Cell-penetrating peptide-modified nano-cerium oxide eye drops, preparation method and application thereof

Abstract

The present invention belongs to the technical field of eye drops, and relates to a cell-penetrating peptide-modified nano cerium oxide eye drops, and a preparation method and application thereof. The nano cerium oxide eye drops contain water-soluble nano cerium oxide, and the surface of the water-soluble nano cerium oxide has a phospholipid polyethylene glycol maleimide-coupled cyclic cell-penetrating peptide, so that the surface of the water-soluble nano cerium oxide has a positive potential; wherein the water-soluble nano cerium oxide contains trivalent cerium and tetravalent cerium. The present invention adds the modification of amphiphilic cell-penetrating peptides to the surface of the water-soluble nano cerium oxide, so that the surface of the water-soluble nano cerium oxide has a higher positive potential, and the affinity of the cell-penetrating peptide itself to the cell membrane effectively prolongs the ocular surface residence time and improves the anterior segment penetration effect, increases the anterior chamber drug concentration of local eye drop treatment, and also provides a guarantee for the stability of nanoparticles in common media, and can be maintained stable and non-aggregated for more than 30 days in such as artificial tears, PBS and pure water.

Description

Technical Field

[0001] The invention belongs to the technical field of eye drops, and specifically relates to a cell-penetrating peptide-modified nano cerium oxide eye drop, a preparation method and an application thereof. Background Art

[0002] The development and progression of age-related cataract is a slow and multifactorial process, in which oxidative stress plays an important role and is the most important risk factor for the disease; oxidative stress refers to a state of excess oxidants relative to antioxidants, which may lead to harm or damage. A large number of antioxidants have shown encouraging results in laboratory settings, but their application is mainly limited to isolated lens experiments or oral, intraperitoneal or intravenous injections to experimental animals; this limits the feasibility of their wider practical application. The current challenge is how to deliver antioxidants to the anterior segment of the eye non-invasively by topical administration, mainly due to the uniqueness of the ocular surface microenvironment, the rapid turnover of the tear film and the dense corneal epithelial barrier, which pose a major obstacle to achieving optimal bioavailability of topically administered drugs.

[0003] Cerium oxide nanoparticles (less than 5 nanometers) have trivalent cerium Ce on their surface. 3+ and tetravalent cerium Ce 4+ The unique coexistence of cerium and tetravalent cerium states with a sufficient number of oxygen vacancies inside their lattice structure results in antioxidant activity through electron transfer between trivalent cerium and tetravalent cerium. In addition, cerium oxide nanoparticles of this size are able to easily recover their redox activity due to the intrinsic memory function of their lattice structure and the ability to exchange electrons with other ions, which enables them to repeatedly eliminate reactive oxygen radicals (ROS), demonstrating their unique self-regeneration properties.

[0004] However, in the use of ocular diseases, applying drugs to the ocular surface and expecting them to pass through the dense corneal barrier into the anterior chamber to treat anterior segment diseases is more challenging than delivering therapeutic substances directly to the closed vitreous cavity with slow fluid turnover. When eye drops are dropped on the ocular surface, the first obstacle encountered is the dynamic and rapidly turning over tear film, which forms the first permeability barrier that limits ocular drug delivery; inside the tear film, its innermost mucus layer plays the most important barrier role due to its negatively charged sugar groups and hydrophobic pore structure. Assuming that particles with positive surface charges can effectively attach to the tear film, what awaits them next is the dense corneal barrier composed of alternating hydrophilic and hydrophobic layers between different layers, making it almost impossible for most drug molecules to enter the anterior chamber through passive diffusion; the corneal epithelium and stroma represent the permeation rate limiting layer of drugs through the cornea. The outer multilayer epithelium of the cornea is hydrophobic, and its cells have tight junctions, forming a dense barrier to hinder the diffusion of hydrophilic drugs. Compared with the epithelium, the stroma is a hydrophilic fibrous layer, which is the main rate-limiting layer for the diffusion of hydrophobic molecules, resulting in hydrophobic molecules being passively transported only through intracellular pathways. Therefore, the molecular size and hydrophilicity / hydrophobicity of the drug are key factors affecting its corneal permeability.

