shown. During shorter incubation periods, Au@PEG-RBD NP associated more abundantly with cells overexpressing ACE2 and L-SIGN compared to Au@PEG-methoxy NP. During longer latency periods, Au@PEG-RBD NP associated with similar amounts of both overexpressing cell types compared to Au@PEG-methoxy NP. Data are presented as mean ± SEM. Data were from two experiments with n=3. Statistical significance was calculated using two-way analysis. P<0.001, P<0.05, no significant difference (ns); P>0.05.

[0034] Figure 25 Additional confocal images of HEK293 cells incubated for 1 hour with Cy5.5-labeled Au@PEG–RBD NPs (red). Au@PEG-RBD NPs preferentially bind to HEK293 cells overexpressing ACE2 (green). Blue = nucleus. White numbers represent the PCC between the gold NP (red) and the receptor (green). Figure 1F A similar image is shown.

[0035] Figure 26 Additional confocal images of HEK293 cells incubated for 1 hour with Cy5.5-labeled Au@PEG–RBD NPs (red). Au@PEG-RBD NPs preferentially bind to HEK293 cells overexpressing L-SIGN (green). Blue = cell nucleus. White numbers indicate the PCC between the gold NP (red) and the receptor (green). Figure 1F A similar image is shown.

[0036] Figures 27A-27B The image shows Au@PEG–RBD NP (red) labeled with Cy5.5 incubated for 1 hour with different levels of ( Figure 27A ACE2 expression and ( Figure 27B Confocal image of HEK293 cells expressing L-SIGN (green). NPs preferentially enter cells overexpressing ACE2 / L-SIGN by overlapping with their receptors. Blue = cell nuclei. White numbers represent the PCC between the gold NP (red) and the receptor (green).

[0037] Figure 28 Additional confocal images of HEK293 cells with low ACE2 expression (green) incubated for 1 hour with Cy5.5-labeled Au@PEG-RBD NPs (red). Cell influx and colocalization with ACE2 were lower compared to ACE2-overexpressing cells. Blue = cell nucleus. White numbers represent the PCC between the gold NP (red) and the receptor (green).

[0038] Figure 29Additional confocal images of HEK293 cells with low L-SIGN expression (green) after 1 hour incubation with Cy5.5-labeled Au@PEG-RBD NPs (red). Cell entry and co-localization with L-SIGN were lower compared to L-SIGN-overexpressing cells. Blue = cell nucleus. White numbers indicate the PCC between the gold NP (red) and the receptor (green).

[0039] Figure 30 The calculation of maximum nickel exposure in hamsters is shown. We assume that two forms of Ni exist in the NP aqueous solution prior to aerosolization. 2+ Ions, (1) free ions in water and (2) chelated ions in NP. Free Ni in water 2+ The quantity was measured directly by ICP-MS. Figure 22 ), while chelated Ni 2+ The amount is based on Figure 2B The inhaled gold content in the lungs measured by ICP-MS was inferred stoichiometrically (per Au@PEG–RBD NP, ~300 Ni). 2+ (Ions). During the 36-hour treatment period, the hamsters inhaled a total of ~12 μg of nickel (free and chelated) after three inhalations, below the theoretically tolerable upper limit of intake. This is based on the human tolerable upper limit of nickel intake (1 mg / day).

[68] We use the FDA's equivalent dosage.

[24] To estimate the upper limit of tolerance dose for hamsters (18.5 μg / 150 g hamster / day). In fact, because the atomized water droplets were removed by the dryer before being delivered to the animal inhalation chamber, the actual intake of free nickel ions was even lower than our theoretical calculation.

[0040] Figure 31 based on Figure 2A The timeline in the figure shows the distribution of Au@PEG–RBD NP (triangles) and Au@PEG–methoxy NP (squares) at the organ level (left) and in the upper / lower respiratory tract (right) after three inhalations in healthy hamsters. Data are expressed as mean ± SEM. Data were from one experiment with n=3. Statistical significance was calculated by two-way ANOVA. No significant difference (ns) was found, P>0.05.

[0041] Figure 32 based on Figure 2AThe timeline in the figure shows the distribution of Au@PEG–RBDNP (triangles) and Au@PEG–methoxy NP (squares) at the organ level (left) and in the upper / lower respiratory tract (right) after three inhalations of LPS-induced ARDS in hamsters. Data are expressed as mean ± SEM. Data were from 3 experiments, n=3. Statistical significance was calculated by two-way analysis. No significant difference (ns) was considered, P>0.05.

[0042] Figure 33 Following the timeline in 2A, the tissue-level distribution of Au@PEG-RBD NP and Au@PEG-methoxy NP in different lung lobes of healthy hamsters and LPS-induced ARDS hamsters is shown. Data are presented as mean ± SEM. Data were from two experiments with n=3. Statistical analysis was determined by two-way analysis. No significant differences were found (ns), P>0.05.

[0043] Figure 34 The graph shows the relationship between the amount of Au@PEG-methoxy NP (squares) and Au@PEG–RBD NP (triangles) in the lungs of healthy and ARDS hamsters and the number of inhalations. (Inhalation 3 graph) Figure 2B The corresponding plots are identical. Data are expressed as mean ± SEM. In one experiment, n=3 per group. Statistical significance of the two NP types was compared using Student... t Verification calculations. P<0.05; P<0.001.

[0044] Figure 35 Confocal reflectance images of lung tissue from ARDS hamsters show weak association between Au@PEG–RBD NPs (green; silver enhancement) and endothelial cells (brown: CDH5). Black numbers represent the Pearson correlation coefficient (PCC) between gold NPs (green) and cell types (brown). Representative images are from three tissue sections from each hamster, with n=3 hamsters per group.

[0045] Figure 36This study demonstrates the specific binding of Au@PEG-RBD NP to L-SIGN and ACE2 receptors in ARDS lungs. Pretreatment with anti-L-SIGN antibody via intratracheal administration reduced the accumulation of Au@PEG-RBD NP in the lungs of LPS-induced ARDS hamsters after the first two inhalations, but not after the third. In contrast, pretreatment with anti-ACE2 antibody via intratracheal administration did not reduce the accumulation of Au@PEG–RBD NP after the first two inhalations, but did prevent lung accumulation after the third inhalation. Data from the “Au@PEG-methoxy”, “Au@PEG–RBD”, “anti-L-SIGN antibody + Au@PEG–RBD”, “anti-ACE2 antibody + Au@PEG–RBD”, and “anti-L-SIGN antibody + anti-L-SIGNs antibody + Au@PEG-RBD NP” groups are compared with… Figure 2F The corresponding groups were identical. Data are expressed as mean ± SEM. In the three experiments, n=3 for each group. Statistical analysis was determined by one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05; P<0.001; P<0.001.

[0046] Figure 37 This illustrates the combined role of L-SIGN and ACE2 receptors in mediating Au@PEG-RBD NP delivery to the lungs of ARDS. LPS-induced ARDS hamsters received a single dose of NP at 12 hours (first time point; top panel) or 36 hours (third time point; bottom panel) post-LPS induction. Anti-L-SIGN antibody, anti-ACE2 antibody, or both antibodies were administered intratracheally 1 hour prior to NP inhalation. The results in the top panel are consistent with those at the first inhalation time point shown in Figure 2f. Data are expressed as mean ± SEM. In each of the three experiments, n=3.

[0047] Figures 38A-38C The protein corona analysis of Au@PEG-methoxy NP and Au@PEG–RBD NP after incubation in BALF extracted from LPS-induced ARDS Syrian hamsters for 30 minutes is shown. Figure 38A The top 10 BALF proteins in the protein crown are shown. Figure 38B The SDS-PAGE gel analysis of the NP protein crown is shown. Figure 38C This study showed that pretreatment with antibodies targeting FcR or SPARC (receptors for both albumins) did not significantly reduce the accumulation of Au@PEG–RBD NP in the lungs of ARDS hamsters. Therefore, the BALF protein crown does not affect the lung delivery of Au@PEG–RBD NP. Data are presented as mean ± SEM. There were 3 groups in each of the 3 experiments. Statistical analysis was determined by one-way ANOVA. No significant difference (ns) was observed, P > 0.05. P<0.05; P<0.001.

[0048] Figure 39 The inhalation regimen for ARDS treatment is shown. According to... Figure 1A , 1B Data from 7 and 8 showed that L-SIGN and ACE2 were upregulated at ~12 hours and ~36 hours after ARDS induction, respectively. We targeted L-SIGN (scheme 1, which is similar to...) Figure 2A (The timelines shown are the same) and ACE2 (Program 2) developed two inhalation protocols with different starting points.

[0049] Figure 40 The quantification of levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6, and IL-8) and anti-inflammatory cytokines (IL-4 and IL-10) in BALF extracted from healthy hamsters treated with Au@PEG–RBD NP (or untreated with Au@PEG–RBD NP) is shown (following the principle of quantitative analysis). Figure 2A (Timeline in the table). Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05.

[0050] Figure 41 This study shows the quantification of TNF-α levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical, with the bottom row showing detailed values ​​and statistical analysis for each group. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.01; P<0.001; P<0.0001.

[0051] Figure 42 This study shows the quantification of TNF-α levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation protocol 2). The bottom row shows detailed values ​​and statistical analysis for each group of each curve in the top row. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. P<0.05; P<0.01; P<0.001; P<0.0001.

[0052] Figure 43 This paper presents a quantification of IFN-γ levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data are from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns). P<0.0001.

[0053] Figure 44 This study shows the quantification of IFN-γ levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation protocol 2). The bottom row shows detailed values ​​and statistical analysis for each group of each curve in the top row. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. P<0.01; P<0.001; P<0.0001.

[0054] Figure 45 This study shows the quantification of IL-6 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical to those in the main graph. The bottom row shows detailed values ​​and statistical analysis for each group. Data are expressed as mean ± SEM. Data are from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. P<0.01; P<0.0001.

[0055] Figure 46 This study shows the quantification of IL-6 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 36 hours after induction (following...). Figure 39 (Inhalation protocol 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. P<0.01; P<0.0001.

[0056] Figure 47 This study shows the quantification of IL-8 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.01; P<0.001; P<0.0001.

[0057] Figure 48This study shows the quantification of IL-8 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation protocol 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. P<0.01; P<0.0001.

[0058] Figure 49 This study shows the quantification of IL-4 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.001; P<0.0001.

[0059] Figure 50 This paper presents a quantification of IL-10 levels in BALF extracted from LPS-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 2A (Timeline in the middle). Top row and Figure 3A The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.001; P<0.0001.

[0060] Figure 51 This study demonstrates the quantification of cytokine levels in BALF following inhalation of a free PEG-RBD conjugate. Following... Figure 39The timeline shown represents BALF extracted from LPS- or HCl-induced ARDS hamsters after three inhalations. Group A data is compared with... Figures 41-48 and Figures 56-63 Data were identical for the untreated group. Data are presented as mean ± SEM. Data were drawn from 3 experiments with n=6. Statistical analysis was performed using student... t The test confirmed that there was no significant difference (ns), P>0.05.

[0061] Figure 52 It shows Figure 3B The quantitative analysis of Western blot data is shown. Data are presented as mean ± SEM. Data were obtained from 3 experiments (n=3). Statistical significance was assessed using two-way ANOVA. No significant difference (ns) was found, P>0.05. P<0.05; P<0.001; P<0.001; P<0.0001.

[0062] Figure 53 The cell counts in the BALF are shown. Red blood cells (RBCs) and other BALF cells were counted using a hemocytometer slide. The proportions of different cell types were calculated using Wright-Giemsa staining. Western blot analysis showed that the increased macrophages were primarily M2 macrophages.

[0063] Figure 54 The tissue repair effects of Au@PEG–RBD NP inhaled after the first and second inhalations are shown in LPS-induced ARDS hamsters. Lung injury scores (left) [lung tissue histological images are shown (right)] indicate that Au@PEG-RBD NP is more effective than Au@PEG-methoxy NP and hydrocortisone in reducing tissue damage. Representative images are from 3 tissue sections per hamster, n=9 hamsters / group. Statistical significance was assessed using one-way ANOVA.

[0064] Figure 55The quantification of cytokine levels in BALF following specific antibody blocking in LPS-induced ARDS hamsters is shown. Anti-L-SIGN antibody was administered intranasally to ARDS hamsters 2 hours prior to each inhalation. BALF was extracted from LPS-induced ARDS hamsters, with treatment initiated 12 hours post-induction (following the timeline in Figure 2a). Data from groups A and B are compared with... Figure 41 , Figure 43 , Figure 45 , Figure 47 , Figure 49 and Figure 50 The corresponding group is the same. The diagram for "third inhalation" is the same as... Figure 3E The corresponding plots are identical. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05; P<0.01; P<0.001; P<0.0001.

[0065] Figure 56 The quantification of cytokine levels in BALF after specific antibody blockade in LPS-induced hamsters is shown. Anti-ACE2 antibody was administered intranasally to ARDS hamsters 2 hours prior to each inhalation. BALF was extracted from LPS-induced ARDS hamsters, where treatment began 36 hours after induction (following...). Figure 39 Inhalation regimen 2). Data from groups A and B are consistent with... Figure 42 , Figure 44 , Figure 46 and Figure 48 The corresponding groups were identical. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05; P<0.01.

[0066] Figure 57Lung function parameters of healthy hamsters (left), LPS-induced ARDS hamsters (middle), and NP-treated LPS-induced ARDS hamsters (right), measured by whole-body plethysmography, are shown. Results indicated that after three inhalations of Au@PEG-RBD NP, peak inspiratory flow rate (PIF), peak expiratory flow rate (PEF), expiratory time (Te), and relaxation time (Tr) all returned to healthy levels. Data are presented as mean ± SEM. In the three experiments, n = 5 ns per group; no significant differences were observed; P > 0.05. P<0.05; P<0.01.

[0067] Figure 58 This study shows the quantification of TNF-α levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 39 Inhalation regimen 1). Top row and Figure 6B The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.05; P<0.01; P<0.0001.

[0068] Figure 59 This study shows the quantification of TNF-α levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation regimen 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. No significant difference (ns), P>0.05; P<0.01; P<0.001; P<0.0001.

