| Objective:Using the donor cell membrane modification strategy and the electroporation technology,our research group successfully synthesized vascular targeted exosomes incorporated with black phosphorus quantum dots(BPQDs@RGD-EXO,BREs).We explored the effect and mechanism of BREs on angiogenesis in vivo and in vitro,aiming to provide new strategy for the treatment of retinal neovascular diseases.MethodsIn order to improve the biocompatibility and vascular targeting ability of black phosphorus quantum dots(BPQDs),we synthesized engineered exosome BREs by using donor cell membrane modification strategies and electroporation technology.Transmission electron microscopy(TEM),nanoparticle tracking analyzer(NTA),Western blot were used to explore the physical characterization of BREs.The targeting ability of BREs was assessed by immunofluorescence staining in vitro and in vivo.The live-dead staining assay and HE staining of retinal tissues were used to explore the biocompatibility of BREs.The angiogenesis assay of HUVECs cells was used to explore the effects of BREs on angiogenesis in vitro.The anti-angiogenic role of BREs in vivo was evaluated by mouse retinal vascular development model and oxygen-induced retinopathy model.Combined RNA-seq and metabolomic analysis were performed to explore the anti-angiogenic mechanism of BREs in vitro and in vivo,screening out differentially expressed genes and metabolites,we used GO gene analysis and KEGG pathway enrichment analysis to find out the relevant pathways and further explore their functions.Quantitative Real-time PCR,Western Blot and retinal immunofluorescence staining were used to verify the potential mechanism.Finally BREs disrupted glucose metabolism,which was further confirmed by evaluating metabolites,Lactic Acid,Pyruvate and ATP production.Results1.Our research group successfully synthesized engineering exosome BREs.The BREs showed a cup-shaped structure with an average size of 105 nm.Western Blot analysis showed that BREs expressed exosome specific markers.The successful modification of RGD on exosomes was confirmed by H NMR and Coomassie blue staining.Inductively coupled plasma-mass spectrometry(ICP-MS)showed that BPQDs quantum dots were successfully incorporated in BREs.The targeting ability of BREs was assessed by fluorescence microscopy.Our data indicated that BREs had good vascular targeting ability in vitro and in vivo.The biocompatibility of BREs was assessed by live/death staining,indicating that BREs had no toxicity to HUVECs cells.HE staining was performed on the retinal tissue of mice and other important organs of mice,which showed that intraocular injections of BREs had no toxicity to mice.2.BREs can inhibit angiogenesis in vivo and in vitro.BREs significantly inhibited the migration,tube-formation and proliferation of HUVECs cells.The anti-angiogenic role of BREs in vivo was evaluated by mouse retinal vascular development model and oxygeninduced retinopathy model.In the mouse retinal vascular development model,our data revealed that the number of tip cells and vascular length were apparently decreased in the sprouting region of the retinal vasculature in BRE-treated retinas,indicating that BREs could inhibit physiological angiogenesis in vivo.In the OIR model,BREs significantly attenuated pathological neovascularization and avascular areas,indicating that BREs could inhibit pathological neovascularization in vivo.3.BREs target glucose metabolism to suppress retinal angiogenesis.We performed RNA seq analysis suggested that c-Myc and HK2 were differentially expressed genes after BREs treatment,these genes were mainly enriched in glucose metabolism.Meanwhile,metabolomics analysis suggested that glycolytic-related metabolites decreased in BREstreated group.These results suggest that BREs target glucose metabolism via the cMyc/HK2 pathway to attenuate retinal angiogenesis.Immunofluorescence and Western Blot indicated that HK2 and c-Myc expression were upregulated in the retina of the OIR model,which could be inhibited by BREs.ConclusionWe designed a vascular targeting and biocompatible BREs and evaluated its antiangiogenic properties in vitro and in vivo.Combined RNA-seq and metabolomic analysis revealed that BREs mainly disrupted glucose metabolism through the c-Myc/HK2 pathway.Our study suggests that engineered BREs show great therapeutic potential in pathological angiogenesis,which could be a promising alternative for anti-VEGF therapy. |