[0005] Therefore, there is an urgent need in this field to solve the problem of drug delivery to the anterior segment of the eye by precisely controlling the properties of polymer nanocarrier particles and simply modifying their surface so that their excellent multifunctionality can be used. Summary of the invention

[0006] Based on the above-mentioned shortcomings and deficiencies in the prior art, one of the objects of the present invention is to at least solve one or more of the above-mentioned problems in the prior art. In other words, one of the objects of the present invention is to provide a cell-penetrating peptide-modified nano-cerium oxide eye drops and a preparation method and application thereof that meet one or more of the aforementioned needs.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A cell-penetrating peptide-modified nano-cerium oxide eye drop, the eye drop containing water-soluble nano-cerium oxide, the surface of which has a cyclic cell-penetrating peptide coupled with phospholipid polyethylene glycol maleimide DSPE-PEG-Mal, so that the surface of the water-soluble nano-cerium oxide has a positive potential;

[0009] Among them, the water-soluble nano cerium oxide contains trivalent cerium and tetravalent cerium.

[0010] As a preferred embodiment, the Zeta potential of the surface of the water-soluble nano-cerium oxide is 20-40 mV.

[0011] As a preferred embodiment, the hydraulic diameter of the water-soluble nano-cerium oxide is 5 to 30 nm.

[0012] As a preferred embodiment, the proportion of water-soluble nano-cerium oxide in the eye drops is 1 to 5 g / L.

[0013] As a preferred embodiment, the structural formula of the phospholipid polyethylene glycol maleimide DSPE-PEG-Mal coupled cyclic cell-penetrating peptide is:

[0014] ;

[0015] Here, n is an integer from 30 to 60.

[0016] The present invention also provides a method for preparing the nano cerium oxide eye drops as described in any of the above schemes, comprising the following steps:

[0017] (1) Synthesis of circular cell-penetrating peptides;

[0018] (2) Annular cell-penetrating peptides were coupled to phospholipid polyethylene glycol maleimide DSPE-PEG-Mal;

[0019] (3) The DSPE-PEG-Mal coupled cyclic cell-penetrating peptide is modified onto nano-cerium oxide to obtain water-soluble nano-cerium oxide; the aqueous solution of water-soluble nano-cerium oxide is nano-cerium oxide eye drops.

[0020] As a preferred solution, the step (1) specifically includes the following steps:

[0021] (a) placing 2-chlorotrityl chloride resin in dichloromethane (DCM) and shaking, and then filtering off DCM to obtain swollen 2-chlorotrityl chloride resin;

[0022] (b) reacting the swollen 2-chlorotrityl chloride resin with amino acid Fmoc-Cys(Trt)-OH in N,N-diisopropylethylamine DIEA and N,N-dimethylformamide DMF, and capping with methanol;

[0023] (c) removing the Fmoc protecting group of the product obtained in step (b);

[0024] (d) coupling the product obtained in step (c) with the amino acid Fmoc-Gln(Trt)-OH, and removing the Fmoc protecting group from the product obtained by the coupling reaction;

[0025] (e) Repeat step (d) and couple the following amino acids in sequence: Fmoc-Arg(pbf)-OH, Fmoc-(2-Nal)-OH, and Fmoc-Phe-OH;

[0026] (f) cleaving the polypeptide from the resin obtained in step (e) and obtaining a protected peptide segment after rotary evaporation;

[0027] (g) dissolving the protected peptide in a solvent DCM, adding benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate PyBop and DIEA for cyclization reaction, and rotary evaporating to obtain a cyclic protected peptide;

[0028] (h) The Trt protecting group and the pbf protecting group of the cyclic protected peptide segment were removed, followed by nitrogen drying and purification to obtain a cyclic cell-penetrating peptide.

[0029] As a preferred embodiment, in the step (2), the annular cell-penetrating peptide and DSPE-PEG-Mal are dissolved in a mixed solution of water and acetonitrile (ACN) for reaction, and then dialyzed, purified, and freeze-dried.

