Benzene-1,4-dicarboxylic acid (BDC), Zirconium (IV) oxide chloride octahydrate (ZrOCl₂$\cdot$8H₂O), N,N-dimethylformamide (DMF) and lipopolysaccharides from Escherichia coli O111:B4 (LPS) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Percoll and erythrocyte lysate were purchased from Solarbio Biotechnology (Beijing, China). The BCA protein assay kit, Cell Membrane Green Fluorescence Staining Kit (DiO), EISA kits for mouse tumor necrosis factor-$\alpha$ (TNF-$\alpha$) (P5318, Beyotime, Shanghai, China) and inflammatory factors interleukin 6 (IL-6) (PI335, Beyotime, Shanghai, China) were purchased from Shanghai Beyotime Biotechnology (Shanghai, China). MTT kit was purchased from Shanghai Jiji Biochemical Technology (Shanghai, China).

RAW264.7 cells and DMEM were purchased from Shanghai FuHeng Biology Co., Ltd. (Shanghai, China). All cell lines were validated by short tandem repeat (STR) profiling and tested negative for mycoplasma. Culture conditions: gas phase: 95% air, 5% carbon dioxide. Temperature: 37 °C, incubator humidity maintained at 70%–80%. Cryoprotectant solution: 90% fetal bovine serum (FBS) (C0226, Beyotime, Shanghai, China), 10% DMSO, prepared fresh and used immediately. Healthy, 8-week-old, male BALB/c mice of SPF (Specific Pathogen Free) grade were purchased from Cavens Laboratory Animal Co., Ltd. (Changzhou, Jiangsu, China). All mice weighed between 20 and 25 grams. The animal experiment was approved under approval number AMUWEC20210417, and all procedures were conducted in strict compliance with the relevant regulations and guidelines set by the Laboratory Animal Welfare and Ethics Committee of Third Military Medical University.

After being anesthetized with 100% carbon dioxide for five minutes, the BALB/c mouse underwent euthanasia to ensure complete cessation of life. After the euthanasia of the BALB/c mouse, the femur and tibia were surgically removed. Subsequently, the bone ends were neatly trimmed, and an RPMI 1640 solution, enriched with 10% FBS and 2 mM ethylene diamine tetraacetic acid (EDTA), was aspirated to flush out the bone marrow cells. To lyse the red blood cells, 20 mL of a 1.6% NaCl solution was added. Utilizing Percoll in combination with density gradient separation techniques, neutrophils were successfully purified from the bone marrow cells. To identify the purified neutrophils and for downstream experimental purposes, flow cytometry was performed. Additionally, the isolated neutrophils were stained with Shanghai Beyotime Biotechnology Wright-Giemsa kit (C0131, Shanghai, China) and examined under a microscope.

In order to obtain the neutrophil membrane, the neutrophils were destroyed with a low osmotic dissolution buffer containing protease inhibitors for 20 minutes. BeyoZonase broad spectrum nucleases (D7121, Shanghai, China) were added to the solution and treated for 10 minutes. The cells were immersed in liquid nitrogen and 37 ℃ water baths for 5 consecutive cycles of cell destruction. After centrifugation at 1000 g for 5 minutes, the precipitate was discarded. The enriched supernatant was centrifuged at 20,000 g for 30 minutes and 80,000 g for 1.5 hours, respectively. The precipitate was collected and washed three times with PBS mixed with protease inhibitors. Under low temperature conditions, ultrasound was used to obtain dispersed cell membranes.

The method used in the reference literature employs solvothermal synthesis to prepare UiO66 nanoparticles. The specific steps are as follows: 200 mg of benzene-1,4-dicarboxylic acid (BDC) and dissolve it in 2 mL of N,N-dimethylformamide (DMF). Add the solution to a round-bottom flask and stir for more than 5 minutes. Then, weigh 42 mg of zirconium oxychloride octahydrate (ZrOCl₂$\cdot$8H₂O) and dissolve it in 6 mL of DMF. Add the 6 mL of DMF containing zirconium oxychloride octahydrate to the above solution and mix the two solutions for 5 minutes. Afterward, add 4 mL of acetic acid. Seal the round-bottom flask containing the mixed solution and place it in an oil bath at 110 °C. Stir and react for 5 hours. Afterward, centrifuge the obtained product and wash it twice with DMF and deionized water, respectively. Dry the product overnight under vacuum and calculate the yield.

