Glycopeptide-Nanotransforrs Eyedrops With Enhanced Permeability And Retention For Preventing Fundus Neovascularization

BackroundPhysiological angiogenesis is critical during eye development and is essential for normal vision.In contrast,pathologic angiogenesis occurs in diseases such as proliferative diabetic retinopathy(PDR)and retinopathy of prematurity(ROP).Due to the high permeability of neovascularization,it often leads to repeated hemorrhage,blurred vision,retinal traction detachment and even complete loss of vision.Under the stimulation of certain inflammatory factors such as IL-4,mature macrophages can differentiate into M2 macrophages.They accelerate the growth of vascular endothelial cells by promoting the secretion of VEGF,or promote the proliferation and migration of vascular endothelial cells by promoting the secretion of MMP-2 and MMP-9 in the extracellular matrix,and further induce abnormal retinal vascular proliferation.It has been reported that reducing M2 macrophages can effectively reduce fundus neovascularization.There are generally two ways to deliver drugs to the fundus,one is invasive treatment such as intravitreal injection,the other is non-invasive treatment such as eyedrops.Intravitreal injection is commonly used in clinic because of its highly targeted local effects and easy to achieve therapeutic concentration.However,it has potential side effects and complications such as increased intraocular pressure,intraocular inflammation,and complicated cataracts.Due to the structure and physiological characteristics of the eyeball itself,only about 5%of the eye drops remain on the surface of the eye after a local eye spotting,and even less drugs can reach the fundus,resulting in poor bioavailability of drugs.Combined with the above problems,there is currently a lack of a non-invasive and efficient drug delivery method that can penetrate the surface to release the drug slowly to the posterior segment of the eye.Therefore,we designed a morphology-transformable co-assembled glycopeptide nanomaterial(MRP@DOX)as a drug delivery system,which can effectively penetrate the corneal and scleral barriers,and then target M2 macrophages with a long-lasting effect.The retention of the drug will ultimately prevent the formation of new blood vessels in the fundus.The properties and mechanism of action were discussed by solution state and in vitro and in vivo experiments.Part Ⅰ Synthesis and Assembly Properties of MRP@DOXObjectivePositively charged co-assembled glycopeptide nanoparticles(MRP@DOX)were synthesized to explore the optimal ratio between different components.Methods1.The cationic peptide RP(sequence:RTTAANLVFF)was synthesized by solidphase synthesis,and the glycopeptide MP(sequence:Mannose-AANLVFF)was synthesized by the Click reaction.The molecular weight and purity of MP and RP were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)and high performance liquid chromatography(HPLC).2.The critical micelle concentration(CMC)of MP and RP was determined by pyrene fluorescence probe spectroscopy,and the assembly behavior of MP and RP was analyzed by transmission electron microscopy(TEM)and dynamic light scattering(DLS).3.MRP co-assembles with different proportions of MP and RP were synthesized.The morphology and charge of the co-assembled bodies were analyzed by TEM and DLS.The secondary structure was determined by circular dichroism(CD)to analyze the assembly rules of different component proportions.4.MRP@DOX was prepared by sequential nanoprecipitation method,the encapsulation efficiency(EE)and drug loading(DL)were calculated by UV-Vis spectrophotometer,and the optimal proportions of MP,RP and DOX was explored.The morphology and diameter of MRP@DOX were characterized by TEM and DLS.Results1.The CMCs of MP and RP were 3.4 μM and 18.5 μM.2.MP assembled into nanofibers with a cross-sectional diameter of about 1.9 ±0.2 nm and a zeta potential of+4.2 mV,and RP into nanoparticles with a diameter of 37.8± 9.0 nm and a zeta potential of+18.1 mV.3.The β-sheet structure content of MP is 40.9%,and the β-sheet structure content of RP is 10.1%.When the molar ratio of MP and RP in MRP changed from 1:10 to 1:100,the content of β-sheet gradually decreased,and MRP approached the nanoparticle structure.4.The optimal molar ratio of MP:RP:DOX is 1:50:50.At this time,MRP@DOX is a nanoparticle with a size of 148.5±7.3 nm and+14.2 mV.ConclusionsGlycopeptides,cationic peptides and DOX can be co-assembled into positively charged nanoparticles(MRP@DOX)in a molar ratio of 1:50:50,which meets the requirements for fundus drug delivery.Part