[0030] As a preferred embodiment, in the step (3), the DSPE-PEG-Mal coupled cyclic cell-penetrating peptide and the polyethylene glycol-modified phospholipid DSPE-PEG are dissolved in chloroform, then mixed with the nano-cerium oxide chloroform dispersion and subjected to rotary evaporation, followed by vacuum drying and purification to obtain water-soluble nano-cerium oxide, which is then dissolved in water to obtain nano-cerium oxide eye drops.

[0031] The present invention also provides the use of the nano cerium oxide eye drops as described in any one of the above schemes or the nano cerium oxide eye drops prepared by the preparation method as described in any one of the above schemes in the preparation of drugs for treating and preventing cataracts.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention adds an amphiphilic (i.e., hydrophilic and lipophilic) cell-penetrating peptide modification to the surface of water-soluble nano-cerium oxide. This modification makes the surface of water-soluble nano-cerium oxide have a higher positive potential. In addition, the affinity of the cell-penetrating peptide itself for the cell membrane effectively prolongs the residence time of the nanomaterial on the ocular surface, while improving the anterior segment permeation effect of the water-soluble nano-cerium oxide and increasing the anterior chamber drug concentration for local eye drop treatment. This performance improvement makes it possible to deliver therapeutic drugs non-invasively to the anterior segment of the eye. The high positive surface potential of the nanoparticles also guarantees their stability in common media, and they can remain stable and non-aggregating for more than 30 days in artificial tears, PBS and pure water. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Synthesis of the cyclic cell-penetrating peptide of Example 1 of the present invention and its reaction with phospholipid polyethylene glycol maleimide DSPE-PEG 2000 -Route map of Mal coupling;

[0035] Figure 2It is a comparison diagram of the hydrodynamic diameters of three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs, and cCPP-CeNPs of the present invention;

[0036] Figure 3 It is a comparison diagram of the Zeta potential of three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs, and cCPP-CeNPs of the present invention;

[0037] Figure 4 The transmission electron microscope TEM image, high-resolution transmission electron microscope HR-TEM image and selected electron diffraction SAED pattern of the hydrophobic nano-cerium oxide of Example 1 of the present invention;

[0038] Figure 5 This is an X-ray photoelectron spectrum of the hydrophobic nano-cerium oxide of Example 1 of the present invention;

[0039] Figure 6 This is an X-ray diffraction analysis XRD diagram of the hydrophobic nano-cerium oxide of Example 1 of the present invention;

[0040] Figure 7 It is a curve diagram showing the change of particle size of three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs dissolved in artificial tears over time;

[0041] Figure 8 It is a color change diagram of aging of aqueous solutions of three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs of the present invention after adding hydrogen peroxide H2O2 solution;

[0042] Fig. 9 It is the SOD simulation activity diagram of three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs of the present invention;

[0043] Fig.10 The EPR spectrum of the water-soluble nano-cerium oxide cCPP-CeNPs in Example 1 of the present invention at 9.867 GHz in the experimental X-band;

[0044] Fig.11 This is a confocal microscope image of the mouse eyeball (mainly the cornea and anterior capsule) 4 hours after the three water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs eye drops labeled with FITC of the present invention were applied; the white triangle arrow points to the anterior capsule of the lens;

[0045] Fig.12The present invention provides slit lamp microscope photographs of mouse eyes after being treated with different eye drops and ultraviolet disease modeling, and stereo microscope imaging comparison diagrams of mouse lenses after being removed from the body. DETAILED DESCRIPTION

[0046] In order to more clearly illustrate the embodiments of the present invention, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other accompanying drawings and other implementation methods can be obtained based on these accompanying drawings without creative work.

[0047] Embodiment 1:

[0048] The preparation method of the cell-penetrating peptide-modified nano-cerium oxide eye drops of this embodiment comprises the following steps:

[0049] (1) Preparation of cerium oxide nanoparticles CeNPs (i.e., hydrophobic nano cerium oxide);

[0050] 0.43 g of trivalent cerium acetate and 3.25 g of oleylamine were dissolved in 15 mL of xylene, the solution was mixed at room temperature and rapidly stirred (speed of 900 r / min) for 12 hours, then heated to 90°C at a heating rate of 2°C / min under an argon atmosphere, 1 mL of deionized water was rapidly injected into the heated solution to initiate a sol-gel reaction, the reaction indication of which was a color change from purple to turbid yellow; the reaction solution was then incubated at 90°C for 3 hours until it became transparent and cooled to room temperature; 100 mL of acetone was then added to precipitate hydrophobic cerium oxide nanoparticles CeNPs, which were harvested by centrifugation and then resuspended in chloroform to a final concentration of 10 mg / mL, and the purified hydrophobic CeNPs were easily dispersed in chloroform to obtain a nano-cerium oxide chloroform dispersion for storage and use.