The particle size, potential of the samples were detected by Malvern laser particle sizer (NS-90, Malvern, Malvern, UK). Mix 20 mg Dex with 100 mg UiO66, disperse in 20 mL anhydrous ethanol, and stir overnight at room temperature. Then centrifuge the mixture at a speed of 80,000 rpm for 10 minutes, collect the supernatant, and resuspend the precipitate with anhydrous ethanol. Continue centrifugation three times, collecting each supernatant. The morphology of the prepared sample was examined using a transmission electron microscope (TEM) (HT7700, Hitachi, Japan). Quantify the content of free Dex in the supernatant using high performance liquid chromatography (HPLC) (HPLC 1260, Agilent, Santa Clara, CA, USA) and record it as WF. Then, the obtained precipitate was vacuum dried at 120 °C for 24 hours to obtain UiO66-Dex, whose weight was recorded as WNP. Determination of drug loading content and encapsulation efficiency. The encapsulation efficiency and loading content of Dex were calculated as follows:

(1)$$\text{Drug Loading content}=(\mathrm{WT}-\mathrm{WF})/\mathrm{WNP}\times 100\%$$

where WT is the total weight of Dex fed, WF is the weight of non-encapsulated free Dex, and WNP is the weight of nanoparticles.

(2)$$\text{Drug Encapsulation efficiency}=(\mathrm{WT}-\mathrm{WF})/\mathrm{WT}\times 100\%$$

where WT is the total weight of Dex fed, and WF is the weight of non-encapsulated free Dex.

The neutrophil membrane proteins were determined by a BCA protein assay kit and adjusted to 5 mg/mL. Subsequently, they were mixed with UiO66-Dex (1 mg/mL) in a volume ratio of 1:1. The above mixture was ultrasonically mixed under low-temperature conditions and then passed through an Avant extruder (Avanti Polar Lipids, Inc.,610000, Alabaster, AL, USA) to coat the neutrophil membrane on the surface of UiO66-Dex. The morphology of the prepared sample was examined using a transmission electron microscope (HT7700, Hitachi, Japan).

To investigate the release of UiO66-Dex@NMP, a dialysis method was employed. Briefly, UiO66-Dex@NMP loaded with 100 µg of Dex was placed into a dialysis bag with a molecular weight cutoff of 3 kDa. The dialysis bag was then immersed in 20 mL of PBS and incubated at 37 °C for various durations while continuously shaking at 100 rpm. At designated time points, 1 mL of the external medium was removed and replaced with the same volume of PBS. The concentration of Dex released into the bulk dialysis medium was determined by high performance liquid chromatography (HPLC).

RAW264.7 cells were added to 96-well plates at 1 $\times$ 10⁵ cells/well and incubated for 24 hours. Dex, UiO66, UiO66-Dex@NMP solutions at 10, 50, 100, 200, and 500 µg/mL were added into the incubator. After incubation for 48 hours, 20 µL of MTT reagent was added to each well and incubation was continued for 4 hours. 200 µL of DMSO was added to dissolve metazan. Absorbance was measured with an enzyme meter (BioTek ELx808, Winooski, VT, USA).

RAW264.7 cells were added into 96-well plates at 1 $\times$ 10⁵ cells/well, and put into the incubator for 24 hours. The wells were divided into Control, SP+PBS, SP+Dex and SP+UiO66-Dex@NMP groups, in which cells were stimulated with viruses except for the Control group, and then different drugs were added respectively, and put into the incubator for 12 hours. Cells were incubated with enzyme-labeled antibody working solution for 3 hours, and then added termination solution for 20 minutes, and the optical density (OD) value was determined by enzyme marker. The levels of inflammatory factors TNF-$\alpha$, IL-6, IL-10 were detected according to the instructions of each kit.

