Eye drops based on ultra-small gold nanoparticles as well as preparation method and application of eye drops

Abstract

The invention discloses eye drops based on ultra-small gold nanoparticles as well as a preparation method and application of the eye drops. The invention provides a surface copper-doped gold nanoparticle and glutathione / cell-penetrating peptide double-ligand synergistic modification strategy. According to the invention, a high-density bionic enzyme active center is constructed by doping copper in a surface area, superoxide anions and hydrogen peroxide can be efficiently removed, and an SOD-CAT cascade anti-oxidation pathway is realized; gSH is anchored to the surfaces of the nanoparticles through thiol bonds, so that the stability is enhanced, and residual free radicals are neutralized; the cell-penetrating peptide CR8 endows the compound with efficient cross-eye barrier penetrating ability, so that the compound can be delivered to the posterior segment of the eye in the form of eye drops. The process is simplified, copper ion leakage is inhibited through GSH coordination passivation and the surface confinement effect, the limitation of traditional invasive treatment is broken through, high biocompatibility, low toxicity and clinical transformation potential are achieved, and a novel noninvasive and efficient nano treatment scheme is provided for posterior ocular segment oxidative stress diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of eye drops, and particularly relates to a preparation method and application of an eye drop based on ultra-small gold nanoparticles. BACKGROUND

[0002] Abnormal accumulation of reactive oxygen species (ROS) is the core pathological mechanism of posterior segment diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). ROS induces oxidative stress, inflammatory response and imbalance of angiogenesis, leading to retinal pigment epithelial cell (RPE) apoptosis, blood-retinal barrier damage and choroidal neovascularization. Traditional antioxidant therapies (such as vitamin C / E, N-acetylcysteine) can partially neutralize ROS, but their poor molecular stability, low corneal penetration efficiency (bioavailability <5%) and inability to target retinal tissue make it difficult to achieve effective concentrations at the lesion site.

[0003] In recent years, nanenzyme technology has provided a new way for ROS clearance by simulating the catalytic activity of natural antioxidant enzymes (such as superoxide dismutase SOD, catalase CAT, glutathione peroxidase GPx). For example, platinum / cerium-doped nanoparticles (such as CeO2) can simulate SOD activity, but their catalytic efficiency is limited and they lack cascade catalytic ability, making it impossible to simultaneously remove O2 - , H2O2 and ·OH. Gold nanoparticles (AuNPs) have been widely studied due to their surface plasmon effect and easy modification characteristics, but single-ligand-modified AuNPs lack targeting ligands and size optimization, resulting in insufficient blood-retinal barrier penetration, insufficient choroid / retina drug accumulation, and simultaneous activation of SOD, CAT or GPx pathways, leading to low ROS clearance efficiency. In the prior art, glutathione (GSH)-modified AuNPs can improve biocompatibility, but their enzyme activity is limited and the problem of posterior eye targeting delivery has not been solved; copper-doped AuNPs have been shown to significantly improve their enzyme-like catalytic activity, but the complex structure of the eye, including tear film mucin adsorption, corneal epithelial tight junction, blood-retinal barrier, and retinal pigment epithelial layer tight junction, still seriously hinders its delivery to the posterior eye. In addition, single-ligand-modified nanenzymes are prone to tear fluid protein adsorption and immune clearance due to fluctuations in cell uptake efficiency and unbalanced surface charge.

[0004] Therefore, there is an urgent need to develop a nanenzyme system with high catalytic activity, efficient intraocular penetration and multi-ROS synergistic clearance capacity to meet the treatment needs of ROS-related posterior segment diseases. SUMMARY

[0005] Based on the above-mentioned shortcomings and deficiencies existing in the prior art, one of the purposes of the present application is to at least solve one or more of the above-mentioned problems existing in the prior art, in other words, one of the purposes of the present application is to provide a preparation method and application of an eye drop based on ultra-small gold nanoparticles to meet one or more of the aforementioned needs. In view of the problems that the existing eye drops are difficult to penetrate the blood-ocular barrier in the eye and the nano-carrier has low reactive oxygen species (ROS) removal efficiency, the present application innovatively proposes a surface copper-doped gold nanoparticle and glutathione / cell-penetrating peptide dual-ligand synergistic modification strategy. The new nanoparticle constructs a high-density biomimetic enzyme active center in the surface region through copper doping, can efficiently remove superoxide anion and hydrogen peroxide, and realize the SOD-CAT cascade antioxidant pathway; GSH is anchored on the surface of the nanoparticle through thiol bond, enhances the stability and neutralizes the residual free radicals; cell-penetrating peptide CR8 endows it with high-efficiency trans-ocular barrier penetration ability, so that it can be delivered to the posterior segment of the eye in the form of eye drops. The present application synchronously realizes copper atom surface doping and dual-ligand in-situ modification by one-pot method, simplifies the process, and uses GSH coordination passivation and surface confinement effect to inhibit copper ion leakage, breaks through the limitation of traditional invasive treatment, has high biocompatibility, low toxicity and clinical transformation potential, and provides a non-invasive and efficient nano-treatment new scheme for oxidative stress diseases in the posterior segment of the eye.

