To investigate the properties of aloe-derived EV nanoparticles, we isolated and purified EV_Aloe from the aloe-homogenized juice (defined as EV_Aloe) by consecutive centrifugation and ultracentrifugation (Figure 1A), and subsequent transmission electron microscopy (TEM) examination (Figure 1B) and nanoparticle tracking analysis (NTA) (Figure 1C) revealed that the EV_Aloe exhibited a classic cup-shaped spherical structure, with an average diameter of 144.5 ± 2.8 nm. The purified EV_Aloe was quantified using a micro-bicinchoninic acid (BCA) protein analysis kit. The results indicated a high abundance of EV nanoparticles in aloe (approximately 500 mg/kg), suggesting that aloe can generate a significant amount of EV nanoparticles. Furthermore, we conducted a duplicate analysis of the protein composition of the purified EV_Aloe using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1D). The findings revealed a plethora of proteins within the EV_Aloe that potentially possess immune-modulating capabilities.

To evaluate the biosafety of EV_Aloe, we conducted cell viability and hemolysis assays. The EV_Aloe exhibited minimal toxicity to macrophages at dosages up to 200 μg/mL (Figure 2A). Hence, we set the EV_Aloe concentration below 200 μg/mL for assessing macrophage uptake and polarization modulation in our cellular study, considering that higher concentrations might induce cellular toxicity and complicate immunological responses. Hemolysis tests conducted on red blood cells incubated with various concentrations of EV_Aloe revealed no observable hemolysis within a wide range of EV_Aloe concentrations (Figure 2B).

To evaluate the impact of EV_Aloe on macrophage immune activation, we first assessed macrophage uptake and internalized EV_Aloe. Employing DID-labeled EV_Aloe, we visualized the phagocytosis and internalization of EV_Aloe on macrophages. Immunofluorescence imaging revealed a dose-dependent increase in EV_Aloe within macrophages (Figure 3A). Additionally, similar outcomes were obtained through flow cytometry analysis, where the fluorescence signal of EV_Aloe was notably higher in the group incubated with 200 μg EV_Aloe than in other groups, indicating a dose-dependent increase in EV_Aloe internalization by macrophages (Figures 3B, C). Importantly, as mentioned earlier, varying concentrations of EV_Aloe showed no evident cytotoxic effects on macrophages. Together, these results suggest that EV_Aloe can be engulfed and internalized by murine macrophages.

Regarding the preceding experiments, we confirmed the internalization and uptake of EV_Aloe by murine macrophages. Subsequently, we assessed the impact of varying EV_Aloe concentrations on the polarization capacity and phenotypic alterations in macrophages. Bright-field microscopy showed a notable transformation in macrophage morphology following EV_Aloe treatment, particularly treated with higher EV_Aloe concentrations, inducing more pronounced alterations in cell shape (Figure 4A). Additionally, flow cytometry analysis of primary macrophages treated with diverse EV_Aloe dosages revealed the most substantial percentage of M2-like tumor-associated macrophages (TAMs) within the group exposed to 200 μg of EV_Aloe (Figures 4B, C). These observations suggest that EV_Aloe exhibits remarkable and dose-dependent immunomodulatory attributes, effectively steering macrophages toward an M2 immune-activating phenotype (Figure 4C).

To assess the translational viability of murine macrophages influenced by EV_Aloe, we conducted a clinical feasibility study employing clinical bronchoalveolar lavage fluid (BALF) as a representative model (Figure 5A). Compared with lung biopsy, BALF is safer and less invasive, with few complications, and the resulting sample is larger than the source bronchus and multiple lung lobes (Mondoni et al., 2022). The information gained from BALF-EVs is regarded to be a complement to lung biopsy pathology (Zareba et al., 2021). To delve into this, we gathered BALF samples from bacterial pneumonia patients (n = 7), supported by confirmed clinical images (Figure 5A). Our investigation focused on discerning the immune impact of EV_Aloe on macrophage cells within BALF under ex vivo conditions. Flow cytometry analysis of macrophages after EV_Aloe incubation showcased a substantial increase in the expression levels of M2-associated surface markers compared to the untreated BALF control (Figures 5B–D). Simultaneously, a correlated decrease in the expression of M1-related protein markers was observed (Figure 5E). These discernible alterations in polarization biomarkers were further authenticated by quantifying the M1/M2 ratio (Figure 5F), signaling the effective repolarization of macrophages due to EV_Aloe treatment. Furthermore, employing enzyme-linked immunosorbent assay (ELISA) to evaluate the inflammatory cytokine profile changes in BALF revealed increased levels of inflammatory cytokines in pristine pleural effusion across all samples, aligning with previous clinical observations of immune BALF (Figure 5G). However, upon EV_Aloe treatment, a significant increase in anti-inflammatory cytokines evidently indicated the efficacy of the treatment. In concert, these results validate the substantial potential of EV_Aloe for clinical research and its profound impact on immune modulation.