Firstly, UiO66-Dex@NMP was incubated with a cell membrane green fluorescence staining kit for 10 minutes. After centrifugation to wash away excess fluorescent dye, DiO-labeled UiO66-Dex@NMP was resuspended in physiological saline solution. RAW264.7 cells were seeded in a confocal dish at a concentration of 1 $\times$ 10⁵/mL and cultured overnight in a cell incubator at 37 °C with 5% CO₂. LPS was added to the RAW264.7 cells at a final concentration of 100 ng/mL, and the cells were further cultured for 2 hours. The culture medium was aspirated, and the cells were washed twice with PBS before replacing it with DMEM medium. After another 4 hours of culture, DiO-labeled UiO66-Dex@NMP was added, and the cells were incubated for another 2 hours. The culture medium was aspirated again, and the cells were washed three times with PBS. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS, and observed under a confocal microscope (LSM800, Zeiss, Germany) to assess the internalization of UiO66-Dex@NMP by the cells.

The animals were situated in a controlled environment with a consistent 12-hour light-dark cycle, allowing them unrestricted access to standard food and water. Subsequently, the mice were gently anesthetized with isoflurane before being intranasally administered 35 µL LPS solution (20 mg/mL) to induce a pneumonia model.

First, UiO66-Dex@NMP was incubated with the DiR fluorescence staining kit for 10 minutes. After that, it was centrifuged and washed three times, and then adjusted to an appropriate concentration with physiological saline. Next, the mice with pneumonia were anesthetized and inhaled the commercially available aerosolized UiO66-Dex@NMP labeled with DiR (MB12482, MeiluneBio, Dalian, Liaoning, China). Four hours after the atomized treatment, the distribution of UiO66-Dex@NMP within the body was examined using a small animal in vivo imaging system (IVIS Spectrum, Perkin Elmer, Waltham, MA, USA).

The SP model mice were established through induction as described in 2.11. SP model mice were randomly assigned to four groups, each containing five animals: the normal control group, the SP model control group, the Dex treatment group, and the UiO66-Dex@NMP treatment group. Two hours after the SP mouse model was successfully established, the control group solely underwent PBS treatment. Conversely, the Dex treatment group and the UiO66-Dex@NMP treatment group were given atomized administrations of Dex and UiO66-Dex@NMP respectively (Dex dosage 1 mg/kg). Administration of atomized medication was conducted using the Atomization Drug Administration Apparatus for Rats and Mice (KW-DM-YWH, Nanjing Calvin Biotechnology Co., Ltd.), with a treatment duration of 25 minutes. Twenty-four hours post-treatment, blood was collected from the mice, and the sera from each group were subsequently frozen for the purpose of cytokines detection.

The levels of inflammatory cytokines TNF-$\alpha$, IL-6 and IL-10 were measured according to the manufacturer’s instructions of the respective kits. At the end of the treatment period, the mice were sacrificed by carbon dioxide asphyxiation, the lungs were isolated and processed for fixation, sectioning, and HE staining. The pathological scores of each group were then compared.

Data analysis was conducted using GraphPad Prism 6.0 software (GraphPad software, San Diego, CA, USA), with measurement data following a normal distribution described as mean $\pm$ standard deviation ($\overline{x}$ $\pm$ s). Multiple group data differences were compared using repeated measures variance and one-way ANOVA analysis. Differences between the two groups were compared using two-way ANOVA analysis. When p 0.05, it indicates that the difference is statistically significant.

Firstly, neutrophils were separated from bone marrow and peripheral blood. The isolated cells were conducted through Wright-Giemsa staining, revealed a distinct purple-blue lobulated nucleus, which is suggestive of the typical morphology associated with neutrophils, as clearly depicted in Fig. 2A. Neutrophils were preactivated by TNF-$\alpha$, flow cytometry was employed to meticulously identify surface markers on the neutrophils. The findings presented in Fig. 2B illustrate that both Ly6G and CD11B positive cells accounting for 96.3% of the whole population. These observations strongly indicate that the neutrophils isolated in this study possess a remarkable degree of purity and a highly differentiated state.

Fig. 2.

Neutrophils separation and characterization of UiO66-Dex@NMP. (A) Wright Giemsa staining of the purified neutrophils. Scale bar = 50 µm. (B) CD11b biomarker analysis of the neutrophil by Flow cytometry. (C) Morphological observation of UiO66 by TEM. Scale bar = 100 µm. (D) Morphological observation of UiO66-Dex by TEM. Scale bar = 100 µm. (E) Morphological observation of UiO66-Dex@NMP by TEM. Scale bar = 500 µm. (F) Particle size analysis of UiO66, UiO66-Dex and UiO66-Dex@NMP. (G) Potentiometric analysis of UiO66, UiO66-Dex and UiO66-Dex@NMP. TEM, transmission electron microscope; FSC, forward scatter; SSC, side scatter.

