Ultra-small gold nanoparticle capable of penetrating blood-eye barrier as well as preparation method and application of ultra-small gold nanoparticle

Abstract

The invention discloses ultra-small gold nanoparticles capable of penetrating a blood-eye barrier and a preparation method and application of the ultra-small gold nanoparticles. The ultra-small gold nanoparticles comprise an atomic precision gold nanocluster (Au22) core; the ligand is covalently modified on the surface of the Au22 core through an Au-S bond; the ligands are glutathione (GSH), and the number of the ligands is 10 to 16; the sequence of the cell-penetrating peptide CR8 is CRRRRRRR, a part of GSH ligands are replaced by Au-S bonds, and the modification quantity is 2-8 CR8 molecules / Au22 clusters. Safety evaluation proves that the preparation has no toxic effect on corneal epithelium and retina nerve cells. Through collaborative design of the atomic precision metal cluster and the cell-penetrating peptide, the technical problem of non-invasive treatment of posterior ocular segment diseases is innovatively solved, and a safe and efficient new treatment strategy is provided for age-related macular degeneration, diabetic retinopathy and other ROS-mediated fundus diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoformulations, and particularly relates to ultra-small gold nanoparticles capable of crossing the blood-eye barrier, a preparation method thereof, and an application thereof. Background Art

[0002] Photodamaging retinopathy is mainly caused by short-wavelength light such as blue light and ultraviolet light. Its pathological mechanism involves the absorption of light energy by lipofuscin in retinal pigment epithelial cells, triggering the Fenton reaction, resulting in the generation of a large amount of reactive oxygen species. Excessive reactive oxygen species attack the mitochondrial DNA of photoreceptor cells and destroy the membrane lipid bilayer structure, ultimately leading to apoptosis and retinal scarring. Epidemiological investigations show that among the approximately 2.2 billion visually impaired people globally, the proportion of light damage-related lesions shows a significant upward trend, with an annual growth rate of 3.2%. It is particularly prevalent in outdoor workers, intensive users of electronic screens, and the elderly who are long-term exposed to strong light environments, seriously affecting the quality of life of patients. Existing clinical intervention methods have significant limitations. First, oral antioxidants such as vitamin C / E are difficult to reach effective concentrations in the lesion area due to low corneal penetration efficiency and insufficient blood-retinal barrier penetration rate. Second, although intravitreal injection of anti-VEGF drugs can deliver drugs locally, there are risks of infection and complications of retinal detachment, and monthly repeated injections are required. At the same time, physical protection means such as blue light-blocking glasses can only reduce the risk of light damage by about 40% and cannot reverse the already formed pathological changes. The current treatment bottlenecks are mainly reflected in the dual biological membrane barriers of the cornea and the blood-retinal barrier, poor targeting of traditional antioxidants and easy inactivation by enzymes, and the lack of treatment strategies that can simultaneously scavenge reactive oxygen species and promote tissue repair.

[0003] Nanozymes have shown unique potential in scavenging reactive oxygen species (ROS). Their catalytic activity stems from the mimicry of natural antioxidant enzymes (such as SOD, CAT). However, traditional nanozymes (such as metal oxides, carbon-based materials) are limited by problems such as low catalytic efficiency and unclear active sites. Although metal-based nanozymes (such as iron, copper-based materials) have improved the ROS scavenging ability through metal active centers (such as the SOD-like activity of Fe3O4 is 80% of that of natural enzymes), they face the risk of oxidative damage or metabolic toxicity caused by ion leakage. Gold cluster nanozymes (Au NCs), by virtue of their atomically precise structure (such as Au 22 、Au 25 ) and surface ligand synergy, have achieved precise regulation of the catalytic mechanism: their clear atomic arrangement and thiol ligand-mediated electron transfer path can directionally activate the SOD / CAT cascade reaction, and the ROS scavenging efficiency is 10-50 times higher than that of traditional metal nanozymes. At the same time, their sub-nano size and modifiable ligands endow them with excellent biocompatibility and tissue permeability, becoming a new benchmark for efficient and safe ROS scavenging, providing a new paradigm for the precise intervention of oxidative stress-related diseases.