[0069] Figure 60 This study shows the quantification of IFN-γ levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 39Inhalation regimen 1). Top row and Figure 6B The corresponding graphs are identical to those in the main graph. The bottom row shows detailed values ​​and statistical analysis for each group. Data are expressed as mean ± SEM. Data are from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. P<0.05; P<0.001; P<0.0001.

[0070] Figure 61 This study shows the quantification of IFN-γ levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation regimen 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. No significant difference (ns), P>0.05; P<0.05; P<0.01; P<0.001; P<0.0001.

[0071] Figure 62 This shows the quantification of IL-6 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 39 Inhalation regimen 1). Top row and Figure 6B The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.05; P<0.001; P<0.0001.

[0072] Figure 63This study shows the quantification of IL-6 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation regimen 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. No significant difference (ns), P>0.05; P<0.01; P<0.001; P<0.0001.

[0073] Figure 64 This shows the quantification of IL-8 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 39 Inhalation regimen 1). Top row and Figure 6B The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.05; P<0.01; P<0.001; P<0.0001.

[0074] Figure 65 This study shows the quantification of IL-8 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 36 hours after induction (following the guidelines). Figure 39 (Inhalation protocol 2). Detailed values ​​and statistical analysis for each group are shown in the bottom row. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was determined by one-way ANOVA. P<0.0001.

[0075] Figure 66 This study shows the quantification of IL-4 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following the guidelines). Figure 2A(Timeline in the middle). Top row and Figure 6B The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.001; P<0.0001.

[0076] Figure 67 This study shows the quantification of IL-10 levels in BALF extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 2A (Timeline in the middle). Top row and Figure 6B The corresponding graphs are identical, with detailed values ​​and statistical analysis for each group shown in the bottom row. Data are expressed as mean ± SEM. Data came from 3 experiments with n=9. Statistical analysis was performed using one-way ANOVA. No significant differences were found (ns), P>0.05; P<0.05; P<0.0001.

[0077] Figure 68 It shows Figure 6C Quantitative analysis of the Western blot data is shown. Data are presented as mean ± SEM. Data are from 3 experiments (n=3). Statistical significance was assessed using two-way ANOVA. P<0.05; P<0.001; P<0.001; P<0.0001.

[0078] Figure 69 The tissue repair effect of inhaled Au@PEG–RBD NP in HCl-induced ARDS hamsters is shown. Histological images of lung tissue (top) [lung injury scores are shown (bottom)] indicate that Au@PEG-RBD NP is more effective than Au@PEG-methoxy NP and hydrocortisone in reducing tissue damage. Representative images are from three tissue sections per hamster, n=9 hamsters / group. Statistical significance was assessed using one-way ANOVA.

[0079] Figure 70 The quantification of cytokine levels in BALF after specific antibody blockade in HCl-induced ARDS hamsters is shown. Anti-L-SIGN antibody was administered intranasally to ARDS hamsters 2 hours prior to each inhalation. BALF was extracted from HCl-induced ARDS hamsters, where treatment began 12 hours after induction (following...). Figure 2A (Timeline in the middle). Data from Group A and Group B and Figure 58 , Figure 60 , Figure 62 , Figure 64 , Figure 66 and Figure 67 The corresponding group is the same. The diagram for "third inhalation" is the same as... Figure 6E The corresponding plots are identical. Data are expressed as mean ± SEM. Data are from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05; P<0.01; P<0.001; P<0.0001.

[0080] Figure 71 The quantification of cytokine levels in BALF after specific antibody blockade in HCl-induced ARDS hamsters is shown. Anti-ACE2 antibody was administered intranasally to ARDS hamsters 2 hours prior to each inhalation. BALF was extracted from HCl-induced ARDS hamsters, with treatment initiated 36 hours after induction (following...). Figure 39 Inhalation regimen 2). Data from groups A and B are consistent with... Figure 59 , Figure 61 , Figure 63 and Figure 65 The corresponding groups were identical. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05; P<0.01; P<0.001; P<0.0001.

[0081] Figure 72This study demonstrates the quantification of ROS levels in BALF cells in ARDS hamsters via specific antibody blocking or free PEG-RBD conjugate. Anti-L-SIGN antibody was administered intranasally to ARDS hamsters 2 hours prior to each inhalation. BALF was extracted from ARDS hamsters, with treatment initiated 12 hours after induction (following...). Figure 2A (Timeline in the middle). Data from the Untreated group, Au@PEG–methoxy group, and Au@PEG-RBD group are compared with... Figure 3C and Figure 6D The corresponding groups were identical. Data are expressed as mean ± SEM. Data were from 3 experiments, n=6. Statistical analysis was performed using one-way ANOVA. No significant difference (ns) was found, P>0.05; P<0.05.

[0082] Table 1. Overview of non-gold nanoparticles (NPs) for ARDS management.

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094] Abbreviations: iv: intravenous injection; ip: intraperitoneal injection; in: intranasal injection; BALF: bronchoalveolar lavage fluid; TNF: tumor necrosis factor; IL: interleukin; IFN: interferon; PEG: polyethylene glycol; NA: not applicable; STAT3: signal transduction and transcription activator 3; NF-κB: nuclear factor κB; SP-A: surfactant protein A; TGF: transforming growth factor; CXCL: chemokine (CXC motif) ligand; MLE: mouse lung epithelial cells; ROS: reactive oxygen species.

[0095] Table 2. Overview of ARDS Therapy Using Gold Nanoparticles (NPs)

[0096] Abbreviations: in: intranasal injection; ip: intraperitoneal injection; it: intratracheal injection; LPS: lipopolysaccharide; BALF: bronchoalveolar lavage fluid; NA: not applicable; TNF: tumor necrosis factor; IL: interleukin; IFN: interferon; P12: a 14-amino acid peptide (sequence: PSHISKYILRWRPK (SEQ ID NO:1)); TLR: toll-like receptor.

[0097] Table 3. Inhalable spike-RBD bound NPs (Note: These are the only spike-RBD bound NPs available in the literature. In all cases, the NPs were used as vaccines, not for treating lung inflammation.)

[0098] Table 4. cDNA sequences of His-tagged spike receptor-binding domains (RBDs) with His tags (in bold underline).

[0099]

[0100] We selected the spike RBD sequence (left column) based on the Procko laboratory plasmid pcDNA3-SARS-CoV-2-S-RBD-8his (Addgene #145145); this plasmid was originally used to express the spike RBD in eukaryotic HEK cells.

[109] In order to [do something] in prokaryotic Escherichia coli ( E. Coli For expression in cells (for convenience and cost reduction, as we require large amounts of protein for efficacy studies), we sought codon optimization and plasmid construction services from GenScript to construct a prokaryotic version of the plasmid pET11a-RBD-8his (right column).

[0101] Table 5. Physicochemical characteristics of other gold nanoparticles

[0102]

[0103] Table 6. Physicochemical characteristics of gold nanoparticles (NPs) incubated in fresh BALF supernatant (from healthy hamsters) at 37°C prior to DLS measurements.

[0104]

[0105]

[0106] Table 7. Lung Injury Scoring Scale

[0107] Table 8. Kinase profile analysis based on 281 kinases, showing the top 50 kinases inhibited by Au@PEG–methoxy nanoparticles.

[0108]

[0109]

[0110] Table 9. Proteomic analysis of differentially expressed proteins

[0111]

[0112] Table 10. Blood cell counts and biochemical parameters of ARDS hamsters after inhalation of nanoparticles (1 month, 6 months and 1 year after inhalation).

[0113]

[0114]

[0115]

[0116]

[0117] Table 11. Previous clinical reports on the treatment of ARDS with corticosteroids.

[0118]

[0119]

[0120]

[0121]

[0122] Detailed Implementation

[0123] Embodiments of the present invention relate to a method for preparing inhalable targeted gold nanoparticles for accelerating lung delivery and treating lung inflammation.

[0124] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" as used herein are intended to include both plural and singular forms. It should be further understood that when the terms "comprising" and / or "including" are used in this specification, the presence of the stated features, steps, operations, elements, and / or components is explicitly stated, but the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof is not excluded.

[0125] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms (e.g., those defined in common dictionaries) shall be interpreted as having the meaning consistent with their meaning in the context of the relevant technology and this invention, and shall not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0126] When the term “about” is used in conjunction with a numerical value in this document, it is understood that the value can range from 90% to 110% of that value, i.e., the value can be + / - 10% of the value. For example, “about 1 kg” means 0.90 kg to 1.1 kg.

[0127] In describing this invention, it should be understood that numerous techniques and steps are disclosed. Each of these techniques has its own advantages, and each technique can be used in conjunction with one or more, or in some cases with all the other disclosed techniques. Therefore, for clarity, the description will avoid unnecessarily repeating every possible combination of the steps. However, upon reading the specification and claims, it should be understood that such combinations are entirely within the scope of this invention and the claims.

[0128] A rapid, non-invasive, and disease-driven nanomedicine for ARDS is provided, based on a gold NP conjugated to multiple recombinant spike receptor-binding domain (RBD) subunit proteins (derived from the SARS-CoV-2 virus), for inhalation delivery to epithelial cells (which comprise approximately 90% of lung cells). This biomimetic approach stems from (i) the rapid infection of lung epithelial cells via SARS-CoV-2 virus inhalation through spike receptors, (ii) the clinical validation of the safety of RBD-containing NPs as a COVID-19 vaccine (see Table 3), and (iii) the discovery of transient and sequential activation of two spike receptors on lung epithelial cells induced by ARDS at different time points after ARDS onset (12 hours for liver / lymph node-specific intercellular adhesion molecule-3-binding integrin (L-SIGN), followed by 36 hours for angiotensin-converting enzyme 2 (ACE2)). The treatment premise is to enhance the inhaled delivery of self-healing gold NP to ARDS lung epithelial cells by binding the recombinant spike RBD protein to activated L-SIGN and ACE2 receptors; these two receptors superimposedly mediate alveolar targeting of the nanomedicine. The two ARDS models selected are lipopolysaccharide (LPS)-induced (corresponding to bacterial infection; see Figure 3) and hydrochloric acid (HCl)-induced (corresponding to inhalation; see Figure 6) Syrian hamsters (which have spike receptors similar to those in humans).

[16] Both of these non-viral ARDS are applicable to a broader patient population. Furthermore, although the spike RBD protein is conjugated to the gold NP, viral blocking mechanisms or COVID-19-related ARDS animal models were not involved. Pharmacologically, the gold core naturally inhibits p38α mitogen-activated protein kinase (MAPK) phosphorylation and polo-like kinase 3 (PLK3) phosphorylation—both therapeutic targets for ARDS—without the need for chemical drugs, biological products, or external physical action. The polyethylene glycol (PEG) chain shell confers stability to the NP in nebulization and bronchoalveolar lavage fluid (BALF), allowing the RBD to be conjugated to the gold core. The overall size of the NP is approximately 94 nm, large enough to inhibit ABB penetration into the systemic circulation.

[17] But small enough to allow for even distribution of alveoli.

[18] In both hamster models, after intermittent inhalation for 6 hours, NP deposits were more abundant in the lungs of patients with ARDS than in healthy lungs, and NP was more effective in treating ARDS than standard steroid treatment. No long-term retention in major organs or toxicity was observed after one year of inhalation.

[0129] According to embodiments of the present invention, a spike receptor-binding domain (RBD)-conjugated gold NP is provided for inhalation delivery to lung epithelial cells and treatment of ARDS in hamsters. The rapid infection of human lung cells by inhaled SARS-CoV2 virus inspired this NP design, and the similarity of spike-associated receptors between humans and hamsters prompted the use of hamsters as a proof-of-concept. The 20 nm gold core resolves the bio-nano interactions in the lungs and naturally inhibits p38α mitogen-activated protein kinase (MAPK) phosphorylation and polo-like kinase 3 (PLK3) without the use of chemical or biological agents. The 20,000 Da PEG chain confers stability to the NP in nebulization and bronchoalveolar lavage fluid (BALF), chemically linking the RBD and the gold core, and maintaining a large overall NP size (e.g., approximately 90 nm) to inhibit entry into systemic circulation by penetrating the air-blood barrier.

[0130] Spike RBDs can rapidly and extensively enter lung epithelial cells overexpressing angiotensin-converting enzyme 2 (ACE2) and liver / lymph node-specific intercellular adhesion molecule-3-binding integrin (L-SIGN), both of which are transiently activated in hamsters after ARDS induction. In lipopolysaccharide (LPS)-induced Syrian hamsters, repeated inhalation of NPs resulted in greater deposition in ARDS-affected lungs than in healthy lungs, and was more effective than corticosteroids in reducing tissue damage, oxidative stress, and inflammation in BALF cells and lung tissue. Furthermore, they inhibited ARDS-related proteins and did not cause gold retention or toxicity in major organs one year after inhalation. Similar therapeutic effects were observed in hamsters with hydrochloric acid (HCl)-induced ARDS.

[0131] method Research Design The aim of this study was to develop a non-invasive, epithelial-targeting gold nanoparticle drug that can rapidly enter lung cells with transiently activated spike-associated receptors, ultimately treating ARDS more effectively than conventional steroids without long-term systemic toxicity and accumulation. The chosen animal model was clinically relevant LPS- or HCl-induced hamster disease. [16,20] All procedures followed the guidelines set forth by the Animal Ethics Committee (AEEC) of the Chinese University of Hong Kong, approval number: 21-098-MIS. Anti-inflammatory efficacy was measured by three outcomes: ARDS-related cytokines in BALF supernatant and lavage lung tissue, ROS levels in BALF cells, and the degree of tissue repair. For efficacy studies, the size of the treatment group was estimated using the Dunnett formula and power calculations (α=0.05, power=0.80). In short, , where μ is the correlation coefficient depending on the size N of each group. For the four different treatment groups, μ is 4.46.

[59] If the outcome (δ) in the advanced treatment group is 1.5 standard deviations (σ) better than that in the control group, then the required size of N is... Or, equivalent to 9 mice per group. Hamsters were randomly assigned to each group. All in vivo mouse studies were conducted according to best practices and are described in the sections on "Animal Models of Acute Respiratory Distress Syndrome" and "NP In Vivo Inhalation". All experiments in this study were performed using 2-3 independent biological replicas, as specified in the illustrations. Blinding was not used in the analysis of histological images. Blinding was used for blood chemistry tests.

