单核细胞靶向肽修饰载甲泼尼龙脂质体的构建及其对急性肺损伤的靶向治疗OA

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.