[0004] As a non-invasive treatment method for posterior segment eye diseases, topical eye drops face complex challenges from multiple physiological barriers. Firstly, the dynamic renewal mechanism of tears leads to rapid drug loss on the ocular surface. Studies have shown that more than half of the active ingredients are washed out by tears within 5 minutes after eye drops are instilled. Secondly, the tight junction proteins between corneal epithelial cells form a high-impedance biofilm barrier, significantly limiting the transmembrane penetration efficiency of drugs. At the same time, although the extensive absorption of conjunctival epithelium can partially uptake drugs, the accompanying capillary and lymphatic drainage will cause a considerable proportion of drugs to enter the systemic circulation, increasing the risk of systemic side effects. In addition, drugs need to further penetrate the blood-retinal barrier composed of tight junctions of retinal capillary endothelium to reach the lesion area. The superimposed effects of these cascading barriers and clearance mechanisms ultimately result in a posterior segment bioavailability of traditional eye drops of less than 0.5%, forcing clinicians to adopt high-frequency drug administration or invasive injection methods, which not only reduce patient compliance but also increase treatment risks.

[0005] Therefore, there is an urgent need in this field to develop new delivery strategies that can significantly prolong the residence time of drugs on the ocular surface, enhance the penetration ability of the cornea and blood-retinal barrier, and reduce the risk of systemic circulation exposure, so as to break through the multiple delivery barriers composed of tear flushing, epithelial tight junctions, conjunctival absorption, and the inner blood-retinal barrier, thereby improving the drug bioavailability of posterior segment lesions and replacing high-risk invasive treatment methods. Summary of the Invention

[0006] Based on the above-mentioned disadvantages and deficiencies in the prior art, one of the objectives of the present invention is to at least solve one or more of the above-mentioned problems existing in the prior art. In other words, one of the objectives of the present invention is to provide a kind of ultra-small gold nanoparticles that can penetrate the blood-eye barrier, its preparation method and application, which can meet one or more of the foregoing requirements.

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

[0008] An ultra-small gold nanoparticle that can penetrate the blood-eye barrier, the ultra-small gold nanoparticle comprises an atomically precise gold nanocluster (Au 22 ) core; glutathione (GSH) ligands, covalently modified on the surface of the Au 22 core through Au-S bonds, and the number of ligands is 10 - 16; cell-penetrating peptide CR8, whose sequence is CRRRRRRR (C is cysteine, R is arginine), replaces part of the GSH ligands through Au-S bonds, and the modification amount is 2 - 8 CR8 molecules / Au 22Cluster. The nano - composite is efficiently transported across the cornea and penetrates the blood - retina barrier through the mediation of the positive charge of the CR8 peptide segment. At the same time, relying on the superoxide dismutase / catalase (SOD / CAT) - like catalytic activity of the gold nanoclusters, it precisely scavenges excessive reactive oxygen species (ROS) in the retinal tissue. The ultrasmall gold nanoparticles are prepared with phosphate - buffered saline as the carrier, and sodium hyaluronate is specially added to prolong the residence time of the drug on the ocular surface, forming a preservative - free single - dose sterile preparation.

[0009] As a preferred embodiment, the hydrated particle size of the ultrasmall gold nanoparticles is 1.8 - 2.5 nm, and the Zeta potential of the material is - 5 to - 10 mV.

[0010] An eye drop containing the ultrasmall gold nanoparticles that can cross the blood - eye barrier, including ultrasmall gold nanoparticles, phosphate - buffered saline, and sodium hyaluronate; it is packaged in a single - dose sterile form; the final concentration of the ultrasmall gold nanoparticles is 0.1 - 5 μg / μL; the pH value of the phosphate - buffered saline is 7.2 - 7.6; the concentration of the sodium hyaluronate is 0.05% - 0.1% (w / v).

[0011] The present invention also provides a preparation method of the ultrasmall gold nanoparticles as described in any of the above - mentioned embodiments, including the following steps:

[0012] (1) Synthesize atomically precise gold nanoclusters Au 22 GSH 18 ;

[0013] (2) Ligand - exchange modification of the cell - penetrating peptide CR8;

[0014] (3) Add excipients to prepare the eye drop.