[0132] Data processing and statistical analysis Prism (GraphPad software) and ImageJ were used for data analysis and graph construction. Statistical analyses are indicated below the charts. Unpaired two-tailed t-tests were performed to determine the statistical significance of two group comparisons. Unpaired one-way ANOVA was performed to determine the statistical significance of multiple group comparisons, with Tukey's test used for post-hoc analysis. Two-way ANOVA was performed to determine the statistical significance of multiple group comparisons with two variables (e.g., NP treatment and time), with Tukey's test used for post-hoc analysis. The normality of the mean sampling distribution was verified by the Shapiro-Wilk test. Homogeneity of variance was verified by Bartlett's test. Results were considered significant at p < 0.05.

[0133] Preparation of spike RBD protein Step 1: Plasmid preparation The plasmid (pET11a-RBD-8xHis) encoding the His-labeled RBD was transformed using a heat-shock method. Specifically, 50 μL of DH5α competent cells (Invitrogen) were mixed with 5 ng of the plasmid, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and then incubated on ice for another 2 min. After adding 950 μL of pre-warmed 2% lysate broth (LB, Sigma, L3022), the cells were incubated at 37°C with orbital oscillation at 225 rpm for 1 h. Then, 100 μL of the transformed cells were seeded onto LB agar plates containing 100 μg / mL ampicillin (J&K Chemicals, 947040) and incubated overnight at 37°C. One colony was taken from each plate and incubated for 16 hours with shaking at 225 rpm at 37°C in 500 mL of LB / c ampicillin (100 μg / mL). Cells were collected by centrifugation at 3600 × g for 15 min, and the plasmid was purified using the Qiagen Plasmid Midiprep Kit according to the supplier's protocol. In one embodiment, 1 μg of plasmid was obtained from 200 mL of bacterial culture.

[0134] Step 2: Plasmid transformation After thawing competent cells on ice (previously stored at -80°C), 100 ng of plasmid was added to 100 μL of competent cells. Cells were placed on ice for 30 min, heat-shocked in a 42°C water bath for 90 s, and then incubated on ice again for 3 min. The mixture was added to 400 μL of fresh LB medium, and the mixture was shaken at 270 rpm for 1 h at 37°C, followed by centrifugation at 5000 × g for 30 s. After discarding the 400 μL supernatant, the remaining 100 μL of cell pellet was resuspended. The bacteria were inoculated onto LB agar plates containing appropriate antibiotics and incubated overnight at 37°C.

[0135] Step 3: Protein expression induction Origami B cells (Sigma; 70836) pre-transformed with the molecular chaperone plasmid pG-KJE8 (Takara Bio Inc.; 3340) were transformed using the RBD expression plasmid pET11a-RBD and incubated overnight on LB agar plates. At noon the following day, one colony was selected and added to 20 mL of fresh LB medium containing appropriate antibiotics. On the third day, 20 mL of the overnight culture was added to 400 mL of fresh LB medium and incubated until the OD600 reached approximately 0.5 (usually within about 3 hours). Induction was performed by adding 0.4 mL of induction buffer containing 1 mM isopropyl β-D-1-thiogalactoside (IPTG; J&K Scientific; 266729) and 4 mg / mL L-arabinose (TCI; A0515). Six hours later, cells were collected by centrifugation (4200×g, 15 minutes) for purification or stored at -80°C.

[0136] Step 4: Protein purification Origami B cells from the previous step were resuspended in 40 mL of protein purification buffer (20 mM Tris, 300 mM NaCl, 10% glycerol, pH 8.0) and lysed using an ultrasonic processor. After centrifuging the lysate at 15000 g for 10 min, the supernatant was collected, filtered through a 0.45 μm syringe filter, and incubated for 1 h at room temperature with 3 mL of Ni-NTA resin (Promega, V8821). Finally, the lysate was transferred (along with the resin) to a blank gravity column. After discarding the liquid stream from the lysate-resin mixture, 18 mL of wash buffer (replenished with 50 mM imidazole protein purification buffer) was added to elute non-specifically bound proteins. Finally, the RBD protein product was eluted by adding 18 mL of elution buffer (protein purification buffer supplemented with 300 mM imidazole), and dialyzed three times at 4 °C with storage buffer (20 mM Tris, 300 mM NaCl, 50% glycerol, pH 8.0). The purity and concentration of the protein product were measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the Bradford assay, respectively.

[0137] Preparation of spike-RBD coupled gold nanoparticles (Au@PEG-RBD NP) Using Turkevich's method 46Citrate-capped gold NPs (cit-AuNPs) with diameters of, for example, 20 nm were synthesized. After bringing 95 mL of HAuCl4 (0.267 mM) (Sigma) to a boil, 5 mL of sodium citrate (1% by weight / volume) (Alfa Aesar) was added under vigorous stirring, and the mixture was kept boiling for 15 minutes. The product was slowly cooled to room temperature (RT) to obtain 20 nm cit-AuNPs. Freshly dissolved thiol (HS)-PEG was added at a 1:1 molar ratio. 20k -Methoxy or HS-PEG 20k -N-N-triacetic acid (NTA) (Biochempeg) at per nm 2 The total concentration of 5 PEG molecules on the NP surface was added to a 20 nm cit-AuNPs solution, and the mixture was stirred overnight at room temperature. After adding 5 mL of NiCl2 (50 mM) (TCI), the mixture was stirred for 1 hour to obtain Au@PEG-NTA-Ni. 2+ NP. 100 μg of His-labeled RBD protein was added to Au@PEG-NTA-Ni 2+ The NP solution was stirred overnight at 4°C to obtain Au@PEG-RBD NP. The NP was dialyzed with Nanopure water by centrifugation and filtration, repeated 3 times (Thermo Fisher Scientific; MWCO 50k).

[0138] According to an embodiment of the present invention, the total size of the inhaled nanoparticles can be greater than 50 nm, and the core (cit-AuNP) can be in the range of 3 nm to 50 nm.

[0139] To prepare untargeted NPs as a control, 20nm cit-AuNPs were prepared only with HS-PEG. 20k The -methoxy group reacts, and then the mixture is purified by centrifugation and filtration as described above. Figure 14 As shown.

[0140] Physicochemical properties of NP Part 1: NP Size and Surface Charge In solution, according to Beer-Lambert's law and 20 nm AuNP at 450 nm (5.41 × 10⁻⁶), 8 M -1 cm -1The molar extinction coefficient at ) was used to determine the concentration of gold NP by ultraviolet-visible-near-infrared spectroscopy (UV-vis-NIR spectroscopy) (Agilent Technologies, Cary 5000). 47 Hydrodynamic diameter (HD) and zeta potential were measured using a DelsaMax PRO light scattering analyzer (Beckman Coulter). For HD measurements, NPs were suspended in freshly collected Nanopure water or bronchoalveolar lavage fluid (BALF) from Syrian hamsters. To test their colloidal stability, NPs were suspended in fresh BALF and incubated at 37°C for up to 24 hours before HD measurements. For zeta potential analysis, NPs were suspended in 1 mM KCl. Reported values ​​represent the mean ± standard deviation (SD) of three independent measurements, accompanied by histograms of NP size prepared from three different batches over three months to ensure batch-to-batch consistency and data reproducibility. AuNPs were visualized via TEM at 100 kV (Hitachi, H7700).

[0141] To characterize the morphology of the entire PEG-coated gold NP (Au core + PEG) and measure its physical diameter, negative staining of the NP was performed for TEM imaging. Briefly, 10 μL of NP solution was dropped onto a formvar / carbon-coated copper grid (200 mesh; Beijing Zhongjingkeyi Technology Co., Ltd.) treated with Harrick plasma and allowed to stand for 30 minutes. The NP droplet was then removed from the grid edge using filter paper. The NP solution was then diluted with Nanopure water to obtain the EM Stainer solution (Nisshin-EM).

[62] The solution was diluted 4-fold (336), and 10 μL of the diluted solution was added to each TEM grid and left for 8 minutes. (EM Stainer is an electronic staining alternative to uranyl acetate.) After removing the EM Stainer solution, the grids were dried at room temperature for 4 hours and then visualized in TEM at 30-50 kV magnification at 100 kV (Hitachi, H7700).

[0142] After atomization, the NPs were characterized in real time. The NP number concentration and aerodynamic particle size distribution were continuously monitored by a condensed particle counter (TSI, CPC3775) and a scanning mobility particle size analyzer (SMPS) (TSI, Classifier 3082), respectively.

[0143] Part 2: Loading of PEG chains, validation of ligand coupling, and ligand stability in biofluids The loading of PEG chains on AuNP was determined by thiol depletion. Specifically, Au@PEG-RBD NP was prepared using 1 mL of 2 nM cit-AuNP, and unreacted free PEG chains were collected, lyophilized, and resuspended in 60 μL of Nanopure water. Next, 20 μL of concentrated PEG sample was mixed with 100 μL of Ellman's assay buffer [1 mM EDTA (Sigma) dissolved in 0.1 mM Na2HPO4 (Sigma); pH=8), and further mixed with 50 μL of Ellman's detection buffer [0.5 mg / mL Ellman's reagent (5,5-dithiobis(2-nitrobenzoic acid) (J&K Scientific)) in the assay buffer]. After 10 minutes, the absorbance of the reaction mixture was read at 412 nm using a Multiskan GO UV absorbance microplate reader (Thermo Fisher Scientific). The concentration of PEG was calculated by subtracting the background absorbance of the sample from the background absorbance of the negative control, referring to the standard calibration curve. The reported data represent the mean ± standard deviation (SD) of three independent experiments.

[0144] To demonstrate the coupling of the spike RBD protein to the gold core, 1 mL of freshly prepared Au@PEG-RBD NP was centrifuged at 4°C (15000×g) for 30 min and resuspended in 1 mL of 10 mM Tris-HCl buffer (pH=8). Next, the NP was centrifuged again and resuspended in 1 mL of 1 M imidazole to release the bound protein. Western blot analysis was performed on the supernatant samples from each step to verify the release of the spike RBD protein from the Au@PEG NP (see reference). Figure 14 (See the diagram in the image). Furthermore, the number of coupled RBD proteins was determined using a customized ELISA.

[0145] To test the extent of ligand dissociation in BALF, newly synthesized Au@PEG-RBD NPs were centrifuged at 4°C (15000×g) for 30 min and resuspended in 1 mL of BALF extracted from healthy Syrian hamsters. After incubation at 37°C for 48 hours, the mixture was centrifuged and resuspended in 1 mL of 1M imidazole to release the bound protein. Finally, the NP solution was centrifuged to separate the isolated protein and gold NPs. The NP-treated BALF before imidazole addition and the supernatant from each subsequent centrifugation step after imidazole addition were subjected to Western blot analysis and our internal ELISA analysis to analyze ligand stability in BALF (see reference). Figures 20A to 20C (Diagram in the diagram).

[0146] Part 3: Binding of NP to recombinant RBD receptor (modified ELISA) Following the published method [Yang et al, small, 2016], 0.1 mL of recombinant ACE2 (10108-H08H-B), L-SIGN (10559-H01H), DC-SIGN (10200-H01H), mannose receptor (16065-H08H1), or MGL protein (10821-H01H) (10 μg / mL; Sino Biological) was added to a 96-well ELISA plate and incubated overnight at 4°C. After washing three times with washing buffer (0.25 mL, 0.05% Tween 20 in PBS), 0.2 mL of blocking buffer (5% milk in PBS) was added to each well, and the plate was shaken for 1 hour. After washing, 0.1 mL of NP (0.2 nM) prepared in dilution buffer was added to the well. After incubation at room temperature for 2 hours, the wells were washed three times. Finally, 0.1 mL of aqua regia was added to the wells, and the amount of gold bound in the acid solution was quantified by ICP-MS.

[0147] For ligand competition detection, we used a similar modified ELISA. 100 μL of 10 μg / mL recombinant spike receptor protein (e.g., ACE2, Sino Biological, 10108-H02H) or L-SIGN (Sino Biological, 10559-H01H) prepared in dilution buffer (e.g., 20 mM Tris, 140 mM NaCl, 10% glycerol, pH=8) was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing three times with wash buffer (e.g., 0.25 mL, 0.05% Tween 20 phosphate-buffered saline (PBS) solution), 0.2 mL of blocking buffer (e.g., 5% milk PBS solution) was added to each well, and the plate was shaken for 1 hour. After three washes, 0.2 mL of dilution buffer containing 3 nM NP and different concentrations (e.g., up to 8 μM) of free recombinant spike RBD protein was added, and the mixture was incubated at room temperature for 2 hours. After three washes, 0.1 mL of aqua regia was added, and the gold content in the solution was quantified by ICP-MS.

[0148] Part 4: Nickel Ion Leakage in Au@PEG-RBD NP Freshly synthesized Au@PEG-RBD NP product was collected from a 2 mL dialysis bag to track nickel concentration in the solvent. Briefly, 1 mL of the dialysis product was centrifuged at 8000 × g for 10 min to collect the supernatant for ICP-MS analysis. Another 1 mL of the dialysis product was stored at 4°C. After one month, the NP solution was centrifuged at 8000 × g for 10 min, and the supernatant was collected for ICP-MS analysis. The nickel concentrations of the two samples were compared to detect potential nickel leakage.

[0149] Part 5: Cytotoxicity of NP A549 alveolar epithelial carcinoma cells (ATCC; CCL-185) and HEK293 cells (ATCC, CRL-1573) were incubated in complete Dulbecco modified Eagle medium [DEM supplemented with 10% fetal bovine serum (Gibco, A5256701) and 1% penicillin / streptomycin (Gibco, 15070063)]. BEAS-2B cells (non-cancerous bronchial epithelial cells; ATCC, CRL-3588) were incubated in LHC-8 medium (Gibco, 12678017). Cells were seeded at a density of 1000 cells / well in 96-well plates. After 12 hours, the medium was replaced with fresh medium containing varying concentrations of Au@PEG-methoxy and Au@PEG-RBD NP (up to 6 nM, 100 μL / well) and incubated for another three days. After two PBS washes, cell viability was measured using an AlamarBlue assay (Invitrogen, DAL1025) according to the manufacturer’s protocol.