[0051] (2) Synthesis of cyclic cell-penetrating peptide CyclicCPP;

[0052] like Figure 1 As shown, the synthesis process of cyclic cell-penetrating peptide CyclicCPP includes the following steps:

[0053] (a) Resin swelling: Place 2-chlorotrityl chloride resin in a reaction tube, add dichloromethane (DCM), the solid-liquid ratio of 2-chlorotrityl chloride resin to dichloromethane (DCM) is 1 g:15 mL, and shake for 30 minutes;

[0054] (b) Grafting the first amino acid, filtering off the solvent through a sand core to obtain swollen 2-chlorotrityl chloride resin, adding a 3-fold molar excess of Fmoc protected amino acid, i.e. amino acid Fmoc-Cys(Trt)-OH, then adding a 10-fold molar excess of N,N-diisopropylethylamine DIEA, followed by adding N,N-dimethylformamide DMF to dissolve, shaking for 30 minutes, and capping with methanol for 30 minutes;

[0055] (c) Deprotection: After the coupling of the first amino acid, deprotect with 20% piperidine / DMF solution for 15 minutes to remove the Fmoc protecting group; use ninhydrin reagent to detect the binding of the first amino acid on the resin, remove the piperidine solution, take a dozen resins, wash them three times with ethanol, add one drop of ninhydrin, potassium cyanide KCN, and phenol solution, heat at 105-110°C for 5 minutes, and turn dark blue for a positive reaction; then wash, wash with DMF twice, methanol twice, and DMF twice in sequence;

[0056] (d) Grafting the second amino acid, i.e., performing a condensation reaction, adding a 3-fold molar excess of Fmoc protected amino acid, i.e., Fmoc-Gln(Trt)-OH, to the product obtained in step (c), then adding a 3-fold molar excess of peptide coupling reagent HBTU, followed by adding a 10-fold molar excess of DIEA, and finally adding DMF to dissolve, and shaking for 45 minutes; then, checking whether the grafting is successful, removing the solution, taking a dozen resins, washing them three times with ethanol, adding one drop each of ninhydrin, potassium cyanide KCN, and phenol solution, and heating at 105-110°C until colorless, which is a negative reaction, indicating that the reaction is complete; then washing again, sequentially washing with DMF once, methanol twice, and DMF twice;

[0057] (e) Repeating the above steps (c) and (d) to sequentially couple the following amino acids: Fmoc-Arg(pbf)-OH, Fmoc-(2-Nal)-OH, and Fmoc-Phe-OH, until the Fmoc protecting group of the last amino acid is removed;

[0058] (f) Resin cleavage: prepare a cleavage solution consisting of a mixture of trifluoroethanol and dichloromethane (DCM) in a volume ratio of 3:7, and cleave the peptide from the resin for 120 minutes. Remove the solvent using a rotary evaporator to obtain a protected peptide segment, i.e., a protected peptide segment.

[0059] (g) Cyclization reaction, dissolving the protected peptide in DCM solvent, adding two-fold molar excess of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate PyBop and ten-fold molar excess of DIEA, reflux at 45°C, react overnight, and remove the solvent by rotary evaporation to obtain a cyclic protected peptide;

[0060] (h) Removing the peptide protecting group, preparing a cutting solution: trifluoroacetic acid TFA 95%, 1,2-ethanedithiol EDT 2%, triisopropylsilane TIS 2% and water H2O 1%, cutting time: 120 minutes, removing the Trt protecting group and pbf protecting group of the cyclic protected peptide segment, then blowing dry with nitrogen, washing with ether six times, and then evaporating at room temperature to obtain a crude cyclic peptide;

