PLGA (Mw, 20 kDa; lactic acid: glycolic acid, 25:75) with a terminal carboxyl group was purchased from Daigang (Jinan, China). Fluorescent, internally labeled, polystyrene microspheres (PS microspheres) of various sizes (3, 10, 25 and 40 μm) were purchased from Unibead Scientific Co., Ltd (Tianjin, China). Rhodamine B and RPMI 1640, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Microfil was purchased from Flow Tech, Inc. (Massachusetts, USA). All other chemicals and reagents were obtained from commercial sources with the analytical reagent available.
Male Kunming mice 4 weeks old, 20-22 g were purchased from Pengyue (Shandong, China), and were housed under specific-pathogen-free conditions, with a 12-h light/12-h dark cycle. All experimental animal protocols were approved by the Animal Protection and Research Ethics Committee of Qingdao Agricultural University.
Acute toxicity evaluation
Mice were randomly assigned to nine groups (n = 6 per group), including one control group with PBS injection and eight experimental groups intravenously administered PLGA (3 mm: PLGA-3, 10 mm: PLGA-10, 25 mm: PLGA-25, or 40 mm: PLGA-40) or PS microspheres (3 mm: PS-3, 10 mm: PS-10, 25 mm: PS-25, or 40 mm: PS-40). For the microspheres groups, 0.5 mL of the microspheres in phosphate-buffered saline (PBS) was injected, while 0.5 mL of PBS was administered to the control group. After administration, we observe the mice daily for abnormal behaviors. Dead mice were immediately weighed and dissected. The lungs were harvested, photographed, and weighed to calculate the lung coefficient, which is defined as the percentage of organ weight to body weight. Lung samples were fixed in 10% (v/v) neutral buffered formalin at 24-26℃. The fixed samples were embedded in paraffin, cut into 5 mm-thick sections, and stained with a hematoxylin and eosin (H&E) staining kit (Jiancheng Biotech, Jiangsu, China) using a standard histological method.
Micro CT imaging
Lung perfusion and casting were performed according to a previous study . Briefly, mice were divided into five groups (PBS, PLGA-3, PLGA-10, PLGA-25, PLGA-40). Mice have injected with PLGA microspheres (250 mg/kg) and the mice were anesthetized with 5% chloral hydrate after injected for 1 hour. After removing the anterior chest wall, a syringe was inserted through the right ventricle, and the lungs were perfused with normal saline to remove blood. The Microfil was then mixed (MV-122: MV diluent: MV curing agent = 4:5:0.5) and perfused through the lung. After solidifying at 4°C overnight, the lungs dehydrated with an ethanol gradient, and finally immersed in methyl salicylate to render the lung tissue transparent. The lungs were scanned on a micro CT scanner (Quantum GX2, PerkinElmer, USA) and established a 3-dimension (3D) model of the pulmonary blood vessels. The scans were acquired using the following settings: field of view (36 mm), energy or intensity (90 kV, 88 mA), and integration time (14 min).
Fluorescence imaging of mice was performed using a small animal in vivo imaging system (IVIS Spectrum, PerkinElmer, USA). DsRed excitation (λex= 500–550 nm) and emission (λem= 575–650 nm) filters were used. Mice were injected with microspheres (3, 10, and 25 mm) in PBS through the tail vein. 30 min after the injection of the microspheres, the mice were anesthetized by isoflurane and imaged using the in vivo imaging system. The heart, lungs, liver, spleen, and kidneys were then removed intact and imaged intact ex vivo. Additionally, different pro-inflammatory mediators, namely tumor necrosis factor-α (TNF-α), and interleukins 1β (IL-1β), and interleukins 8 (IL-8) were measured in the Lung tissue homogenate (PBS, pH 7.4, 1:9) using enzyme-linked immunosorbent assay (ELISA) kits (Meimian, Jiangsu, China).
Dose assessment of blood oxygen saturation and lung injury
The four monosized PLGA and PS microspheres were dispersed in PBS (pH 7.4). The microspheres were vortexed immediately before mice (n = 3 per group) tail vein injection. The mice were anesthetized by 5% chloral hydrate after injection. Arterial hemoglobin oxygen saturation (SpO2) of the mice was monitored by a pulse oximeter (YK-820 miniA, USA) for 10 days. After 10 days, the lung tissues of the mice were collected for histopathological examinations and stained with H&E.
To further verify the maximum injection dose tolerated in mice, we used a small animal in vivo imaging system to quantitatively measure the fluorescence accumulation in the mouse lung tissue. Microspheres with the optimum particle size were used as injection drugs, and the lung tissues of the mice were imaged and fluorescence quantitative analysis at 15, 30, and 60 min after tail vein injection with different doses (125, 250, 375, 500 mg/kg).
Lung injury and post-injury repair
The mice were injected with high-dose (250 mg/kg) PLGA microspheres (3, 10 mm, n = 3 per group) through the tail vein. Lung samples were collected after seven days. One portion was used for western blot analysis, and the other portion was fixed in neutral formalin. The fixed samples were embedded in paraffin, stained with H&E, and subjected to immunofluorescence.
The mice were injected intravenously with PLGA microspheres (10 mm) of different doses (75, 150, 225 mg/kg, n = 3 per group). The lung tissues of the mice were formalin-fixed and paraffin-embedded on day 7 and day 14, stained with H&E, and subjected to immunofluorescence. Image J was used to calculate the microvessel density.
The expression of TNF-α (Abcam, UK, 1:200), IL-1β (Abcam, Cambridge, UK, 1:200), p38 (Abcam, Cambridge, UK, 1:200), vascular endothelial growth factor-A (VEGF-A, Abcam, Cambridge, UK, 1:200), and matrix metalloproteinase-9 (MMP-9, Abcam, Cambridge, UK, 1:200) was detected by western blot analysis. The lung tissues were collected and the protein was isolated by RIPA lysis buffer, quantified by BCA protein assay. After the standard procedure of western blot, protein bands were visualized by enhanced chemiluminescence (ECL) detection reagents and captured by Tanon-4200 Gel Imaging System (Tanon Science, China). The quantitative analysis of western blot was carried out by Image J software.
Immunofluorescence analysis was performed to detect the expression of MMP-9， VEGF, and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31, Bioss, Beijing, China, 1:200). The paraffin sections of the collected samples were deparaffinized with xylene and ethanol, rehydrated, subjected to antigen retrieval, blocked with bovine serum albumin (BSA, 5%, 30 min), and incubated overnight at 4°C with specific primary antibodies. After rinsing with PBS, the samples were incubated with appropriate corresponding secondary antibodies and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China). All images were captured with a fluorescence microscope (CKX53, Olympus, Japan) and analyzed using the NIS Elements Advanced Research software (Nikon, Japan).
All data are expressed as the means ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0. Tests for significant differences were analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.