[0150] Protein crown composition of NP Part 1: Collection of BALF proteins adsorbed onto NPs In one embodiment, 0.1 mL of concentrated NP (300 nM) was incubated for 30 min at 37 °C in 0.9 mL of fresh BALF collected from male Syrian hamsters (8 weeks old). Protein-adsorbed NP was washed by three rounds of centrifugation (13500 rpm at 4 °C) to remove unbound proteins, and the NP was resuspended in PBS containing 0.05% v / v Tween 20 (Sigma). An equal volume of NP-free BALF was used as a control for nonspecific protein adsorption. After centrifugation of the washed NP, the supernatant was discarded, and 25 μL of the remaining NP particles were treated with 10 μL of Laemmli sample buffer (4×) (Bio-Rad) and 5 μL of 4.8 M dithiothreitol (J&K Chemicals; 415951). After incubation at 70°C for 1 hour and shaking to release the adsorbed protein, NP was removed by centrifugation at 13,500 rpm at 4°C. The supernatant containing the desorbed protein was then characterized, purified, and quantified by PAGE according to literature precedent.

[0151] Part 2: PAGE 0.5 μL of mercaptoethanol (Sigma) was added to 40 μL of desorbed protein in Laemmli sample buffer. After heating the mixture at 95 °C and shaking for 5 min, 6 μL of denatured protein and 6 μL of PrecisionPlus protein dual-color standard (Bio-Rad) were loaded into a 4-20% Mini-PROTEAN TGX unstained gel (Bio-Rad) in triglycine SDS electrophoresis buffer (Bio-Rad). After separation by electrophoresis at 200 V for 40 min, the bands were visualized using a ChemiDoc Touch gel imaging system (Bio-Rad).

[0152] Part 3: Tag-free proteomics analysis Liquid chromatography-tandem mass spectrometry (LC-MS / MS): All reagents used were chromatographic grade (Thermo Fisher Scientific). Extracted peptides were lyophilized and resuspended in 10 μL of 0.1% formic acid for separation using an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) equipped with a self-made 150 μm × 15 cm column packed with Acclaim PepMap RPLC C18 (1.9 μm, 100 Å, Dr. Maisch GmbH, Germany). An organic gradient was driven over 120 minutes at a flow rate of 600 nL / min using buffer A (0.1% formic acid aqueous solution) and buffer B (20% 0.1% formic acid aqueous solution and 80% acetonitrile). The gradient elution program was as follows: 4% to 8% B for 3 minutes; 8% to 28% B for 86 minutes; 28% to 40% B for 20 minutes; 40% to 95% B for 1 minute; and 95% to 95% B for 10 minutes. The eluted peptides were directly sprayed into the mass spectrometer. Ten MS / MS data-dependent scans and one high-resolution (60,000 m / z at 400 m / z) full-scan mass spectra were acquired simultaneously to provide the amino acid sequence and mass-to-charge ratio of the selected peptide ions.

[0153] Cellular uptake Part 1: ICP-MS Before transfection, HEK293 cells were stored at 0.5 × 10⁶ cells per well. 6 Cells were seeded at a density of 1000 cells / well in confocal culture dishes or 24-well plates for 12 hours. Next, 0.3 mL of transfection medium was added to each well. Each well contained 1.5 μL of Lipofectamine 2000 (Invitrogen) and 500 ng of plasmid DNA (pcDNA3.1-ACE2-GFP, pCMV6-L-SIGN-GFP, or pCMV-CD81-GFP) prepared in OptiMEM. After 12 hours of transfection, the transfection medium was replaced with DMEM supplemented with 10% FBS and 1% PS. Cells were then incubated with 2 mL of 0.2 nM NP (in complete medium) for 2 hours. After washing twice with phosphate-buffered saline (PBS), cells were harvested by adding 0.5 mL of aqua regia to each well. The dissolved contents were diluted 10 times with Nanopure water, filtered through a 0.2 μm filter, and then measured by ICP-MS (Agilent 7900).

[0154] Part 2: Time-dependent cellular uptake As described in Part 1, after transfection with pcDNA3.1-ACE2-GFP or pCMV6-L-SIGN-GFP, cells were incubated with 2 mL of 0.2 nM NP (in complete medium) for varying times, up to 24 hours. After washing twice with PBS, cells were harvested by adding 0.5 mL of aqua regia to the wells. The dissolved contents were diluted 10-fold with Nanopure water, filtered through a 0.2 μm filter, and then measured by ICP-MS (Agilent 7900).

[0155] Part 3: Confocal Microscopy HEK293 cells (ATCC) were transfected with human ACE2-GFP (Addgene, 154962), pCMV6-L-SIGN-GFP (OriGene; RG226741), or mPA-GFP-CD81-10 (Addgene; 57124) using Lipofectamine 2000 to overexpress the aforementioned fluorescent proteins. Next, the cells were incubated for 2 hours with 2 mL of 0.2 nM Cy5.5-labeled NP (in complete medium). After washing twice with phosphate-buffered saline (PBS), the cells were fixed with 1 mL of 4% paraformaldehyde for 20 minutes and stained with 1 mL of 1 μg / mL DAPI (4',6-diamidinyl-2-phenylindole; Thermo Fisher Scientific) for 10 minutes at room temperature for imaging under a Leica SP8 confocal microscope. The excitation wavelengths for DAPI, GFP, and Cy5.5 were 405 nm, 488 nm, and 651 nm, respectively. The emission wavelength ranges of DAPI, GFP, and Cy5.5 are 415nm-500nm, 500nm-520nm, and 670nm-790nm, respectively.

[0156] Animal models of acute respiratory distress syndrome (ARDS) All procedures followed the guidelines set forth by the Animal Ethics Committee (AEEC) of the Chinese University of Hong Kong. Male Syrian hamsters aged 8 to 12 weeks were randomly assigned to different treatment groups and housed in a temperature- and humidity-controlled environment at the Laboratory Animal Services Centre (LASEC) with a 12-hour light / dark cycle. Before establishing an acute respiratory distress syndrome (ARDS) model, the hamsters were anesthetized by intraperitoneal injection of approximately 0.5 mL of ketamine / toluidine (150 mg / kg body weight and 10 mg / kg body weight, respectively). For the lipopolysaccharide (LPS)-induced ARDS model, 120 μL of 12.5 mg / mL LPS (Santa Cruz Biotechnology; sc-221855B) was intranasally injected into ~150 g hamsters (10 mg LPS / kg body weight). For the HCl-induced ARDS model, 120 μL of HCl (pH=2) was intranasally injected into ~150 g hamsters (800 μL / kg body weight). 48 After induction, hamsters were monitored for respiration and heart rate for 10 minutes, then returned to their cages for 12 hours until the inhalation study was conducted. Food and water were provided to the hamsters when they were not placed in the inhalation chamber.

[0157] Customized ELISA for detecting spike RBD protein and spike receptor The following plan is adapted from our past work.

[21] Synthetic recombinant RBD proteins or spike receptors [ACE2 (Sino Biological, 10108-H02H), L-SIGN (Sino Biological, 10559-H01H), DC-SIGN (Sino Biological, 10200-H01H), mannose receptor (LS Bio, LS-G20979-20) or MGL (Sino Biological, 10821-H01H) proteins] or samples (all prepared in diluted buffer (20 mM Tris, 140 mM NaCl, 10% glycerol, pH=8))] were added to 96-well ELISA plates and incubated overnight at 4°C. After rinsing three times with washing buffer [0.25 mL, 0.05% Tween 20 phosphate-buffered saline (PBS)], add 0.2 mL of blocking buffer (5% milk PBS) to each well and shake the plate for 1 hour. After rinsing, 100 μL of dilution buffer containing primary antibodies [2 µg / mL for RBD (R&D, MAB10540); 2 µg / mL for ACE2 (Proteintentech, 66699-1-Ig); 40 µg / mL for DC-SIGN (Invitrogen, MA5-35828); 20 µg / mL for L-SIGN (Invitrogen, PA5-68454); 5 µg / mL for mannose receptor (Invitrogen, PA5-46994); 3 µg / mL for MGL (Invitrogen, PA5-82781)] was added to each well and incubated at room temperature for 1 hour. After rinsing, 100 μL of horseradish peroxidase (HRP)-conjugated secondary antibody (1 μg / mL dilution buffer; Invitrogen, 31460) was incubated at room temperature for 1 hour. Following washing, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB; Bethyl, E102) was added to the wells and incubated at room temperature in the dark for 15 minutes. Finally, 100 μL of 2N H₂SO₄ was added to stop the reaction. Absorbance was read at 450 nm and 570 nm using a microplate reader.

[0158] NP in vitro binding with lungs The hamster was euthanized and placed on the operating table. After making a 2cm horizontal incision in the skin at the neck, the fascia above the mandibular gland and muscles was dissected. After further removing the mandibular gland and muscles to expose the trachea, a small incision was made at the upper opening of the trachea. Then, a soft rubber tube [modified from BD Vacutainer by removing the metal needle at the end] was inserted. ® [A Safety-Lok™ lancet (BD; 367292)] is inserted into the trachea. After sutures are used to secure the rubber tubing to the trachea, 2 mL of a 0.2 nM NP aqueous solution is injected into the lungs through the tube. After incubation for 30 minutes, the NP is aspirated, and the lungs are washed three times with 2 mL of PBS (using injection and aspiration cycles). Finally, the lungs are dissected and immersed in aqua regia for ICP / MS analysis or 4% PFA for tissue sections.

[0159] NP inhaled into the lungs Below, we will use "100%" to represent the lung gold content when targeting Au@PEG-RBD NP achieves "complete receptor targeting" without any receptor blockade; and "0%" to represent the lung gold content when non-targeting Au@PEG-methoxy NP achieves "receptor-free targeting".

[0160] Part 1: Binding to the lungs via L-SIGN and ACE2 according to Figure 2A The dosage regimen shown involved LPS-induced ARDS hamsters inhaling NP for 2 hours, followed by immediate sacrifice or return to their cages for 10 hours, a process that continued until the third inhalation. To determine whether Au@PEG-RBD NP specifically binds to the lungs in vivo via L-SIGN or ACE2, and to assess the relative contribution of each spike receptor to pulmonary delivery, hamsters received an additional antibody blocking step before each round of NP inhalation. At the designated endpoint time point, hamsters were anesthetized 1 hour before inhaling NP by intraperitoneal injection of saline containing ketamine (150 mg / kg hamster) and toluenethiazide (10 mg / kg hamster), (ii) by intranasal injection of 100 μL of PBS containing primary antibodies against L-SIGN (20 μg / mL; Invitrogen, PA5-68454) or ACE2 (20 μg / mL; Proteintech, 66699-1-Ig), and returned to their cages for 1 hour, (iii) by inhalation of 6 nM Au@PEG-RBD NP for 2 hours, and (iv) by final sacrifice for lung dissection. The significant reduction in lung gold content (measured by ICP-MS) indicates that Au@PEG-RBD NP specifically binds to L-SIGN or ACE2, and that spike receptors play a prominent role in mediating lung accumulation.

[0161]

[0162] Part 2: The Relationship Between L-SIGN and ACE2 in Mediating Lung Delivery To assess the additive or synergistic effects of the two spike receptors in pulmonary delivery, we evaluated the effect of a single dose of Au@PEG-RBD NP on pulmonary accumulation at different stages of ARDS pathogenesis. Prior to NP inhalation, LPS-induced ARDS hamsters received single-antibody blockade of L-SIGN, single-antibody blockade of ACE2, or double-antibody blockade of both receptors. After anesthesia, hamsters were intranasally injected with 100 μL of PBS containing anti-L-SIGN (20 μg / mL), ACE2 (20 μg / mL), or both antibodies for 1 hour. Other inhalation and animal sacrifice procedures were the same. The combined contribution (%) of the two receptors was calculated using the following formula. If the combined contribution of the two receptors equals the sum of the contributions of each receptor, the two receptors additively mediate NP delivery to the lungs. If the combined contribution of the two receptors exceeds the sum of the contributions of each receptor, the two receptors synergistically mediate NP delivery.

[0163] [Gold content (Au@PEG-RBD NP) – Gold content (Au@PEG-RBD NP with dual blocking)] ÷ [Gold content (Au@PEG-RBD NP) – Gold content (Au@PEG-methoxy NP)] × 100% NP (6 nM in Nanopure water) was atomized by an aerosol generator (Classifier 3076, TSI) using compressed air (L / min) flowing at a rate of 3 L / min. The NP in the air mixed with the compressed air was passed through a diffusion dryer (TSI, 3062-NC) at a flow rate of 3 L / min. Syrian hamsters (n=3) placed in an airtight whole-body inhalation chamber (Scivena Technologies, RES644) were exposed to a constant concentration of 9 × 10⁻⁶ NM. 6 NP / cm 3 The hamsters were inhaled in the air for 2 hours. Excess airborne NP was removed using an aerosol filter (Bio-Gene Technologies), and the filter outlet to exhaust pipe (0.19 inch inner diameter, TSI) was immersed in water. The flow rates at the inlet (3.0 L / min) and outlet (>2.9 L / min) of the chamber were continuously monitored using a mass flow meter (TSI4, 140) to ensure limited leakage of airborne NP. After inhalation, the hamsters were immediately euthanized or returned to their cages for further study. The hamsters were euthanized with excess CO2 to collect BALF, lavage lung tissue, and other organs. BALF was extracted in situ by infusing the entire lung three times with 0.5 ml of PBS.

[0164] In vivo blockade of RBD One hour before inhalation, ARDS hamsters were anesthetized by intraperitoneal injection of ketamine / toluidine (150 mg / kg body weight and 10 mg / kg body weight, respectively). After intranasal injection of 100 μL of primary antibody against L-SIGN (20 μg / mL; Invitrogen, PA5-68454) or ACE2 (2 μg / mL; Proteintech, 66699-1-Ig), the hamsters were returned to their cages for one hour, followed by inhalation of 6 nM NP for two hours. The reduction in lung gold content indicated that RBD blocked L-SIGN or ACE2.