[0061] (i) Purification: Take a small amount of crude product and dissolve it in H2O / CAN to obtain a sample. Take a small amount of sample and analyze it on the HPLC analyzer to determine the peak time corresponding to the target peak. Use C18 reverse phase chromatography preparation system: Wavelength: 220nm, FlowRate: 15mL / min, Inj.Vol: 20mL, Column Temp: 25℃, Buffer A: 0.1% TFA in water, BufferB: 0.1%TFA in Acetonitrile to collect the target peak solution. Take a small amount of target peak solution in a 1.5mL centrifuge tube for mass spectrometry confirmation and purity detection, freeze-dry the qualified target peak solution, and obtain the cyclic cell penetrating peptide CyclicCPP. Among them, the specific process of the above purification can refer to the existing technology and will not be repeated here.

[0062] (3) Cyclic cell-penetrating peptide CyclicCPP and phospholipid polyethylene glycol maleimide DSPE-PEG 2000 -Mal coupling;

[0063] CyclicCPP and DSPE-PEG 2000 -Mal was dissolved in a mixed solution of water H2O and acetonitrile ACN, and reacted at room temperature to the end point, so that the cyclic cell-penetrating peptide CyclicCPP and DSPE-PEG2000-Mal were coupled together, and the obtained liquid peptide was re-lyophilized, and then the peptide was dissolved in acetonitrile and water, and dialyzed and purified with a 2000Da dialysis bag, and then lyophilized; a small amount of the finished peptide was taken for matrix-assisted laser desorption time-of-flight mass spectrometry MALDI-TOF identification, and the finished product was stored at -80°C; finally, the purified cyclic cell-penetrating peptide CyclicCPP was coupled with the phospholipid polyethylene glycol maleimide DSPE-PEG 2000 -Mal-coupled product, named CyclicCPP-DSPE-mPEG 2000 , whose structural formula is:

[0064] .

[0065] (4) CyclicCPP-DSPE-mPEG 2000 Modified to nano cerium oxide to obtain water-soluble nano cerium oxide;

[0066] In order to synthesize biocompatible nano-cerium oxide, CyclicCPP-DSPE-mPEG was coated on cerium oxide nanoparticles (CeNPs) by combining a thin film hydration method. 2000 and DSPE-PEG 2000 , so that the nanoparticles are transformed from hydrophobic to hydrophilic and grafted with CyclicCPP-DSPE-mPEG 2000 Specifically, 7.5 mg CyclicCPP-DSPE-mPEG 2000 、22.5mg DSPE-mPEG 2000 Dissolved in 5 mL of chloroform, then mixed with 10 mg / mL of nano-cerium oxide chloroform dispersion, evaporated the solution using a rotary evaporator and dried under vacuum at room temperature for 90 minutes; then added 10 mL of deionized water and ultrasonically treated, and the transparent light yellow suspension was filtered using a 0.2 μm pore size syringe filter; in order to remove free DSPE-PEG 2000 , nanoparticles were obtained by precipitation using 450000g high-speed ultracentrifugation for 2 h, and water-soluble nano-cerium oxide cCPP-CeNPs were thoroughly purified by filtration using an Amicon centrifugal filter with a cut-off molecular weight of 30 kDa;

[0067] The purified cCPP-CeNPs were stored in ultrapure water at 4°C to obtain 3 g / L nano-cerium oxide eye drops.

[0068] Comparative Example 1:

[0069] The water-soluble nano-cerium oxide in this comparative example is different from that in Example 1 in that DSPE-mPEG without coupling to CyclicCPP is used. 2000 ;

[0070] Coating of DSPE-PEG on Cerium Oxide Nanoparticles CeNPs by Combining Thin Film Hydration Method 2000 , so that the nanoparticles change from hydrophobic to hydrophilic; specifically, first 30 mg DSPE-mPEG 2000 Dissolved in 5 mL of chloroform, then mixed with 10 mg / mL of nano-cerium oxide chloroform dispersion, evaporated the solution using a rotary evaporator and dried under vacuum at room temperature for 90 minutes; then added 10 mL of deionized water and ultrasonically treated, and the transparent light yellow suspension was filtered using a 0.2 μm pore size syringe filter; in order to remove free DSPE-PEG 2000 , the nanoparticles were precipitated by ultracentrifugation at 450000 g for 2 h, and the water-soluble nano-cerium oxide PEG-CeNPs were thoroughly purified by filtration using an Amicon centrifugal filter with a cut-off molecular weight of 30 kDa;

[0071] The purified PEG-CeNPs were stored in ultrapure water at 4°C to obtain 3 g / L nano-cerium oxide eye drops;

[0072] For other steps, please refer to Example 1.