II Specific Enzymatic Cleavage of MRP@DOX by legumainObjectiveThe specific enzyme cleavage effect of legumain on MRP@DOX was investigated at the solution state and cell level,and the cleavage site and the morphological changes before and after digestion were explored.Methods1.Incubate RP and legumain for 24 h,use HPLC and MALDI-TOF-MS to detect the enzyme cleavage site of legumain.2.The AAN sequence in the two peptides in MRP@DOX was replaced by the AAA sequence,which was set as the control group(MAP@DOX).The experimental group and the control group were incubated with legumain for 24 h.TEM and DLS were used to observe the dynamic change process of the morphology of the solution nanomaterials at different time points.3.MRP@DOX or MAP@DOX were incubated with legumain for 24 h,and the changes of fluorescence morphology before and after enzyme cleavage were observed by confocal laser scanning microscope(CLSM),and the mode of action of DOX and nanoparticle carrier or nanofiber carrier was speculated.4.M2 macrophages with high expression of legumain in lysosomes were coincubated with MRP@DOX or MAP@DOX for 24 h,and the morphology changes of intracellular nanomaterials were observed by bio-transmission electron microscopy(bio-TEM).Results1.The peak time of RP was 11.6 min and the molecular weight was 1139.7.After co-incubating with legumain,new peaks at 2.9 min and 12.8 min and new molecular weights of 547.3 and 633.3 appeared,suggesting that legumain can recognize the AAN sequence and hydrolyze the amide bond at the carboxy terminus of the amide(N)generates a new product with the sequence LVFF.2.The MRP@DOX nanoparticles in the experimental group were reassembled into nanofibers by the enzyme cleavage of legumain,while the MAP@DOX in the control group maintained the morphology of nanoparticles.3.MRP@DOX not co-incubated with legumain exhibited a fluorescent form of nanoparticles,which proved that DOX was encapsulated in the hydrophobic core of nanoparticles;while MRP@DOX co-incubated with legumain was sheared to form LVFF nanofibers,thus showing fluorescence in the form of nanofibers.The above phenomenon proves that DOX is intercalated in the β-sheet structure of nanofibers.In the control group,the fluorescence morphology was always the nanoparticle morphology,proving that MAP@DOX was not cleaved.4.In M2 macrophages,MRP@DOX nanoparticles in the experimental group formed nanofibers under the action of enzyme cleavage,while MAP@DOX in the control group maintained the shape of nanoparticles.ConclusionsLegumain can recognize the AAN sequence and hydrolyze the amide bond at the carboxyl terminus of asparagine(N),enabling MRP@DOX from the morphological assembly of nanoparticles to nanofibers.Part Ⅲ Exploring the Action Process and Retention Mechanism of MRP@DOX in vitroObjectiveTo investigate the specific targeting effect of MRP@DOX on M2 macrophages,the action process of legumain after entry,and the mechanism of its long-term retention in cells.Methods1.M2 macrophages or HUVECs were co-incubated with MRP@DOX for 1 h,and the cell surface was observed by scanning electron microscope(SEM).2.The M2 macrophages were co-incubated with MRP@DOX,and the fluorescence intensity was detected by CLSM at 1 h and 4 h respectively,and the distribution of MRP@DOX in the cells with time was observed.3.The intracellular metabolic environment was simulated by continuously changing the medium.The retention time and real-time retention of experimental group MRP@DDOX and control group MAP@DOX in M2 macrophages were observed by CLSM at different time points within 24 h;The 6 h time point was taken to calculate the amount of DOX released into the nucleus,and to explore the retention mechanism of nanofibers.4.CCK-8 assay was used to detect the cytotoxicity of DOX,MRP and MRP@DOX on M2 macrophages or HUVECs,and to explore the specific killing effect of MRP@DOX on M2 macrophages.5.The supernatant of M2 macrophages,M2 macrophages co-incubated with MRP@DOX and M2 macrophages co-incubated with MAP@DOX were taken,and the effect of each group on cell proliferation and migration was observed by scratch test.Results1.The SEM results showed that after co-incubation with MRP@DOX,a large number of nanoparticles were attached to the surface of the originally smooth M2 macrophages,but after co-incubation with HUVECs,the cell surface was still smooth,and there was no obvious nanoparticle attachment phenomenon.2.At 1 h,MRP@DOX mainly accumulated in lysosomes,and the colocalization coefficient(CC)of MRP@DOX and lysosome was 0.97.At 4 h,MRP@DOX was evenly distributed in