[0015] As a preferred embodiment, step 1 specifically includes the following steps:

[0016] (a) Prepare an aqueous solution of chloroauric acid trihydrate HAuCl4·3H2O with a concentration of 10 - 30 mM, an aqueous solution of glutathione GSH with a concentration of 40 - 60 mM, an aqueous solution of sodium borohydride NaBH4 with a concentration of 5 - 15 mM, an aqueous solution of sodium hydroxide NaOH

[0017] aqueous solution and an aqueous solution of hydrochloric acid HCl;

[0018] (b) Sequentially add the aqueous solution of chloroauric acid trihydrate HAuCl4·3H2O and the aqueous solution of glutathione GSH to deionized water, and then vigorously stir at a speed of 1000 r - 1200 r for 2 - 5 min; the addition amounts of the aqueous solution of chloroauric acid trihydrate HAuCl4·3H2O and the aqueous solution of glutathione GSH satisfy: the molar ratio of HAuCl4 to GSH is 1:1 - 1:2;

[0019] (c) After the solution turns from light yellow to turbid, add an aqueous solution of sodium hydroxide NaOH to adjust the pH of the solution to 11.8 - 12.2. After adjusting the pH, the solution turns bright yellow;

[0020] (d) Slowly add an aqueous solution of sodium borohydride NaBH4 while adjusting the rotation speed to 450 - 500 r; the addition amount of the aqueous solution of sodium borohydride NaBH4 satisfies: the molar ratio of NaBH4 to HAuCl4 is NaBH4:HAuCl4 = 1:35 - 1:45;

[0021] (e) After reacting for 10 - 20 min, the solution turns orange. After reacting for 30 - 40 min, add HCl to adjust the pH of the solution to 2.4 - 2.6;

[0022] (f) Adjust the rotation speed to 150 - 200 r and slowly react for 8 - 9 h. After purification, atomically precise gold nanoclusters Au 22 GSH 18 .

[0023] As a preferred embodiment, the purification method includes:

[0024] (a) After the reaction is completed, add NaOH to adjust the pH of the solution to 6.2 - 6.8.

[0025] (b) Prepare a weak base water with a pH of 8.0 - 8.6;

[0026] (c) Use the weak base water for ultrafiltration centrifugation to remove unreacted substrates and concentrate; the temperature of the ultrafiltration centrifugation is 4 - 30 °C, the rotation speed of the ultrafiltration centrifugation is 2000 - 4000 rpm, the time of the ultrafiltration centrifugation is 5 - 30 min; the number of ultrafiltration times is 8 - 10 times; the membrane pore size of the ultrafiltration tube is 3 - 50 kDa.

[0027] As a preferred embodiment, step (2) specifically includes the following steps:

[0028] (a) Dissolve CR8 using a NaOH solution with a concentration of 1 - 2 mM, and the concentration of CR8 is 4 - 5 mM.

[0029] (b) Quantify the concentration of GSH, and slowly add CR8 to the atomically precise gold nanoclusters Au in an amount with a molar ratio of CR8:GSH = 1:5 - 1:10, and adjust the rotation speed to 400 - 600 r and react for 1 - 3 h. 22 GSH 18 in, and adjust the rotation speed to 400 - 600 r and react for 1 - 3 h.

[0030] (c) Use weak alkaline water for ultrafiltration centrifugation to remove unreacted substrates and concentrate; the temperature of the ultrafiltration centrifugation is 4 - 30 °C, the rotation speed of the ultrafiltration centrifugation is 2000 - 4000 rpm, the time of the ultrafiltration centrifugation is 5 - 30 min; the number of ultrafiltration times is 8 - 10 times; the membrane pore size of the ultrafiltration tube is 3 - 50 kDa.

[0031] Freeze-dry after purification.