[0073] Comparative Example 2:

[0074] The difference between the water-soluble nano-cerium oxide in this comparative example and that in Example 1 is that Linear CPP-DSPE-mPEG 2000 Replacement of CyclicCPP-DSPE-mPEG 2000 ;

[0075] Specifically, the synthesis process of the linear cell-penetrating peptide LinearCPP differs from the synthesis process of the cyclic cell-penetrating peptide CyclicCPP in that the cyclization reaction of step (g) in Example 1 is omitted;

[0076] Accordingly, the linear cell-penetrating peptide LinearCPP and the phospholipid polyethylene glycol maleimide DSPE-PEG 2000 The product of -Mal coupling was named LinearCPP-DSPE-mPEG 2000 , whose structural formula is:

[0077] ;

[0078] Accordingly, the obtained water-soluble nano-cerium oxide lCPP-CeNPs were stored in ultrapure water at 4 °C, and 3 g / L nano-cerium oxide eye drops were obtained;

[0079] For other steps, please refer to Example 1.

[0080] The nano cerium oxide eye drops and their intermediates of Example 1, Comparative Example 1 and Comparative Example 2 are tested and characterized as follows:

[0081] like Figure 2 As shown in the figure, after the surface of cerium oxide nanoparticles was modified by linear cell-penetrating peptides or cyclic cell-penetrating peptides, the hydrodynamic diameter of nano-cerium oxide did not change significantly or aggregate, and remained at about 15nm. However, after being modified by linear cell-penetrating peptides or cyclic cell-penetrating peptides, the surface potential of nano-cerium oxide changed from negative potential to high positive potential, about +29mV. Figure 3 shown.

[0082] like Figure 4As shown in the figure, the representative transmission electron microscope TEM image, high-resolution transmission electron microscope HR-TEM image and selected electron diffraction SAED pattern of the cerium oxide nanoparticles CeNPs synthesized in Example 1 show similar shapes and sizes and good uniformity. Figure 5 As shown in FIG. 1 , it is shown that trivalent cerium and tetravalent cerium coexist in the cerium oxide nanoparticles CeNPs synthesized in Example 1, wherein trivalent cerium accounts for 43.62% and tetravalent cerium accounts for 56.38%. Figure 6 As shown, it is suggested that the cerium oxide nanoparticles CeNPs synthesized in Example 1 have a pure and typical fluorite cubic structure.

[0083] like Figure 7 As shown, the water-soluble nano cerium oxide of Example 1, Comparative Example 1 and Comparative Example 2 were dissolved in artificial tears, and the particle size change of the water-soluble nano cerium oxide within 30 days was detected. The results showed that it could remain free of aggregation for more than 30 days and had good medium stability. This was due to the high positive charge on the surface of the water-soluble nano cerium oxide. The high positive charge ratio of the nano cerium oxide modified with the cell-penetrating peptide in Example 1 was compared with the high negative charge of the nano cerium oxide in Comparative Example 1, which was mainly aimed at the environment of the ocular surface. There was a tear film on the ocular surface, and there was mucin in the tear film, which was negatively charged. This could increase the residence time of the nanoparticles of Example 1 in the tear film, and at the same time, it was not easy to agglomerate.

[0084] In order to analyze the self-regeneration characteristics of water-soluble nano-cerium oxide, H2O2 solution was added to the aqueous solutions of water-soluble nano-cerium oxide in Example 1, Comparative Example 1 and Comparative Example 2, and the solution color changes were observed after aging for 10 days. Figure 8 As shown, after adding H2O2 solution, the color of the solution changes from transparent to light yellow, and then after standing at room temperature for 10 days, the yellow color automatically fades and the solution returns to transparency; after adding H2O2 solution to the solution again, the solution turns light yellow again, and then after standing at room temperature for 10 days, the yellow color automatically fades and the solution returns to transparency; after adding H2O2 solution to the solution again, the solution turns light yellow again; this characteristic can still be observed with the naked eye after three cycles, indicating that the water-soluble nano-cerium oxide has the characteristic of automatic regeneration.