EV_Aloe was isolated from aloe (bought from the Curacao aloe base of Kangyun Biological Company, Yunnan Province, China) juice by differential centrifugation and then purified using sucrose gradient centrifugation methods. In brief, the aloe was washed with deionized water and then homogenized using a blender. The mixtures were first consecutively centrifuged at 500 g for 10 min, 3,000 g for 10 min and 3,000 g for 30 min, and then, 10,000 g for 1 h to deplete large fibers and cell debris, and then, the supernatant was ultracentrifuged at 150,000 g for 2 h. We resuspended the obtained pellet of EV_Aloe in PBS and stored the solution at −80 °C until further use. For characterization of EV_Aloe, the particle sizes of EV_Aloe were characterized by NTA (Particle Metrix ZetaView, Germany). After screening of size, EV_Aloe was prepared for TEM imaging; 10 μL EV_Aloe was deposited onto the surface of a formvar-coated copper grid, 1% uranyl acetate was then added for 15 s twice, and the sample was allowed to dry for subsequent imaging. The EV_Aloe protein expression was analyzed by SDS-PAGE, the concentrations of which were quantified based on protein concentration using Bicinchoninic Acid Protein Assay (KeyGEN BioTECH) following the manufacturer’s protocol. Loading samples were prepared with 20 µg of protein per well. After the proteins in the loading samples were denatured for 10 min at 95°C, the loading samples were analyzed by SDS-PAGE in a Stain-Free™ Precast Gel (Bio-Rad #4568094). Phase contrast images were captured using an inverted microscope (Olympus CX41, Japan), and fluorescent images were captured by laser confocal microscopy (FV1000MPE, Olympus).

To measure the cytotoxicity of EV_Aloe in vitro, RAW 264.7 cells were incubated with different concentrations of EV_Aloe for 24 h. The cell viability was evaluated by using a CCK-8 assay kit (BS350B, Biosharp). Furthermore, to assess the blood compatibility of EV_Aloe, it was evaluated by hemolysis assay. In brief, pure 0.3 mL red blood cells were dispersed in 6 mL normal saline. Then, 0.1 mL of blood red blood cells were co-incubated with different concentrations of EV_Aloe (12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, and 200 μg/mL) at 37°C for 3 h. Distilled water and saline were regarded as the control. The mixtures were centrifuged, and then, the supernatant was measured at an absorbance of 540 nm. The hemolysis rate was calculated as follows: Hemolysis  % = A EV Aloe − A Negative / A Positive − A Negative × 100 % .

EV_Aloe was stained with 0.5 µM DiD far-red fluorescent probe (C1039, Beyotime) according to the manufacturer’s protocol. The RAW 264.7 macrophage cells were seeded into confocal dishes, and the different concentrations of EV_Aloe (20 μg, 100 μg, and 200 µg) were added for 4 h at 37 °C. Then, the cells were stained with the nucleus with the Hoechst 33258 (C1011, Beyotime). Laser confocal microscopy was used to present the stained cells (FV1000MPE, Olympus). Furthermore, the cells were then centrifuged at 500 g for 3 min and resuspended in PBS for further flow cytometry analysis. Fluorescent signals were assessed using a NovoCyte FACS flow cytometer (ACEA Biosciences, Inc.), and data were analyzed using FlowJo software.

To perform in vitro macrophage repolarization experiments, the initial M0 macrophages were treated with different concentrations of EV_Aloe (20 μg, 100 μg, and 200 µg) for 12 h at 37°C. Afterward, the macrophages were harvested and stained with anti-mouse CD11b-APC/Cyanine7 (BioLegend, Cat. No. 101226, clone M1/70) and anti-mouse CD206-PE (BioLegend, Cat. No. 141706, clone C068C2) and then subjected to flow cytometry. Fluorescent signals were detected using a NovoCyte FACS flow cytometer (ACEA Biosciences, Inc.), and data were analyzed using FlowJo software.

To examine macrophage phenotypic changes in the BALF, macrophages from BALF were separated via magnetic-activated cell sorting (MACS) using magnetic beads. For flow cytometry analysis, the macrophages after EV_Aloe treatment were fixed, permeabilized, and stained with anti-human CD11b-Percp/Cyanine5.5 (BioLegend, Cat. No. 301327, clone ICRF44) and M1 macrophage marker (anti-human CD80-PE, BioLegend, Cat. No. 305207, clone 2D10) and M2 macrophage marker (anti-human CD206-APC monoclonal Abs, BioLegend, Cat. No. 321109, clone 15-2) for flow analysis. All data were analyzed using FlowJo.

An ELISA kit was used to measure the concentrations of inflammatory cytokines and chemokines according to the manufacturer’s instructions. The macrophages from BALF were co-incubated with EV_Aloe for 24 h; then, the supernatant was collected for the detection of macrophage-related cytokines, such as the pro-inflammatory phenotype (IL-10, TNF-α, IFN-γ, and IL-6).