[0006] In order to achieve the above-mentioned purposes of the application, the following technical solutions are adopted in the present application:

[0007] An eye drop based on ultra-small gold nanoparticles, characterized in that it is composed of ultra-small nanoparticles and excipients. The ultra-small nanoparticles comprise gold, copper elements and ligands, the ligands comprise glutathione (GSH) and cell-penetrating peptide (CR8), and the excipients comprise hyaluronic acid and sodium chloride.

[0008] As a preferred scheme, the doping amount of copper elements is 1-10wt% of the number of moles of gold atoms, and the particle size of the ultra-small nanoparticles is 2-5nm, and the surface charge is-40mV to-5mV.

[0009] The present application also provides a preparation method of the ultra-small nanoparticles according to any one of the above schemes, comprising the following steps:

[0010] (1) mixing chloroauric acid (HAuCl4·3H2O), copper salt and glutathione (GSH) to form an Au-Cu-GSH ternary complex;

[0011] (2) adjusting the pH of the solution to 9-11, adding cell-penetrating peptide CR8, and forming a CR8-Au-Cu-GSH complex through Au-S bond and electrostatic interaction;

[0012] (3) adding a reducing agent, reacting at 20-30℃ for 10-24h, and ultrafiltration purification.

[0013] (4) adding excipients to prepare eye drops.

[0014] As a preferred solution, the step (1) specifically comprises the following steps:

[0015] (a) preparing an aqueous solution of HAuCl4·3H2O with a concentration of 10-30 mM, an aqueous solution of copper salt with a concentration of 10-30 mM, and an aqueous solution of GSH with a concentration of 40-60 mM;

[0016] (b) adding the aqueous solution of HAuCl4·3H2O and the aqueous solution of copper salt into deionized water in sequence, and then stirring at a speed of 700 r-900 r for 20-40 min; the adding amount of the aqueous solution of HAuCl4·3H2O and the aqueous solution of copper salt satisfies that the molar ratio of HAuCl4 to Cu is 1:0.01-1:0.1;

[0017] (c) slowly adding the aqueous solution of GSH, and then stirring at a speed of 700 r-900 r for 20-40 min; the adding amount of the aqueous solution of GSH and the aqueous solution of HAuCl4·3H2O satisfies that the molar ratio of HAuCl4 to GSH is 1:1-1:3.

[0018] As a preferred solution, the step (2) specifically comprises the following steps:

[0019] (a) preparing an aqueous solution of CR8 with a concentration of 40-60 mM, and an aqueous solution of NaOH with a concentration of 1-2 mM;

[0020] (b) after the solution changes from light yellow to milky white, adding the aqueous solution of NaOH to adjust the pH of the solution to 9-11, and the solution becomes clear after adjusting the pH; (c) slowly adding the aqueous solution of CR8, and then stirring at a speed of 700 r-900 r for 20-40 min; the adding amount of the aqueous solution of CR8 and the aqueous solution of GSH satisfies that the molar ratio of CR8 to GSH is 1:10-1:20.

[0021] As a preferred solution, the step (3) specifically comprises the following steps:

[0022] (a) preparing an aqueous solution of NaBH4 with a concentration of 80-120 mM;

[0023] (b) slowly adding the aqueous solution of NaBH4, and adjusting the speed to 400-600 r; the adding amount of the aqueous solution of NaBH4 satisfies that the molar ratio of NaBH4 to HAuCl4 is HAuCl4:NaBH4=1:1-1:3;

[0024] (c) after reacting for 10-24 h, the solution gradually changes to orange brown, and is purified by ultrafiltration.

[0025] As a preferred solution, the step (4) specifically comprises the following steps:

[0026] The material is freeze-dried, the freeze-dried complex is re-dissolved in a phosphate buffer (PBS) to a concentration of 0.1-5 mg / ml, sodium chloride (NaCl) is added to adjust the osmotic pressure of the eye drop to be isotonic with the tear (280-320 mOsm / kg), 0.1% hyaluronic acid is slowly added in a ratio of 1:1-1:2, 200-300 r magnetic stirring is carried out for 15-30 min, ultrasonic treatment is carried out for 5-10 min, no visible particles are ensured, and then the mixture is sterilely filtered through a 0.1-0.22 um filter membrane and is divided into portions.