[0165] Immunohistochemistry Tissues were fixed in 10% buffered formalin (3.7% w / v) for 48 hours and stored in PBS (0.1 M, pH 7.5) at 4°C. The fixed tissues were dehydrated in ethanol, cleaned in xylene, and embedded in paraffin blocks. Paraffin-embedded tissue sections (4 μm) were cut and mounted in Superfrost Plus. TMAdhesion onto microscope slides (Thermo Scientific). IHC staining on paraffin sections with antigen retrieval. Dewaxing and rehydration of tissue sections. After placing the slides in citrate buffer (10 mM citric acid, pH=6), microwave the slides at high power (approximately 95-100°C) for 3 minutes, then at low power for 20 minutes. After cooling the slides in the heated solution for 30 minutes, rinse them twice with distilled water and then rinse in PBS for 5 minutes. The slides were blocked with 2.5% normal horse serum (Vector Laboratories) for 2 hours and incubated overnight at 4°C with 60 μL of primary antibody [for ACE2 (Proteintech, 66699-1-Ig), 2 µg / mL; for DC-SIGN (Invitrogen, MA5-35828), 40 µg / mL; for L-SIGN (Invitrogen, PA5-68454), 20 µg / mL; for mannose receptor (Invitrogen, PA5-46994), 5 µg / mL; for MGL (Invitrogen, PAA-82781), 3 µg / mL]. Wash the slides in PBS, treat with 3% H2O2 (Merck Millipore) for 30 minutes, rinse, and incubate with 50 μL of secondary antibody (ImmPRESS HRP polymer assay kit, Vector Laboratories) for 30 minutes. Develop sequentially with 3,3'-diaminobenzidine (DAB) enzyme substrate (ImmPACT™ DAB, Vector Laboratories) for 2 minutes each time. Counterstain the slides with Mayer hematoxylin for 3 minutes, wash in distilled water, dry in 90% ethanol, and mount with xylene-based mounting medium (DPX Mountant; Sigma, 06522). Take bright-field images using a Nikon Eclipse Ni (DS-Ri2) microscope.

[0166] ICP-MS determination of organ-level distribution Organs were removed from hamsters, chopped, and thoroughly digested overnight in 1 mL of aqua regia at room temperature. Blood, BALF supernatant, or BALF cell pellet (0.3 mL) were digested in 0.25 mL of aqua regia. The lysates were diluted to 2% HNO3 by adding Nanopure water containing 10 ppb indium as an internal standard, and then passed through a 0.2 μm hydrophilic syringe filter for ICP-MS measurement (Agilent 7900).

[0167] Organizational Level Distribution Part 1: Silver-enhanced staining Lung tissue sections embedded in paraffin (4 μm thick) were dewaxed in xylene (3 times, 5 min each) and rehydrated with a series of ethanol solutions (100%, 90%, 70%; 2 times, 3 min each) and Nanopure water (5 times, 5 min each). The rehydrated tissue sections were stained with a silver enhancement kit for light and electron microscopy (Ted Pella). The silver enhancement solution, solution A (silver salt) and solution B (initiator), was mixed in a 1:1 ratio before use. One drop of the mixture (~50 μL) was applied to the tissue sections for 20 minutes under normal laboratory illumination. Next, the tissue sections were rinsed with Nanopure water (3 times, 3 min each) and counterstained for 10 minutes with Mayer's hematoxylin (blue-violet nuclear stain; Vector Laboratories) or methyl green (blue-green nuclear stain; Vector Laboratories). Bright-field images were acquired using a Nikon Eclipse Ni (DS-Ri2) microscope.

[0168] Part 2: Confocal Reflectance Microscopy Using a Leica SP8 confocal microscope, at 488 nm excitation and with a 20× objective in reflective mode, confocal reflectance images of tissue sections overlaid with true-color images were obtained. The true-color images were generated by superimposing the red, green, and blue (RGB) channels in transmitted light imaging mode.

[0169] Cellular distribution under TEM Tissue blocks (approximately 1 mm from the right caudate lobe) were fixed using 2.5% glutaraldehyde phosphate buffer (pH 7.2-7.4). 3 Or granular BALF cells were collected for 2 hours and stained with 1% osmium tetroxide for 2 hours. The blocks were gradually dehydrated in a gradually increasing ethanol gradient and propylene oxide, embedded in Epon 812 resin (Electron Microscopy Sciences; EMS), and polymerized at 55°C for 48 hours. Ultrathin sections with a thickness of ~70 nm were deposited on a 200-mesh copper grid (EMS), stained with 4% uranyl acetate (EMS, in 50% methanol / water) and Reynolds lead citrate (Sigma) for observation under TEM at a beam voltage of 100 kV (Hitachi, H7700).

[0170] Anti-ARDS efficacy The LPS-induced or HCl-induced ARDS hamster models were randomly divided into 6 groups [untreated group, Au@PEG-methoxy NP (~9×10⁻⁶)]. 6 NP / cm 3 (Inhalation), Au@PEG-RBD NP (~9×10) 6 NP / cm 3 Before inhalation of Au@PEG-RBD NP (intranasal injection; 200 nM, 200 μL), L-SIGN antibody or ACE2 antibody (intranasal injection; 20 μg / mL, 150 μL), or hydrocortisone (5 mg / kg; SinoPharm, H20023069; intraperitoneal injection) were administered. Hamsters in each group received their respective treatments 12 or 36 hours after induction, while untreated hamsters inhaled compressed air. Typically, BALF supernatant, BALF cells, and lavage lung tissue were extracted from hamsters 2 hours after administration to study anti-ARDS efficacy at different time points (i.e., after the first, second, or third administration). Having established the superior efficacy of Au@PEG-RBD NP in previous studies, we also used free PEG-RBD conjugate as a non-treatment control (inhalation, 2 μM in PBS) to ensure that the effect could be specifically attributed to Au@PEG-RBD NP.

[0171] Part 1: Cytokines in BALF Supernatant and Irrigated Lung Tissue According to the manufacturer's protocol, cytokine (IL-6, IL-8, TNF-α, and IFN-γ) levels in BALF supernatant were determined by ELISA (IL-6: FineTest, EHA0006; IL-8: Krishgen Biosystems, KLH0059; TNF-α: FineTest, EHA0004 and IFN-α: FineTest, EHA0005; IL-4: FineTest, EHA0001; IL-10: FineTest, EHA0008). Cytokine levels in the lavaged lung tissue were determined by Western blotting. 20 μg of lavaged lung tissue was added to a 10% denaturing PAGE gel (BioRad) for electrophoresis, and the gel was then transferred to a polyvinylidene fluoride membrane (BioRad) on ice at 100V for 2 hours. After blocking in 5% BSA (Rockland) Tris-buffered saline-Tween (TBST) buffer for 1 hour, the blot was incubated overnight at 4°C with primary antibodies against IL-6 (Invitrogen, 701028), IL-8 (Invitrogen, AHC0881), TNF-α (Invitrogen, PA1-40281), or IFN-γ (Invitrogen, MM700B). After incubation for 1 hour with 1 μg / mL of anti-rabbit goat secondary antibody [conjugated with horseradish peroxidase (HRP)] (Bio-Rad, 1706515) (diluted in TBST containing 5% skim milk), the membrane was treated with Clarity™ Western ECL substrate (Bio-Rad), and protein bands were visualized using a ChemiDoc Touch imaging system (Bio-Rad).

[0172] Part 2: Cytokines in the lavaged lung tissue Lung tissue was collected after lavage, and cytokine levels were measured by Western blotting. 20 μg of lavage tissue was added to a 10% denaturing PAGE gel (Bio-Rad) for electrophoresis, and then the gel was transferred to a polyvinylidene fluoride membrane (Thermo Fisher) for 10 minutes using a Power-blotter semi-dry transfer system (Thermo Fisher) at 25 V, 4 A. After blocking in 5% BSA (Rockland) Tris-buffered saline-Tween (TBST) buffer for 1 hour, the blot was incubated overnight at 4°C with primary antibodies against IL-6 (Invitrogen, 701028, 0.1 µg / mL), IL-8 (Invitrogen, AHC0881, 0.1 µg / mL), TNF-α (Invitrogen, PA1-40281, 0.1 µg / mL), IFN-γ (Invitrogen, MM700B, 2 µg / mL), IL-4 (Invitrogen, PA5-115416, 1 µg / mL) or IL-10 (Invitrogen, PAA-95561, 1 µg / mL) diluted in TBST containing 5% BSA. After incubation for 1 hour with 1 μg / mL of anti-rabbit goat secondary antibody [conjugated with HRP] (Bio-Rad, 1706515) (diluted in TBST containing 5% skim milk), the membrane was treated with Clarity™ Western ECL substrate (Bio-Rad, 1705060), and the bands were developed using a ChemiDoc Touch imaging system (Bio-Rad).

[0173] Part 3: Histology Tissue sections containing the central and lateral portions of the lung were scored primarily based on the following criteria [Xiong et al, AHM, 2018]: (1) edema and hemorrhage; (2) alveolar septal congestion; (3) alveolar septal / luminal cellular infiltration; and (4) alveolar septal necrosis / cellular debris. Table 7 lists the scoring criteria. The feature of "hyaline membrane formation" was not included in the calculation of lung injury scores because, consistent with prior literature, no hyaline membrane was found in the tissue [Aeffner et al, Toxicologic Pathology, 2015].

[0174] Part 4: Measurement of Reactive Oxygen Species (ROS) Freshly extracted BALF cells were resuspended in PBS, counted using trypan blue, and seeded in 96-well plates at a volume of 100 μL per well (~3000 cells / well). Using a cell ROS detection kit (deep red fluorescence) from Abcam (ab186029), 100 μL of ROS working solution was added to each well, and the plates were incubated at 37°C for 30 minutes. The fluorescence signal of each well was read using a microplate reader. The excitation and emission wavelengths for deep red fluorescence were 650 nm and 675 nm, respectively.

[0175] Part 5: Immune cell counts in BALF Freshly extracted BALF cells were resuspended in 1 mL of PBS containing 2 mg / mL EDTA. The total number of BALF cells was counted on a hemocytometer slide using trypan blue. To classify and count different BALF cell types, the extracted cells were resuspended in 50 μL of PBS containing 2 mg / mL EDTA, placed on a slide to prepare a cell smear, stained with the Wright-Giemsa staining kit (Yeasen, 60529ES01) according to the manufacturer's instructions, and analyzed using an optical microscope based on cell morphology.

[67] .

[0176] Part 6: qRT-PCR and Western blot analysis of macrophage markers RNA was isolated using RNAiso Plus (Takara, #9108) and reverse transcribed to produce cDNA using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, K1622). qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) using the TB SYBR Green Premix Ex Taq Kit (Takara, RR82WR) according to the manufacturer's instructions. Transcriptional levels were analyzed using the ΔΔCT method and normalized to housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gene expression levels were quantified using pre-designed primers purchased from Shanghai Rui Mian Bio-Tech. For Western blotting, BALF cell pellets were lysed by adding 50 μL of Laemmli Sample Buffer (1×) (Bio-Rad, 1610747) for XX minutes. Electrophoresis and blotting procedures were the same as described above. The main antibodies are: for the M1 phenotype, CD86 (1 μg / mL, Thermo Fisher Scientific, MA5-35211) and CD80 (1 μg / mL, Abcam, ab254579); for the M2 phenotype, CD206 (2 μg / mL, Proteintech, 60143-1-Ig) and arginase-1 (2 μg / mL, Thermo Fisher Scientific, MA5-56598).

[0177] Part 7: Whole-body plethysmography for lung function measurement The hamsters were placed in transparent plexiglass full-body volume recording chambers (one animal per chamber; diameter 19.1cm; height 14cm; volume 4014.83cm). 3 Data Sciences, where an airflow of 2.5 L / min is provided by a deflection generator, and food and water are readily available. 。Each chamber is equipped with a Validyne pressure sensor (600-900 mm Hg), a temperature sensor (0-100°C), and a humidity sensor (0-100%). All channel signals from the chambers are collected using an ACQ7700 carrier wave and a UniversalXE signal conditioner connected to a Micro 1401 data acquisition unit (Cambridge Electronic Design). The signals are then acquired and analyzed using Spike 2.

[0178] Mechanism of action of gold NP in treating ARDS Part 1: Changes in protein expression Differentially expressed proteins (DEPs) were identified by comparing ARDS hamsters treated with Au@PEG-methoxyNP with untreated ARDS hamsters. During DEP screening, proteins with a fold change ≥2 and P < 0.05 were considered upregulated, while those with a fold change ≤ 1 / 2 and P < 0.05 were considered downregulated. Pathway mapping of DEPs was performed using the Kyoto Genome and Encyclopedia of Genomes (KEGG) database (http: / / www.genome.jp / kegg / ), and the distribution of proteomics data in biological processes (BP), cellular components (CC), and molecular functions (MF) was obtained by mapping to the Gene Ontology database (https: / / geneontology.org).

[0179] Part 2: Kinase Profile After verifying the aqueous solution (20 μM; 100×) of the stock solution by ICP-MS, Au 20 @PEG 5000 -Methoxy NPs were transferred to anhydrous dimethyl sulfoxide (DMSO) via dialysis and sent to SelectScreen Kinase Analysis Services (Thermo Fisher Scientific) for testing the activity of a 250-kinase library using the Z'LYTE biochemical assay. Specifically, Z'LYTE uses a fluorescence-based conjugate formulation based on the different sensitivities of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. NP samples were screened in wells at a single concentration of 200 nM (1×) in 1% DMSO (final). The IC50 values ​​were calculated for the selected highly potent kinases (with at least 80% inhibitory activity). 50To construct dose-response curves, NP samples were serially diluted 3-fold at 10 points, starting with an initial NP concentration of 200 nM (1×). ATP concentrations were used for detection according to the assay method, as shown in the corresponding table. For the "direct method," which acts through phosphorylation and activation of the synthetic peptide substrate of a given kinase, [ATP] was set to its previously determined apparent K value by Z'LYTE assay. m Value (K) m，表观 For a “cascade” approach where action is achieved through phosphorylation and activation of an inactive downstream kinase of a given kinase, [ATP] is set to 100 μM.

[0180] Part 3: Analysis of Kinase Profiling Data The percentage of phosphorylation (%Pho) was determined by referring to the 0% phosphorylation (or 100% inhibition) control (which contained no ATP and therefore did not show kinase activity) and the 100% phosphorylation control (which contained a phosphorylated peptide with the same sequence as the peptide substrate). Control wells did not contain any kinase inhibitors. The percentage of inhibition was calculated using the following equation: %inhibition = [1 - %phosphorylation NP / %phosphorylation 0% inhibition control] × 100, where the 0% inhibition control contained active kinases. IC50 values ​​were fitted from the dose-response curves based on XLfit model number 205 using IDBS.