[0032] As a preferred embodiment, step (3) specifically includes the following steps:

[0033] Redissolve the freeze-dried complex in phosphate buffered saline (PBS) to a concentration of 0.1 - 5 μg / μL, sonicate for 5 - 10 min to ensure no visible particles to the naked eye, slowly add 0.1% sodium hyaluronate in a ratio of 1:1 - 1:2, stir magnetically at 200 - 300 r for 15 - 30 min, and filter the mixture through a 0.2 - 0.22 μm filter membrane for aseptic filtration and then aliquot.

[0034] As a preferred embodiment, the application of the ultra-small gold nanoparticles as described in any one of the above embodiments or the ultra-small gold nanoparticles prepared by the preparation method as described in any one of the above embodiments in the preparation of drugs for treating posterior segment of the eye ROS-related diseases, and the diseases include: photodamage retinopathy, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR).

[0035] As a preferred embodiment, the ultra-small gold nanoparticles are administered 2 - 3 times a day, and the dose per time is 5 - 10 μL.

[0036] As a preferred embodiment, the ultra-small gold nanoparticles penetrate the cornea and blood-retinal barrier, significantly increasing the concentration of gold nanoclusters in the retinal tissue.

[0037] Compared with the prior art, the present invention has the following advantages and technical effects:

[0038] The ultra-small gold nanoparticles prepared by the method of the present invention have a simple synthesis method, mild conditions, high yield, are easy to industrialize, and have high stability and good biological safety;

[0039] The ultra-small gold nanoparticles prepared by the method of the present invention have a breakthrough improvement in penetration efficiency. For traditional eye drops, due to the corneal barrier and tear flushing, the bioavailability in the posterior segment of the eye is <0.1%; although intravitreal injection is direct, the complication rate is >30%. The present invention utilizes CR8 transmembrane peptide-mediated targeted penetration: through the positive charge adsorption and endocytosis mechanism, it penetrates the cornea and blood-retinal barrier, and the drug concentration in the posterior segment of the eye is greatly increased compared with ordinary eye drops.

[0040] The ultrasmall gold nanoparticles prepared by the method of the present invention have significantly enhanced antioxidant efficacy. Free glutathione (GSH) has a low ROS scavenging rate due to its poor stability, weak penetration ability. The present invention utilizes the enzyme-like cascade activity of Au 22 GSH 18 : SOD / CAT synergistically scavenges O2 - and H2O2, and the ROS scavenging rate is greatly improved.

[0041] The ultrasmall gold nanoparticles prepared by the method of the present invention have revolutionary breakthroughs in safety and compliance. In the prior art, intravitreal injection needs to be repeated every 4 - 8 weeks, and the infection risk is >5%; traditional eye drops need to be administered 6 - 8 times a day. The present invention realizes non-invasive long-term treatment: only 2 drops per day, the complication rate is <1%, and Au 22 GSH 18 -CR8 has an ultrasmall size, and its renal metabolic clearance rate can be >80%, without metal ion leakage or liver and kidney burden.

[0042] The ultrasmall gold nanoparticles prepared by the method of the present invention have the ability to cover multiple indications. Most of the prior art are single drug targets (such as anti-VEGF is only used for neovascular lesions), and cannot solve the widespread damage related to ROS. The present invention can achieve broad-spectrum ROS scavenging: applicable to oxidative stress-related diseases such as light damage, AMD, and diabetic retinopathy. Description of the Drawings

[0043] Figure 1 It is the ultraviolet absorption spectrum and fluorescence emission spectrum diagram of the ultrasmall gold nanoparticles prepared in Comparative Example 1 of the present invention;

[0044] Figure 2 It is the mass spectrum diagram of the ultrasmall gold nanoparticles prepared in Comparative Example 1 of the present invention.

[0045] Figure 3 It is the hydrodynamic diameter of the ultrasmall gold nanoparticles prepared in Comparative Example 1 of the present invention.

[0046] Figure 4 It is the comparison diagram of agarose gel electrophoresis and PAGE gel electrophoresis of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1 of the present invention.

[0047] Figure 5 It is the ZETA potential diagram of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1.

[0048] Figure 6 It is the ultraviolet absorption spectrum and fluorescence emission spectrum diagram of the ultrasmall gold nanoparticles prepared in Example 1.