The transmission electron microscopy (TEM) analyses of UiO66 and UiO66-Dex, depicted in Fig. 2C,D, unveil an average particle size of approximately 100 nm, displaying a distinctive octahedral morphology. The encapsulation of Dex had minimal impact on the particle size and morphology of UiO66-Dex, indicating that Dex is primarily adsorbed within the pores of UiO66-Dex. Fig. 2E showcases the TEM image of UiO66-Dex@NMP, featuring a typical core-shell structure adorned with a cellular membrane on its outer edge. This imagery provides compelling evidence of the successful encapsulation of UiO66-Dex within the cellular membrane, with a mean particle size exceeding 100 nm. Fig. 2F presents the hydrated particle size data for UiO66, UiO66-Dex, and UiO66-Dex@NMP. The presence of a hydration film layer on the surfaces of these nanomaterials results in a measured hydrated particle size that is larger than the size observed through TEM. Furthermore, Fig. 2G illustrates the surface potential analysis of UiO66, UiO66-Dex, and UiO66-Dex@NMP. In aqueous solution, UiO66 and UiO66-Dex exhibit a positive charge, whereas UiO66-Dex@NMP carries a negative charge. This change in electric charge is attributed to the coating with neutrophil membranes.

To further characterize the structure of UiO66-Dex@NMP, the ultraviolet absorption was detected. As depicted in Fig. 3A, free Dex (curve in red), UiO66-Dex (curve in black) and UiO66-Dex@NMP (curve in green) all exhibit comparable absorption peaks at identical wavelengths at near 250 nm. Three different absorption curves demonstrate the successful encapsulation of Dex within UiO66-Dex@NMP.

Fig. 3.

Dex loading and release of UiO66-Dex@NMP. (A) Representative UV-visible absorption peaks of dexamethasone (Dex), UiO66-Dex, and UiO66-Dex@NMP in PBS. (B) In vitro release profile of UiO66-Dex@NMP in PBS over 72 hours. (C) Dex encapsulation rate test. (D) Dex loading test. UV, ultraviolet-visible.

As shown in Fig. 3B, the cumulative release rates of Dex from UiO66-Dex and UiO66-Dex@NMP after 72 hours were 32.67% and 18.33%, respectively. Compared with UiO66-Dex, UiO66-Dex@NMP exhibited lower release rates at every time point, indicating that its release effect is more pronounced and sustained after undergoing biomimetic modification. This characteristic will effectively reduce unnecessary drug leakage and, in turn, lower the risk of side effects potentially caused by Dex. In Fig. 3C,D, the optimum loading rate was achieved when the mass ratio of Dex to UiO66 was 1:5, and the Dex encapsulation rate at UiO66-Dex@NMP was 93.65 $\pm$ 2.74% with a drug loading of 9.88 $\pm$ 0.83%.

As shown in Fig. 4A, at a concentration of 100 µg/mL or less, UiO66-Dex@NMP showed no significant toxicity to RAW264.7 cells, and the cell survival rates were all over 90%. The levels of inflammatory factors secreted by RAW264.7 cells were shown in Fig. 4B–D, and UiO66-Dex@NMP significantly reduced the TNF-$\alpha$, IL-6, IL-10 levels. This indicated that UiO66-Dex@NMP had a significant inhibitory effect on the secretion levels of inflammatory factors.

Fig. 4.

Analysis of cellular toxicity and in vitro anti-inflammatory effects of UiO66-Dex@NMP. (A) RAW264.7 cytotoxicity assay. (B) RAW264.7 levels of the inflammatory factor TNF-$\alpha$ secreted by cells. (C) RAW264.7 levels of the inflammatory factor IL-6 secreted by cells. (D) RAW264.7 levels of the inflammatory factor IL-10 secreted by cells. TNF-$\alpha$, tumor necrosis factor-$\alpha$; IL-6, interleukin 6; SP, severe pneumonia. Statistical analysis was performed using two-tailed Student’s t-tests. ns p $>$ 0.05; * p 0.05; ** p 0.01; *** p 0.001; **** p 0.0001.