[0181] Part 4: In vitro validation of proteomics and kinaseomics data Non-cancerous BEAS-2B bronchial epithelial cells were cultured at a rate of 3 × 10⁶ cells per culture dish. 6Cells were seeded at a density of 10 μL in 10 cm culture dishes and supplemented with 10 mL of complete DMEM. At ~80% confluence, the medium was replaced with fresh complete DMEM and supplemented with: (i) 200 μL of PBS, (ii) 200 μL of PBS containing 1 mg / mL LPS, (iii) 200 μL of PBS containing 1 mg / mL LPS + 10 nM Au@PEG-methoxyNP, or (iv) a physical mixture of 2 mg / mL LPS (in 100 μL of PBS) and 50 μM VX-702 (in 100 μL of DMSO; MCE, HY-10401). After 6 hours of incubation, cells were washed three times with PBS according to the manufacturer's protocol and harvested using a nuclear / cytosol separation kit (Abcam, ab289882). The levels of Elavl1, p-p38α, and total p38α were determined by Western blotting. Incubate the blot with primary antibodies against Elavl1 (Proteintech, 11910-1-AP, 0.5 µg / mL), p-p38α (Invitrogen, MA5-15177, 0.1 µg / mL), total p38α (Cell Signaling, 9218, 0.1 µg / mL), or β-tubulin (Abcam, ab108342, 0.1 µg / mL). For secondary antibodies and blot imaging, please refer to the above protocol.

[0182] Biocompatibility Part 1: Immunogenicity After three rounds of Au@PEG-RBD NP inhalation, hamsters were returned to their cages for 1, 14, or 28 days. Serum or BALF was collected and centrifuged at 4000 rpm for 10 minutes. 0.1 mL of the supernatant was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. A commercially expressed mammalian RBD protein (SinoBiological; 40592-V08H) was used as a positive control. After washing three times with 0.25 mL of wash buffer (0.05% Tween 20 PBS), the plate was incubated with 0.2 mL of blocking buffer (5% milk PBS) with shaking for 1 hour. After rinsing, 0.1 mL of 0.4 μg / mL hamster-specific HRP-conjugated IgA and IgG antibodies (Brookwood Biomedical, sab3003A and sab3003G) was added to the wells and incubated at room temperature for 2 hours. After three washes, 0.2 mL of TMB (3,3',5,5'-tetramethylbenzidine; Thermo Scientific; 34022) substrate was added and incubated for 30 minutes. The absorbance at 450 nm was then measured using a microplate reader.

[0183] Part 2: Long-term toxicity After three consecutive days of NP inhalation, ARDS-induced hamsters were returned to their cages for one, six, or twelve months. Upon euthanasia, fresh blood samples were sent to the PathLab clinic (Kowloon, Hong Kong) for hematological and biochemical parameter testing. Major internal organs were dissected, and the remaining gold content was determined by ICP-MS. Histological examination was also performed to assess morphological changes in the tissues.

[0184] Data processing and statistical analysis Prism (GraphPad software) and ImageJ were used for data analysis and graph construction. Statistical analyses are indicated below the charts. The Shapiro-Wilk test verified the normality of the mean sampling distribution. Bartlett's test verified the homogeneity of variance. Results are in... P A value <0.05 is considered significant.

[0185] result Example 1 - Au@PEG-RBD NP and its binding with activated ACE2 and L-SIGN We tracked the expression of six spike receptors in the lungs of LPS-induced ARDS (a mimicking bacterial infection, the most common cause of disease) in hamsters using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). Four receptors [dendritic cell-SIGN, mannose receptor, macrophage galactose C-type lectin (MGL), and transmembrane serine protease 2 (TMPRSS2)] were at healthy basal levels (see reference). Figures 7 to 12 Both receptors are transiently activated within 48 hours after ARDS induction. L-SIGN expression in alveolar epithelial cells surges 12 hours after induction, then returns to baseline levels after 24 hours. ACE2 expression increases 36 hours after induction, then returns to baseline levels after 12 hours (see reference). Figure 1A and Figure 1B This temporally dissociated activation of the two receptors mediated pulmonary delivery of Au@PEG-RBD NP at different time points following ARDS onset.

[0186] To prepare Au@PEG-RBD NPs, we reacted a 20 nm citrate-terminated gold core with approximately 20 kDa thiol-PEG-methoxy and thiol-PEG-nitrotriacetic acid (NTA) in a 1:1 molar ratio via gold-sulfur bonds, and then used Ni 2+ Ions act as chelating agents to link recombinant histidine (His8)-labeled RBD proteins (see reference). Figure 1C (and Figure 13). We used imidazole (competing with His8) to release RBD protein from Au@PEG-RBD NP and blotted with both anti-His antibody and anti-RBD antibody (see Figure 13). Figure 14 The connection of the RBD was verified. As measured by dynamic light scattering, the hydrodynamic size of the Au@PEG-RBD NP was approximately 94 nm, the polydispersity index was approximately 0.05, and the zeta potential was -15 mV; the hydrodynamic particle size distribution was consistent across multiple batches (see reference). Figure 15 According to Elman assay, NP has approximately 600 PEG chains and approximately 300 RBD proteins (see Table 5 and...). Figure 14 Adding only thiol-PEG-NTA to the gold core to achieve complete RBD loading leads to NP aggregation, but this can be reversed by ultrasonic treatment (see [reference]). Figure 16 Negative staining was used to perform TEM imaging of Au@PEG-RBD NP, and the results showed that its physical size was approximately 74 nm (including a gold core of approximately 20 nm and a PEG shell of approximately 25 nm thickness, such as...). Figure 17 (As shown). The NP remains stable after atomization [its size in air (measured by a scanning mobility particle size analyzer (SMPS)) and is nearly identical to its hydrodynamic size (see Table 5 and). Figure 18The sample remained stable after incubation for 24 hours in BALF freshly extracted from healthy Syrian hamsters. [Due to BALF adsorption, the size increased slightly to approximately 120 nm (see Table 6 below).] Figures 19A to 19C [Proteomics analysis shown]; Western blotting and ELISA confirmed that no RBD protein was detected in gold NP after BALF incubation (see reference). Figures 20A to 20C Our internal NP binding analysis showed that Au@PEG-RBD NPs bind to recombinant ACE2 and L-SIGN receptors, with dissociation constants ( ). Kd The concentrations were 0.23 nM and 0.83 nM (measured by ELISA), respectively, approximately 100 times higher than free RBD (refer to...). Figure 13C (See Figure 21); This enhanced binding of NPs may stem from a "multivalent effect," where the curvature of the NP allows the local surfaces of multiple ligands to interact simultaneously with cell surface receptors, a result consistent with other gold NPs targeting receptors. Under competition from increased free RBD concentration, the binding of Au@PEG-RBD NPs to ACE2 and L-SIGN receptors decreased in a dose-dependent manner, further demonstrating receptor-specific binding (see Figure 21). Inductively coupled plasma mass spectrometry (ICP-MS) analysis showed that repeated rinsing of Au@PEG-RBD NPs with water and refrigeration for one month did not lead to the release of free Ni. 2+ Ion leaching (reference) Figure 22 NP showed no toxicity in vitro against A549 alveolar epithelial carcinoma cells, non-cancerous BEAS-2B bronchial epithelial cells, or HEK293 cells (exhibiting epithelial morphology) (see reference). Figure 23 Furthermore, it showed no immunogenicity in healthy hamsters after intratracheal instillation. For up to 28 days following instillation, levels of BALF IgA (a marker of pulmonary mucosal immunity) and serum IgG (a marker of humoral immunity) remained low (see reference). Figure 1D Because Au@PEG-RBDNP does not contain adjuvants. As a non-targeting control, we prepared an RBD-free Au@PEG-methoxy NP with similar physicochemical parameters and stability to Au@PEG-RBD NP, but it did not bind to L-SIGN and ACE2 (see Tables 5 and 6). Figure 21A ).

[0187] We validated the binding of two NP types to the receptors for ACE2 and L-SIGN in in vitro and in vitro experiments. ICP-MS analysis showed that Au@PEG-RBD NP bound to HEK293 cells overexpressing ACE2 and L-SIGN more readily than to unoverexpressed HEK293 cells; however, Au@PEG-methoxy NP showed low binding to all cell types after 1 hour of in vitro incubation (see reference). Figure 1EThis preferential cell binding occurs only during shorter incubation periods, up to 12 hours; during longer incubation periods (24 hours), Au@PEG-RBD NP binds similarly to HEK293 cells, independent of L-SIGN or ACE2 expression levels (see reference). Figure 24 Confocal immunofluorescence images confirmed that Cyanine 5.5-labeled Au@PEG-RBD NPs preferentially entered HEK293 cells overexpressing ACE2 and L-SIGN [both fused to green fluorescent protein (GFP)] after 1 hour of incubation (see reference). Figure 1F The Pearson colocalization coefficient (PCC) between the two receptors and Au@PEG-RBD NP was 0.7-0.8, indicating a high degree of overlap. HEK293 cells with lower receptor expression levels also showed intracellular localization of Au@PEG-RBD NP, but in smaller amounts and with weaker overlap with the receptor (PCC: 0.3-0.4, e.g., ...). Figures 25 to 29 (As shown). Given the moderate efficiency of the commercial transfection reagents used, not all cells expressed ACE2 or L-SIGN. Overall, these results support our claim that cells expressing L-SIGN / ACE2 preferentially take up Au@PEG-RBD NP. ICP-MS analysis of extracted lung tissue from healthy hamsters via intratracheal instillation showed that Au@PEG-RBD NP bound 48% more in vitro than Au@PEG-methoxy NP (see reference). Figure 1G Because ACE2 is expressed only in a small number of epithelial cells (primarily type II; accounting for approximately 10% of all epithelial cells), the increase in binding is relatively mild. Confocal reflectance imaging of silver-enhanced gold NPs (which can reflect light) and IHC binding showed that Au@PEG-RBD NPs preferentially enter alveolar cells expressing ACE2, while Au@PEG-methoxy NPs enter less frequently in all cell types (unrelated to ACE2 expression) (see reference). Figure 1H Given the low expression of L-SIGN in healthy hamsters, we did not investigate the co-localization of NP and L-SIGN.

[0188] Example 2 - Intrapulmonary distribution of inhaled Au@PEG-RBD NP In a typical inhalation experiment, our dosage regimen consisted of three 2-hour inhalations of Au@PEG-RBD NP, for the following reasons: ARDS hamsters were inhaled with atomized NP for 2 hours, then immediately euthanized or returned to their cages for 10 hours. This process continued until the third inhalation, which was the earliest point at which Au@PEG-RBD NP reduced pro-inflammatory cytokines to baseline levels (see reference). Figure 2AShorter inhalation times (e.g., 20 minutes) will reduce animal discomfort because food or water should not be provided during inhalation, but it requires higher NP concentrations to achieve the same therapeutic effect, which can clog the aerosolizer. For 150g hamsters, our inhalation dose is 9 to 10 million NP / mL of compressed air (SMPS measurements as shown in Table 5), which complies with FDA guidelines.

[24] Based on our past results, a reasonable equivalent dose of ~3 million NP / mL is a safe concentration for 25g mice.

[25] According to the FDA's equivalent dose for humans, the cumulative nickel intake from three inhalations in hamsters is below the safety limit (see reference). Figure 30 ).

[0189] In healthy and LPS-induced ARDS hamsters, both NP types accumulated in the lower respiratory tract (lungs) rather than the upper respiratory tract (nasal cavity, larynx, and trachea), were evenly distributed across the five lung lobes, and were undetectable in major organs (see reference). Figures 31 to 33 Both NP types showed similar deposition patterns in the lungs of healthy hamsters, but Au@PEG-RBD NP was deposited at a higher level in the lungs of ARDS than Au@PEG-methoxy NP. The higher accumulation of Au@PEG-RBD NP in the lungs of ARDS compared to healthy lungs demonstrates selective delivery of the disease (see reference). Figure 2B and Figure 34 At the tissue level, 60%-80% of both NP types were present in the lavaged lung (obtained by extracting BALF from the whole lung), with the remainder present in the BALF (containing infiltrating immune cells and supernatant). Furthermore, in healthy hamsters, 20%-30% of the NP types diffused from the BALF supernatant into the lavaged lung tissue; in ARDS hamsters, 10% diffused from the BALF supernatant into the infiltrating BALF cells (see reference). Figure 2C Confocal reflectance imaging and IHC showed that in ARDS lungs, Au@PEG-RBD NP deposition was higher than Au@PEG-methoxy NP, and Au@PEG-RBD NP bound more strongly to epithelial cells [Pearson correlation coefficient (PCC): 0.824 vs. 0.183]; both NP types hardly bound to infiltrating endothelial cells (PCC ~0.1-0.2) (see reference). Figure 2D and Figure 35In ARDS lungs, the overlap between Au@PEG-RBD NP and L-SIGN (PCC: 0.889 vs. 0.336) and Au@PEG-RBD NP and ACE2 (PCC: 0.793 vs. 0.281) was higher than that between Au@PEG-methoxy NP and L-SIGN and Au@PEG-methoxy NP and ACE2 (refer to...). Figure 2E ).

[0190] To verify the specific binding of Au@PEG-RBD NP to the lungs in vivo via L-SIGN or ACE2, we intranasally injected anti-ACE2 or L-SIGN antibodies into LPS-induced ARDS hamsters before each round of NP inhalation. We represent "100%" as the lung gold content when Au@PEG-RBD NP achieves "complete receptor targeting" without any receptor blockade; and "0%" as the lung gold content when Au@PEG-methoxy NP achieves "receptor-free targeting". We detected a sharp decrease in the accumulation of Au@PEG-RBD NP in the ARDS lungs due to L-SIGN blockade. Specifically, during the first and second inhalations (12 and 24 hours after LPS induction), the accumulation of Au@PEG-RBD NP was similar to that of Au@PEG-methoxy NP, indicating antibody blockade. However, during the third inhalation, when L-SIGN was no longer activated by LPS (36 hours after LPS induction), this reduction was not significant. These data match the initial activation of L-SIGN and the baseline level of ACE2 (see reference). Figure 1A Conversely, when we block ACE2, the reduction in NP deposition in the lungs occurs during the second and third inhalations, but not during the first inhalation (see [reference]). Figure 2F and Figure 36 These results confirm the ultimate basal level of L-SIGN and the activation of ACE2 (see [reference]). Figure 1A In summary, L-SIGN primarily facilitates the delivery of Au@PEG-RBD NPs to the lungs in the initial stages following an ARDS attack, while ACE2 primarily facilitates the delivery of Au@PEG-RBD NPs to the lungs in the later stages.