[0027] As a preferred solution, the copper salt is copper nitrate, copper sulfate or copper chloride.

[0028] As a preferred solution, the purification method is:

[0029] (a) preparing alkaline water with a pH of 9-11;

[0030] (b) using alkaline water to remove unreacted substrates by ultrafiltration centrifugation and to concentrate; the ultrafiltration centrifugation temperature is 4-30 DEG C, the ultrafiltration centrifugation speed is 2000-4000 rpm, the ultrafiltration centrifugation time is 5-30 min; the ultrafiltration times are 8-10 times; the membrane pore size of the ultrafiltration tube is 3-50 kDa.

[0031] As a preferred solution, the ultra-small nanoparticle has a concentration of 0.1-5 mg / mL.

[0032] As a preferred solution, the application of the eye drop based on the ultra-small gold nanoparticle in the preparation of a drug for treating ROS-related diseases in the posterior segment of the eye, the diseases including: light-induced retinal degeneration, age-related macular degeneration (AMD), uveitis, diabetic retinopathy (DR).

[0033] Compared with the prior art, the present application has the following advantages and technical effects:

[0034] The eye drop based on the ultra-small gold nanoparticle of the present application has the advantages of simple synthesis method, easy operation, mild conditions, high yield, easy industrial production, high stability, good biological safety and great clinical potential.

[0035] The eye drop based on the ultra-small gold nanoparticle of the present application has the advantages of simple synthesis method, easy operation, mild conditions, high yield, easy industrial production, high stability, good biological safety and great clinical potential. 2+ / Cu +The synergistic effect of redox cycle and Au(0) surface plasmon resonance realizes the superoxide dismutase (SOD) and catalase (CAT) double enzyme cascade activity, can simultaneously remove superoxide anion (O2) and hydrogen peroxide (H2O2), avoids the accumulation of intermediate product toxicity caused by single enzyme activity, and experiments prove that the ROS removal efficiency is significantly improved. In addition, the endogenous antioxidant pathway provided by the GSH ligand can also repair the oxidative damage of retinal pigment epithelial cells.

[0036] The eye drop based on the ultra-small gold nanoparticles of the method of the application breaks through the biological barrier of the posterior segment of the eye by using the transmembrane transport capacity and charge regulation function of CR8 transmembrane peptide.

[0037] The eye drop based on the ultra-small gold nanoparticles of the method of the application has revolutionary breakthroughs in safety and compliance. The intravitreal injection therapy in the prior art needs to be repeated every 4-8 weeks, has high infection risk, and has poor patient compliance. The traditional eye drop needs to be administered 6-8 times a day, and only not more than 5% of the dose can reach the posterior end of the eye, and the effect on the posterior end of the eye is poor. The application can realize non-invasive long-acting treatment: the eye drop is only 2 times a day, the complication rate is less than 1%, and the size of the eye drop based on the ultra-small gold nanoparticles is very small, so even a small amount of the eye drop enters the body, it can be quickly metabolized, and the safety is good.

[0038] The eye drop based on the ultra-small gold nanoparticles of the method of the application has multi-indication covering capacity. Most of the prior art is a single drug target (such as anti-VEGF only for neovascular lesions), which cannot solve the ROS-related extensive damage. The application can realize broad-spectrum ROS removal: suitable for a plurality of oxidative stress-related diseases such as light damage, AMD, diabetic retinopathy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The normalized absorption and emission spectrum diagram of the ultra-small nanoparticles prepared in the comparative examples and examples of the application.

[0040] Figure 2 The agarose gel electrophoresis comparison diagram of the ultra-small nanoparticles prepared in the comparative examples and examples of the application.

[0041] Figure 3 The ZETA potential diagram of the ultra-small nanoparticles prepared in the comparative examples and examples of the application.

[0042] Figure 4 The metal element content of the ultra-small nanoparticles prepared in the examples measured by ICP-MS.

[0043] Figure 5 The Cu 2p XPS spectrum of the ultra-small nanoparticles prepared in the examples. Figure 6 Cu2p orbital electron binding energy spectrum of the ultra-small nanoparticles prepared in the examples of the present application.

[0044] Figure 7 Hydrated particle size of the ultra-small nanoparticles prepared in the comparative examples and examples of the present application.

[0045] Figure 8 UV absorption stability of the ultra-small nanoparticles prepared in the comparative examples and examples of the present application under incubation at 37℃ in artificial tear solution.