[0049] Figure 7 It is the hydrodynamic diameter of the ultrasmall gold nanoparticles prepared in Example 1.

[0050] Figure 8 To study the stability of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1 under incubation at 37 °C under different pH conditions.

[0051] Figure 9 To study the stability of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1 under incubation at 37 °C under artificial tear conditions.

[0052] Figure 10 To study the superoxide dismutase (SOD)-mimicking activity and catalase (CAT)-mimicking activity of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1.

[0053] Figure 11 To study the silver-stained HE section images of the eyes taken at 3 h and 6 h after the ultrasmall gold nanoparticles prepared in Comparative Example 1 were instilled into mice.

[0054] Figure 12 To study the silver-stained HE section images of the eyes taken at 3 h and 6 h after the ultrasmall gold nanoparticles prepared in Example 1 were instilled into mice.

[0055] Figure 13 To study the OCT imaging of healthy mice and mice with light-damaged retinas treated with PBS, Comparative Example 1, Example 1, and commercially available sodium hyaluronate eye drops on the third and seventh days.

[0056] Figure 14 To study the statistically obtained outer nuclear layer and total retinal thickness of healthy mice and mice with light-damaged retinas treated with PBS, Comparative Example 1, Example 1, and commercially available sodium hyaluronate eye drops on the third and seventh days.

[0057] Figure 15 To study the silver-stained eye section results of healthy mice and mice with light-damaged retinas treated with PBS, Comparative Example 1, Example 1, and commercially available sodium hyaluronate eye drops on the third and seventh days.

[0058] Figure 16 For PBS, Au 22 GSH 18 and commercially available sodium hyaluronate eye drops, the standard structural diagram of photoreceptor cells in the outer nuclear layer (ONL) was observed in the retinas of mice with light-damaged retinas. Detailed implementation manners

[0059] To more clearly illustrate the embodiments of the present invention, the specific implementation manners of the present invention will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings, and other implementation manners can be obtained.

[0060] In the following specific examples, chloroauric acid was purchased from Shanghai Merck Chemical Technology Co., Ltd.; sodium borohydride (NaBH4) was purchased from Shanghai Fulian Chemical Reagent Co., Ltd.; cell-penetrating peptide CR8 was purchased from Hefei Sen'er Biotechnology Co., Ltd.; superoxide dismutase activity detection kit and catalase activity detection kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Instruments for exploring the optical properties and morphology of ultra-small gold nanoparticles that can cross the blood-eye barrier mainly include the PerkinElmer fluorescence / phosphorescence / luminescence spectrophotometer LS-55 from the United States, the Shimadzu ultraviolet-visible absorption spectrometer UV-2600 from Japan, the Malvern nano-particle size and ZETA potential analyzer from the United Kingdom, the Thermo Scientific inductively coupled plasma mass spectrometer (iCAP RQ) from Germany, etc.

[0061] Example 1

[0062] The preparation method of the ultra-small gold nanoparticles that can cross the blood-eye barrier in this example includes the following steps:

[0063] (1) Synthesize atomically precise gold nanoclusters Au 22 GSH 18

[0064] At room temperature, prepare 500 μl of an aqueous solution of chloroauric acid trihydrate (HAuCl4·3H2O) with a concentration of 20 mM, 300 μl of an aqueous solution of glutathione (GSH) with a concentration of 50 mM, and 25 μl of an aqueous solution of sodium borohydride (NaBH4) with a concentration of 10 mM. Add the HAuCl4 aqueous solution and the GSH aqueous solution to 9.2 ml of deionized water successively, and then stir vigorously at a speed of 1200 r for 2 min. After the solution changes from light yellow to turbid, add 1 M NaOH aqueous solution to adjust the pH of the solution to 12. After adjusting the pH, the solution becomes bright yellow. Then slowly add 25 μl of the reducing agent NaBH4 aqueous solution, and adjust the rotation speed to 500 r during this period.

[0065] The solution turns orange after reacting for about 15 min, and after reacting for 30 min, add 1 M HCl aqueous solution to adjust the pH of the solution to 2.5.

[0066] Then adjust the rotation speed to 200 r again and react slowly for 8 h.