[0191] Since L-SIGN and ACE2 are activated at different peak time points after ARDS onset, they are unlikely to synergistically mediate pulmonary delivery of Au@PEG-RBD NP. To investigate the relationship between the two receptors, we investigated how antibody blocking of L-SIGN, ACE2, or both receptors affected pulmonary delivery of a single inhaled dose of Au@PEG-RBD NP at different time points after ARDS onset. At the first time point (12 hours after LPS induction, when L-SIGN expression peaked and surpassed ACE2), blocking L-SIGN and ACE2 alone reduced pulmonary accumulation of Au@PEG-RBD NP by 83% and 10%, respectively, while blocking both receptors reduced it by 95%. Conversely, at the third time point (36 hours after LPS induction, when ACE2 expression peaked and surpassed L-SIGN), blocking L-SIGN and ACE2 alone reduced pulmonary accumulation by 10% and 80%, respectively, while blocking both receptors reduced it by 92%. Therefore, these two receptors synergistically mediate pulmonary delivery (see reference). Figure 37 ).

[0192] Furthermore, we confirmed that the BALF protein corona does not affect receptor targeting. For both NP types, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis revealed that the most abundant corona proteins were albumin, actin, pulmonary surfactant protein, and hemoglobin subunits (mass ratio >70%), with similar amounts of bound proteins; the key difference was that albumin bound Au@PEG-RBD NPs at a significantly higher rate than Au@PEG-methoxy NPs (47.74% vs. 24.81%) (see reference). Figure 38A Finally, we tested whether blocking albumin-associated receptors would affect the lung accumulation of NP. We injected ARDS hamsters intranasally with antibodies against Fc or cysteine-rich acidic secretory protein (SPARC) (both of which are albumin receptors).

[26] It did not significantly reduce the accumulation of Au@PEG-RBD NP in the lungs (see reference). Figure 38C ).

[0193] The therapeutic effects of Au@PEG-RBD NP in LPS-induced ARDS hamsters For the efficacy study, we used the same dosage regimen as the biodistribution study in the previous section: for Au@PEG-RBD NP, three inhalations over 2 hours each (refer to...). Figure 2A and Figure 39 The treatment group consisted of Au@PEG-RBD NP, Au@PEG-methoxy NP (with the same gold dosage as Au@PEG-RBD NP), hydrocortisone (intraperitoneal injection, 5 mg drug / kg hamster / 12 hours, 3 times) as positive control drugs, and the untreated group (n=9).

[0194] In untreated LPS-induced ARDS hamsters, the levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6, and IL-8) in BALF supernatant peaked 12-24 hours post-induction, but returned to baseline levels in healthy hamsters 96 hours post-induction. Figure 3A and Figure 40 In all test groups, Au@PEG-RBD NP most rapidly and significantly reduced four cytokines in BALF after a single inhalation; Au@PEG methoxy NP and hydrocortisone also inhibited the same markers, but required 2-3 doses (see reference). Figure 3A and Figures 41 to 48 Furthermore, after three inhalations of Au@PEG-RBD NP, it most significantly and persistently increased the levels of anti-inflammatory cytokines IL-4 and IL-10 (both of which contribute to lung tissue repair) in BALF cells (see reference). Figure 3A and Figure 49 and Figure 50 As a non-therapeutic negative control, the gold-free free RBD-PEG conjugate (72 μmol RBD / kg hamster; RBD dose the same as Au@PEG-RBD NP) was ineffective (see reference). Figure 51 By excluding PEG or RBD as sources of efficacy, the efficacy can be attributed to Au@PEG-RBD NP. Therefore, we infer that Au@PEG-RBD NP is more effective than Au@PEG-methoxy NP due to its greater cellular delivery to the gold nucleus. Au@PEG-RBD NP did not alter BALF cytokine levels in healthy hamsters, which is evidence of ARDS-specific treatment (see reference). Figure 40 ). By protein blotting (refer to) Figure 3B and Figure 52 ) and intracellular reactive oxygen species (ROS) in BALF cells (refer to Figure 3C Based on the levels of four pro-inflammatory cytokines and two anti-inflammatory cytokines in the lavaged lung tissue, similar therapeutic trends were detected. By counting the extracted BALF cells using Wright-Giemsa staining, we found that Au@PEG-RBD NP reduced red blood cells (a hemorrhage marker) and total white blood cells (an immune cell recruitment marker).

[27] ) and neutrophils (the most abundant cell type in BALF, whose activation and infiltration of the lungs promote tissue damage, are hallmarks of ARDS).

[28] ) quantity (refer to) Figure 53Furthermore, Western blot analysis of BALF cells showed that Au@PEG-RBD NP downregulated M1 macrophage markers (CD80 and CD86) but upregulated M2 macrophage markers (CD206 and arginase-1), providing evidence of repolarization of BALF macrophages from a pro-inflammatory to an anti-inflammatory phenotype. Overall, these results suggest that the reduction in ROS levels in BALF cells is primarily mediated by immune cells.

[0195] Next, we performed histological examination of the central and lateral portions of the ARDS lungs. In contrast to healthy lungs, ARDS lungs lacked reticular structure due to edema and congestion caused by infiltration of immune cells and erythrocytes. The first inhalation did not reduce tissue damage in all groups, but after three inhalations, reticular tissue structure was restored and hemorrhage eliminated in both NP types, with fewer immune cells in the Au@PEG-RBD group compared to the Au@PEG-methoxy group; significant hemocyte infiltration remained in the lungs of the untreated and hydrocortisone groups, indicating ABB leakage. Lung tissue damage severity score.

[29] Au@PEG-RBD NP was shown to be the most effective, producing therapeutic effects after two inhalations. (Reference) Figure 3D , Figure 54 (See Table 7). Tissue recovery was slower than that of cytokine and ROS inhibition because time is needed to resolve inflammation and regenerate epithelial cells. In another competitive experiment, we administered an anti-L-SIGN antibody intranasally before inhaling Au@PEG-RBD NP and detected a decrease in efficacy to the level of Au@PEG-methoxy NP (see Table 7). Figure 3E and Figure 55 , Figure 56 and Figure 72 This indicates that, compared to Au@PEG-methoxy NP, Au@PEG-RBD NP produces faster and stronger efficacy by targeting L-SIGN activated during ARDS.

[0196] We validated the efficacy of Au@PEG-RBD NP using whole-body plethysmography (WBP), a non-invasive tool for measuring respiratory function in unrestrained animals. Regarding hamsters, recent reports have used WBP to investigate the disease mechanisms of COVID-19-related lung injury.

[30] While it has shown good response to drugs, its application in non-viral ARDS remains limited. In LPS-induced ARDS mice, impaired lung function due to inflammatory response and vascular leakage leads to lung tissue hardening, decreased compliance, and increased respiratory effort.

[31] In hamsters, we also found that LPS induction led to unstable respiratory patterns, increased respiratory rate, decreased tidal volume, and reduced peak inhalation volume. Furthermore, LPS induction enhanced peak expiratory flow and reduced relaxation time, consistent with data from previous COVID-19-induced ARDS in mice.

[32] Furthermore, LPS induction increases the "enhanced pause" (Penh), an indicator of airway resistance.

[31] It is worth noting that three inhalations of Au@PEG-RBD NP restored these lung function parameters to healthy levels (see reference). Figure 3F and Figure 57 While this WBP study has scientific implications, its translational application has two limitations. Patients with ARDS cannot undergo pulmonary function testing because they are under sedation and intubated (receiving non-invasive ventilation). Furthermore, pulmonary function testing is only meaningful after the acute phase has passed.

[0197] like Figure 34 As shown, although untargeted NPs initially deposited more slowly in the lungs, they later reached the same lung concentrations as targeted NPs. Then, as... Figure 3E and Figure 6E As shown, an anti-L-SIGN antibody was injected intranasally before inhaling Au@PEG-RBD NP, and its efficacy decreased to the level of Au@PEG-methoxy NP. This result indicates that Au@PEG-RBD NP produces a faster and stronger therapeutic effect by targeting L-SIGN upregulated during ARDS compared to Au@PEG-methoxy NP. Figure 51 As shown, the gold-free free RBD-PEG conjugate (72 μmol RBD / kg hamster; RBD dose was the same as Au@PEG-RBD NP) did not reduce ARDS, allowing us to rule out PEG or RBD as a source of efficacy. In summary, this suggests that the anti-ARDS efficacy originates from the gold core, and RBD is merely a targeting ligand.

[0198] Example 3 - Mechanism of the therapeutic effect of gold NP on ARDS Kinase profiling unbiasedly identified potential pharmacological targets for the 20 nm gold NP. Notably, the ~20 nm Au@PEG methoxy NP inhibited only 5 out of 281 kinases, with inhibition rates ≥70%, two of which are associated with ARDS: p38α (inhibition rate 92%) and PLK3 (inhibition rate 70%) (see reference). Figure 4A(See Table 8). The half-maximal inhibitory concentrations (IC50) of Au@PE methoxy NP against Gp38α and PLK3 were 0.019 nM and 0.031 nM, respectively (refer to Table 8). Figure 4B This indicates a potent inhibitory effect. p38α is a clinical trial drug target for ARDS; SB-681323 (a p38α inhibitor) reduces the levels of IL-6, IL-8, and TNF-α receptors in the serum of ARDS patients.

[33] Although PLK3 has not yet undergone clinical testing, its expression is upregulated in rodents with ARDS.

[34] Its gene knockout can prevent death from sepsis.

[35] Inhibiting it can reduce kidney damage.

[36] Therefore, PLK3 may be a therapeutic target for ARDS. The 20nm gold NP did not inhibit other p38 isoforms (p38β, p38δ, or p38γ) or other kinases in representative signaling pathways (e.g., AKT-mTOR, RAF-MEK-ERK, and JAK), demonstrating its limited off-target effects, consistent with data from the 3nm gold NP.

[37] Western blot analysis confirmed that Au@PEG-RBD NP and Au@PEG-methoxy NP inhibited PLK3 and p38α phosphorylation (p-p38α) in BALF cells and lavaged lung tissue after LPS-induced ARDS inhalation, with the former producing a faster inhibitory effect (see reference). Figure 4C Another remedial study showed that intratracheal instillation of anisomycin (a p38α activator) prior to inhalation of Au@PEG-RBD NP eliminated its efficacy; Western blot and ELISA analyses of lavaged lungs and BALF cells showed no cytokine inhibition (see reference). Figure 4D and Figure 4E This confirms that the therapeutic effect of gold NP originates from p38α inhibition. These results match our previous report on the inhibition of p38α by 3nm gold NP in fibrotic kidneys.

[37] (No commercial activator has had its role validated through similar remedial studies.) Next, unbiased proteomics analysis determined the changes in protein expression in ARDS lungs after Au@PEG-methoxyNP treatment. Of the 2488 proteins screened, we detected only 26 differentially expressed proteins (DEPs) in the "Au@PEG-methoxyNP" group, with expression levels ≥2-fold higher or ≥50% lower than the untreated group (P<0.05). These 26 DEPs are too few to enrich the Kyoto Genome and Genome Encyclopedia pathways or reveal gene ontology terms (see [reference]). Figure 4FThis confirms the specificity of gold NP in targeting ARDS inflammation. Nevertheless, we still identified DEPs associated with ARDS, including the upregulation of Thbs1 (which protects against lung injury).

[38] Ephx2

[39] and Srsf1

[40] Downregulation (a therapeutic target for ARDS), Elavl1

[41] and Sod2

[42] Down-regulation (up-regulation in ARDS), and Pclb3

[43] and Strap

[44] Downregulation (positive regulators of IL-8, IL-6, or TNF-α) (see reference) Figure 4G (See Table 9). Incidentally, Elavl1 (a downstream nuclear factor of p38α) binds to the mRNA of inflammatory cytokines and promotes their protein expression; p38α inhibition hinders the nuclear-cytoplasmic translocation of Elavl and inhibits cytokines. [45,46] Here, Au@PEG-methoxyNP inhibited total Elavl1 (nuclear and cytoplasmic) in LPS-stimulated BEAS-2S bronchial epithelial cells and moderately blocked the nuclear-cytoplasmic translocation of Elavl1; conversely, VX702 (a p38α inhibitor) strongly blocked this translocation without inhibiting total Elavl1 (see reference). Figure 4H and Figure 4I Therefore, Au@PEG methoxy NP can act as a p38α inhibitor to block Elavl1 nuclear-cytoplasmic translocation and can interact directly with Elavl1, independent of p38α.

[0199] Example 4 - Long-term toxicity of inhaled Au@PEG-RBD NP In the lungs of ARDS hamsters treated with Au@PEG-RBD NP, gold content remained high after 1 month, decreased by half after 6 months, and became undetectable after 12 months (see reference). Figure 5A Gold was not detected in any other major organs at any time point. Twelve months later, histological examination (see reference) Figure 5B and Figure 5C The histological morphology of the lungs and major organs, blood cell count, liver and kidney function, and serum total protein (see Table 10) were basically normal. One month later, only the serum lactate dehydrogenase level was slightly higher than normal (possibly due to tissue repair).

[25] However, it later returned to normal. The results indicate that Au@PEG-RBD NP has long-term clearance ability and limited in vivo toxicity.

[0200] Example 5 - The therapeutic effect of Au@PEG-RBD NP in hydrochloric acid-induced ARDS hamsters We validated the therapeutic effect of Au@PEG-RBD NP in a second hamster disease model. HCl-induced ARDS mimics the aspiration of gastric contents into the lungs, another common cause.

[20] LPS and HCl induce similar inflammatory responses in terms of the following kinetics and extent: (i) upregulation of ACE2 and L-SIGN, and (ii) pro-inflammatory cytokines throughout the ARDS lung (see reference). Figure 6A Following the same treatment plan (refer to...) Figure 2A In HCl-induced ARDS hamsters, Au@PEG-RBD NP downregulated BALF supernatant (reference). Figure 6B and Figures 58 to 67 ) and lavage lung tissue (refer to Figure 6C and Figure 68 Pro-inflammatory cytokines in BALF cells upregulated anti-inflammatory cytokines and ROS levels in BALF cells. Figure 6D ) and lung tissue damage (refer to Figure 69 Furthermore, in HCl-induced ARDS hamsters, tracheal blockade with anti-L-SIGN antibody reversed Au@PEG-RBDNP to a level of efficacy similar to Au@PEG-methoxy NP (see reference). Figure 6E , Figure 70 and Figure 71 Au@PEG-RBD NP has a faster onset of action than Au@PEG-methoxy NP and hydrocortisone (single dose vs. three doses).

