Abstract
OBJECTIVE To construct methylprednisolone(MPS)-loaded liposomes modified with a monocyte-targeting peptide(MP-MPS-Lipo)and evaluate their targeted therapeutic efficacy and safety against acute lung injury(ALI).METHODS ① Preparation and characterization:MP-MPS-Lipo was prepared using the thin-film hydration method.Particle size,polydispersity index(PDI),and zeta poten-tial were characterized by dynamic light scattering.Free MPS was separated via ultrafiltration-centrifu-gation,and the MPS concentration was quantified by ultra-performance liquid chromatography(UPLC)to calculate encapsulation efficiency and drug loading.Serum stability was evaluated using dynamic light scattering.In vitro drug release was assessed via dialysis combined with UPLC,and cytotoxicity was determined using a CCK-8 assay.② In vitro uptake by inflammatory monocytes:An inflammatory WEHI 274.1 monocyte model was established via co-stimulation with lipopolysaccharide(LPS,100 μg·L-1)and interferon-γ(IFN-γ,20 μg·L-1).Cells were assigned to a control group or treated with conventional liposomes(RhB-Lipo),PEGylated liposomes(RhB-PEG-Lipo),or peptide-targeted liposomes(RhB-MP-Lipo),all labeled with 1%rhodamine B(RhB).Flow cytometry was used to quantify the percentage of RhB-positive cells.A C-C chemokine receptor type 2(CCR2)antagonist(RS504393 50 μg·L-1)was added for receptor blockade to verify the targeting mechanism.③ Tissue distribution of RhB-MP-Lipo in ALI mice:An ALI model was induced in BALB/c mice via the intratracheal instillation of LPS(5 mg·kg-1).Model mice were intravenously injected with RhB-labeled liposomes(RhB-Lipo,RhB-PEG-Lipo,or RhB-MP-Lipo)at an RhB dose of 100 μg·kg-1.At 4 h post-injection,the fluorescence intensities of the heart,liver,spleen,lung,and kidney were analyzed using ex vivo fluorescence imaging,and the lung-to-liver fluorescence ratio was calculated to evaluate tissue distribution.④ In vivo efficacy and safety of MP-MPS-Lipo in ALI mice:Mice were randomly divided into five groups:control,model,model+MPS,model+PEGylated MPS-loaded liposomes(PEG-MPS-Lipo),and model+MP-MPS-Lipo.At 6 h post-modeling,the treatment groups received corresponding formulations intravenously(1 mg·kg-1 MPS equivalent),while the control and model groups received equal volumes of saline.A second identical dose was administered 24 h after the first injection,for a total of two doses.At 48 h post-modeling,bronchoalveolar lavage fluid(BALF),serum,and major organs were collected.Total cells and neutro-phils in the BALF were counted using flow cytometry,and inflammatory cytokines(TNF-α,IL-6,IL-1β,and IL-10)were quantified by ELISA.Pulmonary pathological damage was assessed via HE staining,and inducible nitric oxide synthase(iNOS)expression was detected by immunofluorescence.⑤ Mice were randomly divided into four groups:control,MPS,PEG-MPS-Lipo,and MP-MPS-Lipo.The treat-ment groups received daily intravenous injections of the corresponding formulations(1 mg·kg-1 MPS equivalent)for 7 consecutive days while the control group received normal saline.At 24 h after the final administration(day 8),serum and major organs were harvested.HE staining was performed to examine pathological changes,and serum biochemical parameters(alanine aminotransferase,aspartate amino-transferase,high-density lipoprotein cholesterol,uric acid,and creatinine)were measured to evaluate hepatic function,renal function,and lipid metabolism.RESULTS ① MP-MPS-Lipo had an average size of(84.9±3.2)nm,a PDI of(0.19±0.03),and a zeta potential of(-10.0±0.2)mV,with an encapsulation efficiency of(88.41±0.03)%and a drug loading of(11.88±0.03)%.The particle size remained stable at 100-110 nm after 48 h of serum incubation.In vitro cumulative release was(8.10±0.03)%at 4 h and(54.23±0.50)%at 96 h.Viabilities of both monocytes and endothelial cells remained>80%at MPS concentrations≤2 g·L-1.② The monocyte uptake rate of RhB-MP-Lipo was(75.83±2.01)%,significantly higher than that of RhB-PEG-Lipo.This cellular uptake was markedly inhibited following CCR2 antagonist pretreatment.③ At 4 h post-injection,the lung-to-liver fluores-cence ratio for the RhB-MP-Lipo group was 0.48±0.11,approximately twice that of the RhB-PEG-Lipo group.④ Compared with the LPS model group,MP-MPS-Lipo treatment markedly reduced the propor-tion of neutrophils as well as the concentrations of pro-inflammatory cytokines(TNF-α,IL-6,and IL-1β)in the BALF,but elevated the anti-inflammatory cytokine IL-10.Alveolar architecture was largely restored,and the iNOS fluorescence signal was noticeably weakened.⑤ No obvious histopathological toxicity was observed in the major organs across the treatment groups,and serum biochemical parame-ters remained within normal physiological ranges.CONCLUSION MP modification significantly enhances the monocyte uptake of liposomes and facilitates targeted pulmonary accumulation in ALI,thereby improving the anti-inflammatory efficacy of methylprednisolone.关键词
急性肺损伤/单核细胞/细胞搭便车/靶向递送/脂质体/甲泼尼龙Key words
acute lung injury/monocytes/cellular hitchhiking/targeted delivery/liposomes/methyl-prednisolone分类
医药卫生