[0046] Figure 9 Particle size stability of the ultra-small nanoparticles prepared in the comparative examples and examples of the present application under incubation at 37℃ in artificial tear solution.

[0047] Figure 10 Superoxide dismutase (SOD) mimetic activity and catalase (CAT) mimetic activity of the ultra-small nanoparticles prepared in the comparative examples and examples of the present application.

[0048] Figure 11 Total antioxidant capacity of the ultra-small nanoparticles prepared in the comparative examples and examples of the present application.

[0049] Figure 12 Silver-stained HE section images of the ultra-small nanoparticles prepared in the comparative examples of the present application after ocular delivery to mice at 3h and 6h.

[0050] Figure 13 Silver-stained HE section images of the ultra-small nanoparticles prepared in the examples of the present application after ocular delivery to mice at 3h and 6h.

[0051] Figure 14 HE sections of the eyes of mice treated with PBS, the ultra-small nanoparticles prepared in the comparative examples and examples of the present application after one week of ocular delivery to mice to study the integrity of the cornea and retina of the mice.

[0052] Figure 15 OCT imaging images of healthy mice and light-damaged retinal mice treated with PBS, the comparative examples, the examples and commercially available sodium hyaluronate eye drops at the third day and the seventh day.

[0053] Figure 16 Outer nuclear layer and total retinal thicknesses of healthy mice and light-damaged retinal mice treated with PBS, the comparative examples, the examples and commercially available sodium hyaluronate eye drops at the third day and the seventh day.

[0054] Figure 17 Silver-stained eyeball section results of healthy mice and light-damaged retinal mice treated with PBS, the comparative examples, the examples and commercially available sodium hyaluronate eye drops at the third day and the seventh day. DETAILED DESCRIPTION

[0055] In order to more clearly illustrate the embodiments of the present application, the specific embodiments of the present application will be described below with reference to the accompanying drawings. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings and other embodiments can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0056] In the following specific examples, chloroauric acid is purchased from Shanghai Mayre Chemical Technology Co., Ltd.; sodium borohydride (NaBH4) is purchased from Shanghai Fulian Chemical Reagent Co., Ltd.; cell-penetrating peptide CR8 is purchased from Hefei Senrui Biological Technology Co., Ltd.; superoxide dismutase activity detection kit and catalase activity detection kit are purchased from Beijing Solabio Technology Co., Ltd. The instruments for exploring the optical properties and morphology of the eye drop based on ultra-small gold nanoparticles mainly include PerkinElmer fluorescence / phosphorescence / luminescence spectrophotometer LS-55, Japan Shimadzu ultraviolet-visible absorption spectrometer UV-2600, British Malvern nanoparticle size and ZETA potential analyzer, German ThermoScientific inductively coupled plasma mass spectrometer (iCAPRQ), etc.

[0057] Example 1

[0058] The preparation method of the ultra-small nanoparticles of the present embodiment comprises the following steps:

[0059] (1) Mix chloroauric acid, copper nitrate and glutathione (GSH) to form Au-Cu-GSH ternary complex

[0060] Prepare a 20mM HAuCl4·3H2O aqueous solution, a 10mM copper nitrate aqueous solution and a 50mM GSH aqueous solution at room temperature, then add 500ul of the prepared HAuCl4·3H2O aqueous solution and 100ul of the copper nitrate aqueous solution into 8.8ml of deionized water in sequence, and then stir at a speed of 800r for 30min; then slowly add 375ul of the GSH aqueous solution, and continue to stir at a speed of 800r for 30min.

[0061] (2) Adjust the pH of the solution to 10, add the cell-penetrating peptide CR8, and form the CR8-Au-Cu-GSH complex through Au-S bond and electrostatic interaction Prepare a 50mM CR8 aqueous solution and a 1mM NaOH aqueous solution, add the NaOH aqueous solution to adjust the pH of the solution to about 10 after the solution changes from light yellow to milky white, and the solution becomes clear after adjusting the pH; then slowly add 25ul of the CR8 aqueous solution, and then stir at a speed of 800r for 30min.

[0062] (3) Add reducing agent, react at 27°C for 10 h, and purify by ultrafiltration

[0063] Prepare a 100 mM NaBH4 aqueous solution, and slowly add 200 ul of the NaBH4 aqueous solution into the solution at a pH of 10, and adjust the rotation speed to 500 r; the solution gradually turns orange-brown after about 10 h of reaction; use alkaline water to remove unreacted substrate by ultrafiltration centrifugation and concentrate; the ultrafiltration centrifugation temperature is 27°C, the ultrafiltration centrifugation rotation speed is 3500 rpm, the ultrafiltration centrifugation time is 15 min; the ultrafiltration frequency is 10 times; the membrane pore size of the ultrafiltration tube is 10 kDa.