[0067] (2) Purify the material

[0068] After reacting for 8 h, 1 M aqueous NaOH solution was first added to adjust the pH of the solution to 6.5. Then, weak alkaline water with a pH of 8.3 was prepared, and the unreacted substrate was removed by ultrafiltration centrifugation using the weak alkaline water. The temperature of ultrafiltration centrifugation was 25 °C, the rotation speed was 3750 rpm, and the ultrafiltration centrifugation time was 15 min; the number of ultrafiltration times was 8 times; the membrane pore size of the ultrafiltration tube was 3 kDa, and finally it was concentrated.

[0069] (3) Ligand exchange modification of cell-penetrating peptide CR8

[0070] CR8 was dissolved in a 1 mM aqueous NaOH solution to a concentration of 5 mM. The concentration of GSH was quantitatively determined by ICP, and CR8 was added to the reaction in an amount with a molar ratio of CR8:GSH = 1:10 to GSH. The system was made up to 10 ml with 1 mM NaOH, and the rotation speed was adjusted to 500 r, and the reaction was carried out at room temperature for 2 h. The unreacted substrate was removed by ultrafiltration centrifugation using weak alkaline water and concentrated. The ultrafiltration conditions were the same as those in step (2), and after purification, it was freeze-dried.

[0071] (4) Adding excipients to prepare eye drops

[0072] 6 mg of the freeze-dried complex was redissolved in 1 ml of phosphate buffer (PBS), sonicated for 5 min to ensure no visible particles to the naked eye, 1 ml of 0.1% sodium hyaluronate was slowly added, and magnetic stirring was carried out at 200 r for 15 min. The mixture was aseptically filtered through a 0.2-μm filter membrane and then aliquoted.

[0073] Comparative Example 1:

[0074] The difference between the ultra-small gold nanoparticles in this comparative example and those in Example 1 is that: Au without ligand exchange was used 22 GSH 18 .

[0075] The specific implementation steps can refer to steps (1) and (2) of Example 1

[0076] The ultraviolet absorption spectra and fluorescence emission spectra of the ultra-small gold nanoparticles that can cross the blood-eye barrier prepared in this example and the comparative example were characterized, and their morphologies were also characterized.

[0077] Figure 1 Shown are the ultraviolet absorption spectrum and fluorescence emission spectrum of the ultra-small gold nanoparticles prepared in Comparative Example 1. From the ultraviolet absorption spectrum, it can be seen that the prepared Au 22 GSH 18 has no absorption peak at 520 nm, indicating that the synthesized nanoparticles are of small size. From the fluorescence emission spectrum, it can be obtained that Au 22 GSH 18The emission center is located at 925 nm, and through the charge transfer effect between the ligand and the gold core, its tail extends to 1300 nm, covering the starting band of the NIR-II window (900 - 1700 nm).

[0078] Figure 2 It is the mass spectrum of the ultrasmall gold nanoparticles prepared in Comparative Example 1. The obtained Au 22 GSH 18 The good match between the sample peaks and the theoretically simulated pattern confirms the atomically precise structure.

[0079] Figure 3 It is the hydrodynamic diameter of the ultrasmall gold nanoparticles prepared in Comparative Example 1. The results demonstrate the ultrasmall size of the obtained material. The ultrasmall size below 5 nm enables its rapid metabolic clearance even after entering the body.

[0080] Figure 4 It is the agarose gel electrophoresis and PAGE gel electrophoresis diagrams of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1. It can be seen that due to the modification of the positively charged CR8 through ligand exchange, the negative potential of the modified gold nanoparticle complex becomes smaller, and at the same time, the electrophoresis stratification results also prove the successful modification of the cell-penetrating peptide CR8.

[0081] Figure 5 It is the ZETA potential diagram of the ultrasmall gold nanoparticles prepared in Comparative Example 1 and Example 1. Similarly, due to the introduction of the positively charged cell-penetrating peptide CR8, the negative potential carried by the ultrasmall gold nanoparticles becomes smaller, further proving the successful modification of the positively charged cell-penetrating peptide CR8.