After UiO66-Dex@NMP was labeled with the cell membrane fluorescent dye DiO, it was co-incubated with mouse macrophage RAW264.7 cells and then observed under confocal microscopy. Fig. 5A demonstrates that RAW264.7 cells activated by LPS induction exhibited significantly increased green fluorescence compared to inactivated RAW264.7 cells (Fig. 5B). Consequently, UiO66-Dex@NMP displays a higher targeting efficiency towards activated macrophages in an inflammatory environment, thereby reducing the risk of nonspecific Dex delivery. As shown in Fig. 5C, the in vivo imaging of SP model mice after inhalation of DiR-labeled UiO66-Dex@NMP revealed that the fluorescent signal was primarily concentrated in the lungs. As depicted in Fig. 5D, indicated that apart from the lungs, there was no significant specific enhancement of fluorescent signals in other organs. Inhalation delivery further enhances the accumulation of therapeutic drugs in the lungs, thereby reducing unintended drug diffusion to other parts of the body.

Fig. 5.

In vitro and in vivo targeting. (A) Confocal fluorescence imaging of DiO-labeled NMPs after incubation with activated macrophages (blue, nuclei; green, UiO66-Dex@NMP). Scale bar = 40 µm. (B) Fluorescence quantification of UiO66-Dex@NMP per unit area on macrophages, p 0.001. (C,D) In vivo targeting effect of DiR-labeled UiO66-Dex@NMP administered by atomization.

Fig. 6A–C shows the serum cytokine levels in normal mice and SP model mice after receiving corresponding treatments. Compared to the normal control group, mice in the SP+PBS group exhibited significantly elevated serum levels of TNF-$\alpha$, IL-6, and IL-10 (with extremely significant differences). However, after treatment with atomized Dex and UiO66-Dex@NMP, the serum levels of TNF-$\alpha$, IL-6, and IL-10 in the mice were significantly reduced. Furthermore, compared to the mice treated with atomized Dex, those treated with UiO66-Dex@NMP showed significantly lower serum levels of TNF-$\alpha$ and IL-6, with a moderate reduction in IL-10 although the difference was not significant. Therefore, atomized UiO66-Dex@NMP treatment can significantly reduce the levels of inflammatory cytokines in the serum of SP model mice, which may have a certain interventional effect on the inflammatory cytokine storm caused by SP. In addition, the neutrophil membrane has a certain adsorption effect on cytokines in the inflammatory environment, also known as “cytokine sponge” [15, 23]. This is of great significance in reducing the inflammatory cytokine storm in SP. It is crucial to acknowledge that the influence of nanoparticles on inflammatory factors, including TNF-$\alpha$, IL-6, and IL-10, may differ significantly both in vitro and in vivo, resulting in inconsistent effects across various experimental conditions and models.

Fig. 6.

Anti-severe pneumonia Therapy. The cytokines level after indicated treatments: (A) TNF-$\alpha$; (B) IL-6; (C) IL-10; (D) HE–stained examination of lung tissues after indicated treatments. Scale bar = 100 µm (above); scale bar = 350 µm (below). Statistical analysis was performed using two-tailed Student’s t-tests. ns p $>$ 0.05; * p 0.05; ** p 0.01; *** p 0.001; **** p 0.0001.

The histological sections in Fig. 6D, revealed various degrees of pathological changes in the lung tissue of the SP model group. These included alveolar mice, collapse of alveolar walls, infiltration of inflammatory cells, dilation and congestion of capillaries, among other changes. In contrast, the lung tissue of the normal control group did not exhibit these changes, indicating the successful establishment of the SP model. When compared to the model group, both the Dex and UiO66-Dex@NMP inhalation treatment groups showed a reduction in the severity of SP-related pathological changes in the lung tissue. Furthermore, compared to inhaled Dex treatment, inhaled UiO66-Dex@NMP treatment further mitigated the pathological damage to the lungs caused by SP. Therefore, inhaled UiO66-Dex@NMP treatment demonstrated the most favorable therapeutic effect in SP, with significant improvements in both inflammatory cytokine levels and pathological lung injury.