cells,and the colocalization coefficient of MRP@DOX and lysosome was 0.72.It is speculated that MRP@DOX mediates endocytosis after binding to cell surface ligands,first entering the cell lysosome,then forming nanofibers under the action of legumain,escaping from the lysosome and releasing DOX into the nucleus.3.Compared with the MAP@DOX control group,MRP@DOX group had a longer retention time in the cells and more DOX into the nucleus.A large number of expelled nanoparticles were found around the cells of the control group,while no obvious nanoparticles were found around the cells in the MRP@DOX group,suggesting that the nanofibers could achieve intracellular retention by inhibiting exocytosis.4.MRP has no cytotoxicity to M2 macrophages and HUVECs.DOX has a killing effect on both M2 macrophages and HUVECs.MRP@DOX encapsulated with DOX shows dose-dependent killing to M2 macrophages.However,there was no obvious cytotoxicity to HUVECs,demonstrating that MRP@DOX reduced the cytotoxicity of DOX against M2-type macrophages.And the killing effect on M2 macrophages was better than that of the MAP@DOX control group that maintained the nanoparticle morphology.5.M2 macrophages can promote endothelial cell proliferation and migration,which can be inhibited by co-incubation of MRP@DOX or MAP@DOX.The effect of MRP@DOX group is better than that of MAP@DOX control group.ConclusionsMRP@DOX can precisely target M2 macrophages.After entering the cells,lysosome escape is achieved through morphology transformation,and DOX can be successfully released into the nucleus.Nanofibers remain in cells for a long time by inhibiting cell exocytosis.MRP@DOX selectively kills M2 macrophages and inhibits the proliferation of endothelial cells induced by M2 macrophages.Part Ⅳ Evaluation of the Effect of MRP@DOX on Reducing Retinal neovascularization in a mouse OIR modelObjectiveTo investigate the ocular access route of MRP@DOX in mice,to detect the effect of MRP@DOX on reducing retinal neovascularization in mouse OIR model,and to evaluate the biosafety of MRP@DOX on eye tissue and heart.Methods1.MRP@DOX was used to instill the eyes of mice,and frozen sections were used to observe the fluorescence distribution of eye tissue,and to analyze the way of MRP@DOX entering the eyes.2.OIR model of mice was established.The 7-day-old mice were placed in a hyperoxic environment,restore the normal oxygen environment on the 12th day.The mice were instilled with MRP@DOX once a day,and all mice were sacrificed on the 17th day.Stretched preparation of retina,use CLSM to observe the area of the central non-perfusion area of the retina,and calculate the number of peripheral retinal neovascular branches within per unit area;use the paraffin section HE staining to calculate the number of cell nuclei that break through the retinal inner limiting membrane per unit area;use immunohistochemical staining to observe VEGF unit area optical density.3.MRP@DOX was used to instill eyes in mice for 28 days,the corneal appearance was observed and tissue sections were made to detect corneal thickness,which was used to test the safety of MRP@DOX on eye tissue;blood routine tests were used to detect the levels of white blood cells and platelets,and blood biochemical tests The levels of creatine kinase(CK)and lactate dehydrogenase(LDH),and cardiac tissue sections were used to observe the myocardial state to test the safety of MRP@DOX on cardiac tissue.Results1.MRP@DOX is distributed in all layers of the cornea and retina.2.In the mouse OIR model,MRP@DOX can reduce the central retinal nonperfusion area,reduce the number of peripheral retinal neovascular branches per unit area,reduce the number of nuclei that break through the retinal inner limiting membrane per unit area,and reduce the expression of VEGF.And the effect is better than that of the MAP@DOX control group that cannot be converted into nanofiber morphology.3.After local instillation of DOX solution,corneal edema,thickening and stromal disorder appeared in mice,while the eye tissue was normal after MRP@DOX instillation,which proves that MRP@DOX can prevent corneal edema and inflammatory reaction caused by DOX,reducing the cytotoxicity of DOX to ocular tissues;the levels of leukocytes,platelets,creatine kinase(CK)and lactate dehydrogenase(LDH)were normal,and the cardiac tissue sections were normal,proving that MRP@DOX can prevent DOX-induced cardiotoxicity.ConclusionsMRP@DOX reaches the retina through both corneal and sclera pathways.In the mouse OIR model,MRP@DOX can reduce retinal neovascularization.MRP@DOX has good biocompatibility.