[0201] According to embodiments of the invention, a key finding is the identification of time-dependent transient upregulation of ACE2 and L-SIGN in ARDS lungs during disease onset. This unique pathophysiological insight inspired the design of Au@PEG-RBD NPs to enhance delivery to lung cells overexpressing ACE2 and L-SIGN.

[0202] The NP according to embodiments of the present invention differs from previous ARDS nanomedicines in that it uses ligands applied to target endothelial cells (e.g., adhesion molecules) or immune cell membranes (e.g., macrophages) to homing to sites of inflammation, as shown in Table 1. When combined with inhalation (a method compared to infusion) 36 and intraperitoneal injection 37 When combined with a less invasive delivery route, NPs are evenly distributed in lung tissue, accelerating their accumulation in ARDS lungs. Another key finding is that 20nm gold cores, rather than spike RBDs or PEGs, can serve as a therapeutic agent for ARDS without the need for additional chemical drugs, biological agents, or physical interventions.

[0203] The embodiments of the present invention contribute to the growth of the list of self-treatment applications of gold NP, from the treatment of psoriasis.38 To resolve kidney fibrosis 34 In ARDS, previous reports primarily used gold NPs only as drug carriers, such as the PI2 peptide for blocking Toll-like receptors. 22 Curcumin as an antioxidant 39 Dexamethasone as an anti-inflammatory agent 40 In addition to its recognized anti-inflammatory properties... 41 and antioxidants 42 In addition to its characteristics, the gold NP of the present invention also helps to identify two therapeutic targets of gold NP in ARDS: one is a clinically tested target (p38α), and the other is a newly emerging target (PLK3).

[0204] Current research findings will facilitate the translational application of gold nanoparticles in alleviating ARDS. Firstly, past gold nanomedicines were preventative, administered via injection before disease induction. 43 In this study, such as Figure 3A and Figure 6B As shown, hamsters inhaled NP 12 hours after disease induction (at the peak of ARDS severity). This time point is clinically significant because the acute nature of ARDS makes timely diagnosis and prevention challenging.

[0205] Furthermore, after one year of inhalation, Au@PEG-RBD NP does not remain in the lungs or other major organs, thus alleviating concerns about the toxicity of classic gold ion therapy. 44 Further research is needed to understand how gold NP is cleared from the lungs.

[0206] Finally, compared to classic organic NPs (such as liposomes and micelles), gold NPs offer a more convenient chemical approach to tracking their distribution in vivo. Previous distribution studies focused on anti-ARDS efficacy have now revealed useful insights into lung NP interactions. Figures 2B to 2E and Figure 5A As shown, this paper uses ICP-MS and confocal reflectance imaging to quantify and visualize the distribution of gold NPs at the organ and tissue levels, respectively.

[0207] At the cellular level, IHC and confocal reflectance imaging were combined to demonstrate NP entry into specific lung cell types. While flow cytometry can quantify gold NPs in different lung cell types, as shown in mice... 19 However, due to the scarcity of commercially available hamster reactive antibodies, similar studies are currently not feasible. 45 .

[0208] According to embodiments of the present invention, NP exhibits anti-inflammatory efficacy against severe ARDS. NP is readily synthesized, exhibits anti-ARDS efficacy without drug loading, and does not show significant extrapulmonary retention or systemic side effects.

[0209] Compared to existing technologies, the non-invasive inhalable NP of this invention targets lung cells more uniformly. Overall, this approach provides a safer and more effective solution for pulmonary delivery and ARDS management.

[0210] in conclusion We present a disease-driven, non-invasive gold nanomedicine for ARDS. This drug is based on our observation of transient upregulation of ACE2 and L-SIGN in the lung epithelium shortly after ARDS onset (12-36 hours). We leveraged ARDS pathophysiology to inform the dosing regimen by cleverly aligning the dose with the timing of ACE2 or L-SIGN upregulation due to ARDS. Specifically, L-SIGN primarily facilitates lung delivery of Au@PEG-RBD NPs from ~12 hours after ARDS onset, while ACE2 primarily facilitates delivery from ~36 hours; these two receptors act synergistically to mediate lung targeting. Therefore, we achieve rapid delivery to inflamed endothelial cells without excessively long delivery times, thus avoiding long-term accumulation and potential safety risks. The inhalable NPs are widely distributed in ARDS lung tissue and effectively target the epithelium, producing therapeutic effects within 6 hours of inhalation. As a treatment approach, we initiated the first dose 12 hours after disease establishment, when the pro-inflammatory response in the ARDS lungs peaked (Figures 3 and 6), in contrast to previous prophylactic studies that started 4–6 hours after ARDS induction (Tables 1 and 2). This is clinically relevant for acute conditions like ARDS, where timely prediction is challenging. One year after inhalation, NP did not remain in the lungs or other major organs, alleviating concerns about long-term toxicity.

[47] .

[0211] Another key finding is that gold NP has a self-healing effect on ARDS, which expands its emerging applications for other important diseases, such as psoriasis.

[48] and kidney fibrosis

[37] In ARDS, past reports have used gold NPs solely as drug carriers, such as the PI2 peptide, to block Toll-like receptors.

[29] Curcumin as an antioxidant

[49] Dexamethasone as an anti-inflammatory agent

[50] Our contribution lies in identifying two therapeutic targets for gold NP in ARDS (one clinically tested target (p38α) and the other a target with preclinical evidence (PLK3)), revealing the therapeutic mechanism of gold NP beyond its established anti-inflammatory effects.

[51] and antioxidants

[52] Characteristics. Notably, Au@PEG-RBD NP is more effective than corticosteroids in relieving ARDS, a key standard of care with clinically validated efficacy in ARDS (Table 11). Most reported clinical outcomes of corticosteroid treatment are based on survival data, days without ventilation, and length of ICU stay, and changes in essential pathological markers are generally not reported. Nevertheless, some clinical studies have reported pathological markers in addition to clinical outcomes, showing serum IL-6 levels on day 3 after treatment with hydrocortisone and methylprednisolone in ARDS patients. [53,54] Lung injury score [55,56] These past clinical results match our preclinical efficacy data, which showed that in both ARDS hamster models tested, three doses of Au@PEG-RBD NP reduced IL-6 levels in the lungs (BALF and lavage tissue) and lung tissue damage scores, compared to less efficacy with hydrocortisone (total duration 30 hours).

[0212] Given the limitations of this study, we used prokaryotes to produce spike RBD proteins when constructing this lung nanomedicine because (i) gram-scale RBD proteins are required, and prokaryotes can express proteins rapidly and economically (Table 4), and (ii) the prokaryotic RBD products have only slightly lower affinity for ACE2 and L-SIGN than commercial eukaryotic RBDs. Figures 13A to 13D However, for clinical translation, protein expression should be performed using a mammalian system to avoid allergic reactions in humans. Our treatment regimen requires three inhalation sessions, each lasting two hours, which is a feasible duration for patients with severe ARDS who are frequently intubated and therefore require sedation and bed rest.

[57] To shorten inhalation time (e.g., ~20 minutes) and improve patient compliance, we need to prepare more concentrated NP liquid stock solutions for nebulization on a larger reaction scale, while simultaneously addressing the technical challenges of protein-induced NP aggregation. Furthermore, the scarcity of commercial hamster reactive antibodies limits the use of flow cytometry.

[25] Quantifying the distribution of inhaled NPs in different lung cell types is challenging.

[58] Finally, prior to clinical trials, further validation studies are needed to demonstrate the efficacy and toxicology of our spike-coupled gold NP in large animal models.

[0213] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated herein in their entirety by reference, including all figures and tables, provided that they do not contradict the express teachings of this specification.

[0214] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art can make various modifications or changes based on these embodiments and implementations, which will be included within the spirit and scope of this application. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any and / or all other elements or limitations of any other invention or implementation thereof, and all such combinations are covered within the scope of this invention, but are not limited thereto.

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Claims

1. A method for preparing inhalable targeted gold nanoparticles for accelerating lung delivery and treating lung inflammation, the method comprising: Plasmid preparation; Perform plasmid transformation; Inducing protein expression; Protein purification; and Gold nanoparticles (Au@PEG-RBDNP) bound to spike RBD were prepared.

2. The method according to claim 1, wherein the preparation of plasmids comprises: DH5 Competent cells are mixed with plasmids; Incubate the mixture on ice; The mixture was subjected to thermal shock; The mixture was incubated on ice; Add preheated lysozyme to the mixture; and The cells in the mixture are incubated using orbital oscillations.

3. The method according to claim 2, further comprising spreading the transformed cells onto LB agar plates containing ampicillin and incubating the cells.

4. The method of claim 3, further comprising selecting a community of the cells and causing the selected community to grow in LB / ampicillin under oscillation.

5. The method of claim 4, further comprising collecting cells by centrifugation and purifying the plasmid to obtain a plasmid encoding an RBD of the His marker (pET11a-RBD-8xHis).

6. The method of claim 1, wherein plasmid transformation comprises: Thawing on ice previously at -80 Competent cells stored below; The plasmid was added to the competent cells to obtain a mixture; Keep the cells of the mixture on ice; The cells in the mixture were heat-shocked in a water bath and then incubated again on ice. and Add fresh LB medium and shake the mixture.

7. The method of claim 6, further comprising centrifuging the cells of the mixture and discarding the supernatant.

8. The method of claim 7, further comprising resuspending the remaining precipitated cells.

9. The method of claim 8, further comprising inoculating the bacteria onto an LB agar plate containing antibiotics and incubating the bacteria.

10. The method of claim 1, wherein inducing protein expression comprises: Origami B cells, which were pre-transformed with molecular chaperone plasmid pG-KJE8, were transformed using the RBD expression plasmid pET11a-RBD. The product obtained by culturing on LB agar plates; Select a single community from the results; The community was added to fresh LB medium containing antibiotics; Add the overnight culture to the fresh LB medium; and The cultured product until OD 600 It reaches approximately 0.

5.

11. The method of claim 10, further comprising induction by adding an induction buffer containing isopropyl β-D-1-thiogalactopyranoside and L-arabinose.

12. The method of claim 11, further comprising collecting cells by centrifugation for purification or at -80°C. Download and save.

13. The method of claim 1, wherein the protein purification comprises: Origami B cells were resuspended in protein purification buffer and lysed using an ultrasonic processor to produce lysates. Centrifuge the lysate; Collect the supernatant of the lysate; Filter the supernatant using a syringe filter; The supernatant was incubated with Ni-NTA resin to obtain a pyrolysis product-resin mixture; and The pyrolysis-resin mixture was transferred together with the resin into a blank gravity chromatography column.

14. The method of claim 13, further comprising discarding the liquid flowing from the lysate-resin mixture and adding a washing buffer to elute nonspecific binding proteins.

15. The method of claim 14, further comprising eluting the RBD protein product by adding an elution buffer and dialyzing it against a storage buffer.

16. The method of claim 15, further comprising determining the purity and concentration of the protein product by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and the Bradford assay, respectively.

17. The method of claim 1, wherein the preparation of spike RBD-bound gold nanoparticles (Au@PEG-RBDNP) comprises: Citrate-capped gold nanoparticles (cit-AuNP) with a diameter of approximately 20 nm were synthesized. After boiling HAuCl4, add sodium citrate while stirring vigorously and keep the mixture boiling. The product was cooled to obtain cit-AuNP; and The freshly dissolved thiol (HS)-PEG was mixed at a 1:1 molar ratio. 20k -Methoxy or HS-PEG 20k -N-N-triacetic acid (NTA) (Biochempeg) at per nm 2 The total concentration of 5 PEG molecules on the surface of the nanoparticles was added to the cit-AuNP solution to obtain the resulting mixture.

18. The method of claim 17, further comprising stirring the resulting mixture, adding NiCl2, and then stirring to obtain Au@PEG-NTA-Ni. 2+ Nanoparticles (Au@PEG-NTA-Ni) 2+ NP).

19. The method of claim 18, further comprising adding His-tagged RBD protein to Au@PEG-NTA-Ni 2 + The NP solution was stirred to obtain Au@PEG-RBD nanoparticles (Au@PEG-RBD NP).

20. The method of claim 19, further comprising dialyzing the obtained Au@PEG-RBD NP onto Nanopure ultrapure water by centrifugal filtration.

21. A nanoparticle composition for inhalation delivery to lung epithelial cells, comprising: The gold core has a diameter between approximately 3nm and 50nm. A polyethylene glycol (PEG) shell covering the gold core; and Multiple recombinant spike receptor-binding domain (RBD) subunit proteins that bind to the PEG shell, The nanoparticle composition has a total size greater than 50 nm and is configured to inhibit p38α mitogen-activated protein kinase (MAPK) phosphorylation and inhibit polo-like kinase 3 (PLK3).

22. The nanoparticle composition of claim 21, wherein the total size of the nanoparticles is about 94 nm.

23. The nanoparticle composition according to claim 21, wherein the gold core has a diameter of about 20 nm.

24. The nanoparticle composition of claim 21, wherein the PEG shell is composed of 20000 Da PEG chains.

25. The nanoparticle composition of claim 21, configured to bind to liver / lymph node-specific intercellular adhesion molecule-3-binding integrin (L-SIGN) and angiotensin-converting enzyme 2 (ACE2) receptors on lung epithelial cells.

26. A method for treating an individual with acute respiratory distress syndrome (ARDS), the method comprising intermittently inhaling a therapeutically effective amount of the nanoparticle composition of claim 21 into the individual's lung epithelial cells.

27. The method of claim 26, wherein the ARDS is a non-viral ARDS.

28. The method of claim 26, wherein the ARDS is induced by inhalation of lipopolysaccharide (LPS) or hydrochloric acid (HCl).

29. The method of claim 26, wherein the nanoparticles have therapeutic efficacy in the absence of a virus blocking mechanism.

30. The method of claim 26, wherein the therapeutic effect is achieved by the superimposed binding of the nanoparticles to the L-SIGN receptor and the ACE2 receptor.

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