[0064] Comparative Example 1

[0065] The ultra-small nanoparticles of the present comparative example are different from those of Example 1 in that: no copper nitrate aqueous solution is added during step (1) of Example 1, no CR8 aqueous solution is added during step (2) of Example 1, and the amount of copper nitrate aqueous solution and CR8 aqueous solution is replaced with an equal amount of deionized water solution, and the remaining implementation steps can refer to step (3) of Example 1.

[0066] Comparative Example 2

[0067] The ultra-small nanoparticles of the present comparative example are different from those of Example 1 in that: no copper nitrate aqueous solution is added during step (1) of Example 1, and the amount of copper nitrate aqueous solution is replaced with an equal amount of deionized water solution, and the remaining implementation steps can refer to steps (2) and (3) of Example 1.

[0068] Comparative Example 3

[0069] The ultra-small nanoparticles of the present comparative example are different from those of Example 1 in that: no CR8 aqueous solution is added during step (2) of Example 1, and the amount of CR8 aqueous solution is replaced with an equal amount of deionized water solution, and the remaining implementation steps can refer to steps (1) and (3) of Example 1.

[0070] The following tests and characterizations are performed on the nanoparticles of Example 1 and Comparative Examples 1, 2, and 3 described above, as follows:

[0071] Figure 1The ultraviolet absorption spectrum and fluorescence emission spectrum of the ultrasmall nanoparticles prepared in the examples and the comparative examples are shown in Figure 1. From the ultraviolet absorption spectrum, it can be seen that the AuNPs prepared in Comparative Example 1 have a broad spectrum of absorption, indicating that the size of the prepared ultrasmall nanoparticles is very small. From the fluorescence emission spectrum, it can be seen that the maximum emission wavelength of the gold nanoparticles is about 950 nm. Since the ligand, cell-penetrating peptide CR8, has no obvious characteristic absorption, the CR-AuNPs prepared in Comparative Example 2 still have a broad spectrum of absorption, but the fluorescence emission spectrum shows that the emission of the gold nanoparticles is red-shifted, with a maximum emission wavelength of about 1000 nm, which preliminarily indicates the successful modification of CR8. The AuCuNPs prepared in Comparative Example 3 still have a broad spectrum of absorption, indicating that the size of the ultrasmall nanoparticles after copper doping has not changed much. However, from the fluorescence emission spectrum, it can be seen that the emission peak of the gold nanoparticles after copper doping changes significantly, becoming sharp and the maximum emission wavelength being red-shifted to about 1050 nm, which preliminarily indicates the successful doping of copper elements. The CR-AuCuNPs prepared in the examples still have a broad spectrum of absorption, similar to the ultrasmall nanoparticles prepared in Comparative Example 3, but the fluorescence emission spectrum changes more obviously, being more sharp and the maximum emission wavelength being further red-shifted to about 1100 nm, which preliminarily proves the successful modification of CR8 and the successful doping of copper elements.

[0072] Figure 2 The agarose gel electrophoresis diagram of the ultrasmall nanoparticles prepared in the examples and the comparative examples is shown in Figure 2. It can be seen that due to the introduction of the positively charged cell-penetrating peptide CR8 through ligand exchange, the negative potential of the nanoparticle complex after modification is smaller. At the same time, doping copper will destroy the surface oxide layer, regulate the electronic structure and change the ligand adsorption state, so that the absolute value of the Zeta potential of the gold nanoparticles is further reduced, thereby further moving in the positive direction. The electrophoresis layering result further proves the successful modification of the cell-penetrating peptide CR8 and the successful doping of copper elements.

[0073] Figure 3 The ZETA potential diagram of the ultrasmall nanoparticles prepared in the examples and the comparative examples is shown in Figure 3. Similarly, due to the introduction of the positively charged cell-penetrating peptide CR8, the negative potential of the ultrasmall nanoparticles is smaller. At the same time, doping copper will destroy the surface oxide layer, regulate the electronic structure and change the ligand adsorption state, so that the absolute value of the Zeta potential of the nanoparticles is reduced. Therefore, it is found that the absolute value of the potential of the ultrasmall nanoparticles prepared from the comparative examples to the examples gradually decreases, and the absolute value of the potential of the ZETA ultrasmall nanoparticles prepared in the examples is the lowest, which further proves the successful modification of the positively charged cell-penetrating peptide CR8 and the successful doping of copper elements.