[0085] like Fig. 9 As shown, the water-soluble nano-cerium oxide of Example 1, Comparative Example 1 and Comparative Example 2 were respectively subjected to superoxide dismutase SOD simulation activity analysis, and the results showed that different concentrations of nano-cerium oxide showed different inhibition rates, so nano-cerium oxide can effectively remove superoxide anions in a dose-dependent manner, and the data are presented in the form of mean ± SD (n = 3), **** indicates a significant difference p < 0.0001.

[0086] like Fig.10As shown, through the EPR spectrum analysis of the water-soluble nano-cerium oxide cCPP-CeNPs in Example 1 at the experimental X-band 9.867 GHz, it can be seen that nano-cerium oxide can effectively remove hydroxyl free radicals in a dose-dependent manner. Fig. 9 The results show that the nano-cerium oxide of the present invention can imitate the activities of superoxide dismutase and catalase and effectively exert an antioxidant effect.

[0087] In addition, in order to synthesize water-soluble nano-cerium oxide PEG-CeNPs, lCPP-CeNPs, and cCPP-CeNPs conjugated with fluorescent dye FITC, FITC-conjugated DSPE-PEG was additionally added during the above-mentioned film hydration process. 2000 Add to the above mixture at a mass ratio of 1:50, and continue the subsequent synthesis and purification process, and finally obtain PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs with FITC labeling on the surface. Then, treat mice with cataracts. Before treatment, first verify whether the material does have the corneal barrier penetration effect. By using PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs with FITC labeling on the surface, verify their distribution in the anterior segment of the mouse eye, and use FITC aqueous solution freeFITC as a negative control; Fig.11 As shown, green fluorescence could only be observed on the anterior surface of the mouse cornea in the FITC aqueous solution-treated group; in contrast, in the eyes of mice treated with PEG-CeNPs, significant fluorescence was observed from the corneal epithelium to the corneal endothelium; in addition, after lCPP-CeNPs treatment, a weaker fluorescence signal was observed in the anterior capsule of the mouse lens, while a stronger fluorescence signal was observed after cCPP-CeNPs treatment, indicating that cCPP-CeNPs had the best penetration ability; among them, DAPI was used to mark the nuclei of the corneal layers and lens epithelial cells, and Merge indicated the co-localization of the fluorescent material with the cells.

[0088] From the above test characterization results, it can be seen that the above PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs, after a series of in vitro and cell experiments, have been characterized, confirming that after hydrophilic modification and surface engineering, the above three nanoparticles still maintain good in vitro free radical scavenging ability. Therefore, further in vivo treatment of cataracts in mice was carried out. First, the experimental eye drops were prepared: the drug concentration of PEG-CeNPs, lCPP-CeNPs and cCPP-CeNPs aqueous solutions was configured to 3 mg / mL; 3.6 kJ / m 2The total irradiation dose of medium-wave ultraviolet UVB induced early cataracts in mice, and 6-week-old female C57BL / 6 mice were randomly divided into 6 groups for experiments. The above groups were treated with PBS, PEG-CeNPs, lCPP-CeNPs, cCPP-CeNPs and commercially available Pirenoxine sodium eye drops, respectively. Eye drops were started three days before UV modeling, four times a day, and continued until the seventh day after modeling. On the seventh day after modeling, compared with the other four groups, whether it was observed in vivo under a slit lamp microscope or observed under a stereo microscope after dissection of the mouse eyes, it was obvious that the area of ​​subcapsular opacity in the cCPP-CeNPs group was significantly reduced, as shown in Figure 2. Fig.12 As shown, it is shown that the cCPP-CeNPs of the present invention have effective UVB damage resistance. Therefore, the nano cerium oxide eye drops of the present invention can be used in the preparation of drugs for treating and preventing cataracts.