[0074] Figure 4The metal element content of the ultra-small nanoparticles prepared in the examples was measured by ICP-MS. It can be seen that the copper metal element content of the ultra-small nanoparticles obtained in the examples accounts for 4.55%, and the gold element content accounts for 95.45%, further proving the successful doping of copper elements.

[0075] Figure 5 The Cu 2p XPS spectrum of the ultra-small nanoparticles prepared in the examples. Through XPS energy spectrum analysis of Cu elements, it was found that there were two peaks at 931.5 eV and 954.3 eV, respectively, which belonged to Cu 2p 3 / 2 and Cu 2p 3 / 1 , and no Cu(II) satellite peak was found, indicating that Cu 2+ has been successfully reduced to Cu(0) or Cu(I).

[0076] Figure 6 The hydrated particle size of the ultra-small nanoparticles prepared in the examples and the comparative examples. The results prove that the obtained materials are all ultra-small in size, all below the kidney filtration threshold of 5 nm, and the ultra-small size makes it easier to pass through the tight junction of the blood-ocular barrier and is quickly metabolized and cleared even after entering the body.

[0077] Figure 7 The ultraviolet absorption stability of the ultra-small nanoparticles prepared in the examples and the comparative examples under artificial tear 37℃ incubation. It can be seen that they all have good stability, and the ultraviolet absorption is stable for a long time, which is conducive to the material to maintain good and long-lasting therapeutic effect in the eye.

[0078] Figure 8 The particle size stability of the ultra-small nanoparticles prepared in the examples and the comparative examples under artificial tear conditions at 37℃ incubation. The results also show that they have good long-term stability under this condition and can maintain stable particle size for a long time, further proving that the material can maintain good and long-lasting therapeutic effect in the eye.

[0079] Figure 9 The superoxide dismutase SOD mimetic activity and catalase CAT mimetic activity of the ultra-small nanoparticles prepared in the examples and the comparative examples. The results show that different concentrations of ultra-small nanoparticles show different inhibition rates, and the introduction of cell penetrating peptide CR8 does not affect the original enzyme-like activity of the nanoparticles. At the same time, through the construction of high-density biomimetic enzyme activity center in the surface region by copper doping and the synergistic effect of Au(0) surface plasmon resonance, the superoxide dismutase (SOD) and catalase (CAT) double enzyme cascade activity is realized, so the SOD enzyme activity and CAT enzyme activity of the doped ultra-small nanoparticles are greatly improved, which can effectively scavenge superoxide anion in a dose-dependent manner.

[0080] Figure 10The total antioxidant activity of the ultra-small nanoparticles prepared in the comparative examples and the examples showed that copper doping could greatly improve the total antioxidant capacity of the material by synergizing the multi-enzyme activity, optimizing the electronic structure and forming a broad-spectrum ROS scavenging capacity.

[0081] Figure 11 The intraocular pressure (IOP) of the mice during treatment with PBS, the ultra-small nanoparticles prepared in the comparative examples and the examples was counted. The biological safety of the material in vivo was evaluated by measuring the intraocular pressure (IOP) of the mice during treatment. The change in IOP in the eyes of the mice treated with the ultra-small nanoparticles prepared in the comparative examples and the examples was negligible, which indicated the biological safety of the material and its potential to avoid side effects such as glaucoma.

[0082] Figure 12 The eyes of the mice treated with PBS, the ultra-small nanoparticles prepared in the comparative examples and the examples were taken for HE sectioning after one week of eye drop treatment to study the integrity of the cornea and retina of the mice. The results showed that the cornea and retina of the mice after different treatments still had good structural integrity, which further proved that the material had excellent ocular biocompatibility and emphasized its potential as a non-invasive penetrating eye drop.

[0083] Figure 13 The eyes of the mice treated with PBS, the ultra-small nanoparticles prepared in the comparative examples and the examples were taken for silver-stained HE sectioning at the 6h time point after eye drop delivery. The results showed that most of the ultra-small nanoparticles without cell penetrating peptide modification were retained in the cornea layer and could not break through the cornea barrier to deliver to the back of the eye. Only a small part could be delivered to the back of the eye through the conjunctiva-sclera pathway, but it would still be intercepted by the choroid and could not be delivered to the retina. In contrast, the penetration ability of the original nanomaterials was greatly improved after modification with the cell penetrating peptide CR8. The distribution in the retina tissue at the back of the eye was increased by tens of times compared to the group without cell penetrating peptide. The great improvement in penetration ability proved its ability to deliver to the back of the eye through eye drops and perform treatment.

[0084] The characterization of the ultra-small nanoparticles prepared in the above comparative examples and examples through a series of in vitro and in vivo experiments confirmed their potential as non-invasive eye drops for treating posterior eye diseases. The ability of the ultra-small nanoparticles prepared in the examples to deliver to the posterior eye was greatly improved by modifying the cell-penetrating peptide CR8. The SOD-like and CAT enzyme activities of the ultra-small nanoparticles prepared in the examples were greatly enhanced by doping copper, and it was also proved that the prepared ultra-small nanoparticles had excellent ocular biocompatibility. Therefore, further in vivo treatment of mouse photoreceptor-damaged retinas was carried out. First, the experimental eye drops AuNPs, CR-AuNPs, AuCuNPs and CR-AuCuNPs were configured, and the drug concentration of the solution was configured to be 3 ug / uL; in order to construct a photoreceptor-damaged retina model, a dark box was used, which was equipped with a blue light source at the top and a mirror on the four sides. 6-week-old female BALB / c mice were subjected to dark adaptation for 16 h, and 0.5% tropicamide solution was applied locally to the mouse cornea to ensure pupil dilation. Then, exposure to strong blue light for 12 h, followed by returning to darkness for 12 h, the light cycle was repeated for one month. After that, 6-week-old female BALB / c mice that had constructed a photoreceptor-damaged retina model were randomly divided into 6 groups, and the above groups were treated with PBS, AuNPs, CR-AuNPs, AuCuNPs, CR-AuCuNPs eye drops, respectively, and were locally instilled into the photoreceptor-damaged retina mouse model twice a day (3 ug / uL x 5 uL). Healthy mice were used as positive controls (three eyeballs per group). On the 3rd and 7th day after administration, optical coherence tomography (OCT) was performed to evaluate the outer nuclear layer (ONL) and total retinal thickness of mice from each treatment group. As shown in Figure 14 and Figure 15 In order to make a more fair comparison, the total retinal thickness at 500 um from the fovea and the outer nuclear layer thickness at 300 um were unified, and the quantitative results are shown in Figure 16 The data are presented as mean ± SD (n = 3), * indicates a significant difference p < 0.05, ** indicates a significant difference p < 0.01, *** indicates a significant difference p < 0.001, and ns indicates no significant difference. The photoreceptor-damaged retina mice showed similar ONL and total retinal thickness after treatment, but were significantly thinner than healthy mice. This reduction in ONL and total retinal thickness may be due to tissue damage caused by strong blue light exposure. After 7 days, mice treated with CR-AuCuNPs showed significant recovery of ONL and total retinal thickness, indicating that they can effectively alleviate retinal degeneration by preventing photoreceptor apoptosis. In order to further confirm the anti-apoptotic efficacy of CR-AuCuNPs, HE staining of eyeballs was performed on the 3rd and 7th day after different treatments. As shown in Figure 17As shown, significant inflammatory cell infiltration and disruption of the standard structure of photoreceptors in the ONL were observed in the retinas of light-damaged retinal mice treated with PBS, AuNPs, CR-AuNPs, AuCuNPs eye drops, which were characterized by morphological changes, disordered arrangement, and uneven thickness reduction. In contrast, CR-AuCuNPs can effectively reduce inflammatory cell infiltration, prevent photoreceptor apoptosis, and greatly improve the structural arrangement of the ONL at 3 days and 7 days. Overall, these results demonstrate that CR-AuCuNPs can not only be effectively delivered to the posterior of the eye, but also significantly reduce retinal degeneration by preventing cell apoptosis. Therefore, the ultra-small gold nanoparticle-based eye drops of the present application can be applied in the preparation of drugs for the treatment and prevention of light-damaged retinas.

[0085] In view of the numerous embodiments of the present application, the raw materials and amounts involved can be selected according to actual needs within the limited range, and the experimental data of each embodiment are numerous and not suitable for enumeration here. However, the content to be verified and the final conclusion obtained by each embodiment are close. Therefore, the verification content of each embodiment is not described one by one here.

[0086] The above only describes the preferred embodiments and principles of the present application in detail. For those skilled in the art, the specific implementation manner according to the idea provided by the present application will have changes, and these changes should also be considered as the protection scope of the present application.

Claims

1. An eye drop based on ultra-small gold nanoparticles, characterized by: The super small nanoparticles and adjuvants are composed of gold elements, copper elements and ligands, the ligands include glutathione GSH and cell penetrating peptide CR8, and the adjuvants include hyaluronic acid and sodium chloride.

2. The eye drop based on ultra-small gold nanoparticles according to claim 1, characterized in that: The doping amount of copper elements is 1-10wt% of the number of moles of gold atoms, the particle size of the super small nanoparticles is 2-5nm, and the surface charge is-40mV to-5mV.