[0089] In view of the numerous embodiments of the scheme of the present invention, the raw materials and dosage involved can be selected according to actual needs within the limited range, and the experimental data of each embodiment is huge and numerous, which is not suitable for listing and explaining one by one here, but the content required to be verified and the final conclusion obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be explained one by one here.

[0090] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A cell-penetrating peptide-modified nano-cerium oxide eye drop, characterized in that: The eye drops contain water-soluble nano-cerium oxide, and the surface of the water-soluble nano-cerium oxide has a cyclic cell-penetrating peptide coupled with phospholipid polyethylene glycol maleimide DSPE-PEG-Mal, so that the surface of the water-soluble nano-cerium oxide has a positive potential; Among them, water-soluble nano cerium oxide contains trivalent cerium and tetravalent cerium; The Zeta potential of the surface of the water-soluble nano-cerium oxide is 20-40 mV; The hydrodynamic diameter of the water-soluble nano-cerium oxide is 5 to 30 nm; The structural formula of the phospholipid polyethylene glycol maleimide DSPE-PEG-Mal coupled cyclic cell-penetrating peptide is: ； Here, n is an integer from 30 to 60.

2. The nano cerium oxide eye drops according to claim 1, characterized in that The proportion of water-soluble nano-cerium oxide in the eye drops is 1-5 g / L.

3. The method for preparing the nano cerium oxide eye drops according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Synthesis of circular cell-penetrating peptides; (2) Annular cell-penetrating peptides were coupled to phospholipid polyethylene glycol maleimide DSPE-PEG-Mal; (3) The cyclic cell-penetrating peptide coupled with DSPE-PEG-Mal is modified onto nano-cerium oxide to obtain water-soluble nano-cerium oxide; the aqueous solution of water-soluble nano-cerium oxide is nano-cerium oxide eye drops.

4. The preparation method according to claim 3, characterized in that: The step (1) specifically includes the following steps: (a) placing 2-chlorotrityl chloride resin in dichloromethane (DCM) and shaking, and then filtering off DCM to obtain swollen 2-chlorotrityl chloride resin; (b) reacting the swollen 2-chlorotrityl chloride resin with amino acid Fmoc-Cys(Trt)-OH in N,N-diisopropylethylamine DIEA and N,N-dimethylformamide DMF, and capping with methanol; (c) removing the Fmoc protecting group of the product obtained in step (b); (d) coupling the product obtained in step (c) with the amino acid Fmoc-Gln(Trt)-OH, and removing the Fmoc protecting group from the product obtained by the coupling reaction; (e) Repeat step (d) and sequentially couple the following amino acids: Fmoc-Arg(pbf)-OH, Fmoc-(2-Nal)-OH, and Fmoc-Phe-OH; (f) cleaving the polypeptide from the resin obtained in step (e) and rotary evaporating the polypeptide to obtain a protected peptide segment; (g) dissolving the protected peptide in DCM solvent, adding benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBop) and DIEA for cyclization reaction, and rotary evaporating to obtain a cyclic protected peptide; (h) The Trt protecting group and the pbf protecting group of the cyclic protected peptide segment were removed, followed by nitrogen drying and purification to obtain a cyclic cell-penetrating peptide.

5. The preparation method according to claim 4, characterized in that: In the step (2), the cyclic cell-penetrating peptide and DSPE-PEG-Mal are dissolved in a mixed solution of water and acetonitrile (ACN) for reaction, and then dialyzed, purified, and freeze-dried.

6. The preparation method according to claim 5, characterized in that: In the step (3), the DSPE-PEG-Mal coupled cyclic cell-penetrating peptide and the polyethylene glycol-modified phospholipid DSPE-PEG are dissolved in chloroform, then mixed with the nano-cerium oxide chloroform dispersion and subjected to rotary evaporation, followed by vacuum drying and purification to obtain water-soluble nano-cerium oxide, which is then dissolved in water to obtain nano-cerium oxide eye drops.

7. Use of the nano cerium oxide eye drops as described in any one of claims 1 to 2 or the nano cerium oxide eye drops prepared by the preparation method as described in any one of claims 3 to 6 in the preparation of drugs for treating and preventing cataracts.

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