3. The eye drop based on ultra-small gold nanoparticles according to claim 1, characterized in that: The concentration of the super small nanoparticles is 0.1-5mg / mL.

4. A method of preparing the eye drop based on ultra-small gold nanoparticles according to claim 1 or 2 or 3, characterized in that, The method comprises the following steps: (1) mixing chloroauric acid HAuCl4·3H2O, copper salt and glutathione GSH to form an Au-Cu-GSH ternary complex; (2) adjusting the pH of the solution to 9-11, adding penetrating peptide CR8, and forming a CR8-Au-Cu-GSH complex through Au-S bond and electrostatic interaction; (3) adding a reducing agent, reacting at 20-30℃ for 10-24h, and purifying by ultrafiltration; (4) adding adjuvants to prepare eye drops.

5. The preparation method according to claim 4, characterized in that, In step (1), the following steps are specifically included: (a) preparing an aqueous solution of HAuCl4·3H2O with a concentration of 10-30mM, an aqueous solution of copper salt with a concentration of 10-30mM, and an aqueous solution of GSH with a concentration of 40-60mM; (b) adding the aqueous solution of HAuCl4·3H2O and the aqueous solution of copper salt into deionized water in sequence, and then stirring at a speed of 700r-900r for 20-40min; the addition amount of the aqueous solution of HAuCl4·3H2O and the aqueous solution of copper salt satisfies that the molar ratio of HAuCl4 to Cu is 1:0.01-1:0.1; (c) slowly adding the aqueous solution of GSH, and then stirring at a speed of 700r-900r for 20-40min; the addition amount of the aqueous solution of GSH and the aqueous solution of HAuCl4·3H2O satisfies that the molar ratio of HAuCl4 to GSH is 1:1-1:

3.

6. The preparation method according to claim 4, characterized in that, The specific steps of step (2) are as follows: (a) preparing an aqueous solution of CR8 with a concentration of 40-60mM and an aqueous solution of NaOH with a concentration of 1-2mM; (b) after the solution changes from light yellow to milky white, adding the aqueous solution of NaOH to adjust the pH of the solution to 9-11, and the solution becomes clear after adjusting the pH; (c) slowly adding the aqueous solution of CR8, and then stirring at a speed of 700r-900r for 20-40min; the addition amount of the aqueous solution of CR8 and the aqueous solution of GSH satisfies that the molar ratio of CR8 to GSH is 1:10-1:

20.

7. The preparation method according to claim 4, characterized in that, Step (3) specifically comprises the following steps: (a) preparing an aqueous solution of NaBH4 with a concentration of 80-120mM; (b) slowly adding the aqueous solution of NaBH4, and adjusting the speed to 400-600r; the addition amount of the aqueous solution of NaBH4 satisfies that the molar ratio of NaBH4 to HAuCl4 is NaBH4:HAuCl4=1:1-1:3; (c) after reacting for 10-24h, the solution gradually changes to orange brown, and the solution is purified by ultrafiltration; The purification method is: (a) preparing an alkaline water with a pH of 9-11; (b) removing unreacted substrate and concentrating by using alkaline water ultrafiltration centrifugation; the ultrafiltration centrifugation temperature is 4-30℃, the ultrafiltration centrifugation speed is 2000-4000rpm, the ultrafiltration centrifugation time is 5-30min; the ultrafiltration times is 8-10 times; the membrane pore size of ultrafiltration tube is 3-50kDa.

8. The preparation method according to claim 4, characterized in that, Step (4) specifically comprises the following steps: The material is freeze-dried, the freeze-dried complex is re-dissolved in phosphate buffer (PBS) to a concentration of 0.1-5mg / ml, sodium chloride (NaCl) is added to adjust the osmotic pressure of the eye drop to be equal to that of the tear (280-320mOsm / kg), 0.1% hyaluronic acid is slowly added in a ratio of 1:1-1:2, 200-300r magnetic stirring is carried out for 15-30min, ultrasonic treatment is carried out for 5-10min, no visible particles are ensured, then the mixture is sterile filtered through a 0.1-0.22um filter membrane and is divided into small packages.

9. The method of claim 4, wherein: The copper salt is copper nitrate, copper sulfate or copper chloride.

10. Use of the ultra-small gold nanoparticle-based eye drop according to claim 1 or 2 for the preparation of a medicament for treating ROS-related diseases in the posterior segment of the eye, including: Photoreceptor degeneration, age-related macular degeneration (AMD), uveitis, diabetic retinopathy (DR).

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