[0082] Figure 6 It is the ultraviolet absorption spectrum and fluorescence emission spectrum diagrams of the ultrasmall gold nanoparticles prepared in Example 1. Since the cell-penetrating peptide CR8 used for ligand exchange has no obvious characteristic absorption, the obtained Au 22 GSH-CR8 complex still only shows the absorption peak of Au 22 .

[0083] Figure 7 It is the hydrodynamic diameter of the ultrasmall gold nanoparticles prepared in Example 1. The results demonstrate that the modification of the cell-penetrating peptide CR8 does not affect the ultrasmall size of the material itself. The ultrasmall size below 5 nm after modification enables its rapid metabolic clearance even after entering the body.

[0084] Figure 8To study the stability of the ultra-small gold nanoparticles prepared in Comparative Example 1 and Example 1 under incubation at 37 °C under different pH conditions. It can be seen that they have good long-term stability under conditions ranging from strong acidity to weak alkalinity, and the characteristic absorption is stable for a long time. The pH of the eye ranges from the ocular surface tear film to the aqueous humor in the eye is generally 6.5 - 7.8. According to existing research, the intraocular pH may fluctuate locally due to oxidative stress and metabolic disorders after eye injury. Therefore, these two materials still have good stability within the fluctuating range.

[0085] Figure 9 To study the stability of the ultra-small gold nanoparticles prepared in Comparative Example 1 and Example 1 under incubation at 37 °C in artificial tears. The results show that they have good long-term stability under this condition, and the characteristic absorption is stable for a long time, which is beneficial for the material to maintain a good and lasting therapeutic effect in the intraocular environment.

[0086] Figure 10 To study the superoxide dismutase (SOD) mimicking activity and catalase (CAT) mimicking activity of the ultra-small gold nanoparticles prepared in Comparative Example 1 and Example 1. The results show that different concentrations of the ultra-small gold nanoparticles exhibit different inhibition rates, and the introduction of the cell-penetrating peptide CR8 does not affect the 22 original enzyme-like activity. Therefore, the ultra-small gold nanoparticles can effectively scavenge superoxide anions in a dose-dependent manner, and the data are presented in the form of mean ± SD (n = 3).

[0087] Figure 11 Silver staining and HE sectioning of the eyes of mice were performed at 3 h and 6 h after instillation of the ultra-small gold nanoparticles prepared in Comparative Example 1. The results show that most of the ultra-small gold nanoparticles remained in the corneal layer and could not break through the corneal barrier to reach the posterior part of the eye. Only a small part could reach the posterior part of the eye through the conjunctiva-sclera pathway, but they would still be intercepted by the choroid and could not reach the retina.

[0088] Figure 12 Silver staining and HE sectioning of the eyes of mice were performed at 3 h and 6 h after instillation of the ultra-small gold nanoparticles prepared in Example 1. The results show that compared with the ultra-small gold nanoparticles prepared in Comparative Example 1, after modification with the cell-penetrating peptide CR8, the penetration ability of the original nanomaterial was greatly improved, and the distribution in the retinal tissue at the posterior part of the eye was increased by dozens of times compared with the Comparative Example 1 group. The significant improvement in penetration ability proves its ability to be delivered to the posterior part of the eye by instillation for treatment.

[0089] Figure 13After one week of instilling the ultra-small gold nanoparticles prepared in PBS, Comparative Example 1 and Example 1 into the eyes of mice, the eyes were taken for HE sectioning 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, demonstrating that the prepared ultra-small gold nanoparticles had excellent ocular biocompatibility and highlighting their potential as permeable eye drops for posterior eye drug delivery.

[0090] The ultra-small gold nanoparticles prepared in the above Comparative Example 1 and Example 1 were characterized by a series of in vitro and in vivo experiments, which confirmed that they both had good in vitro free radical scavenging ability. In addition, by modifying the cell-penetrating peptide CR8, the ability of the ultra-small gold nanoparticles prepared in Example 1 to be delivered to the posterior eye was greatly improved. Further, it was proved that the prepared ultra-small gold nanoparticles had excellent ocular biocompatibility. Therefore, further in vivo treatment of light-damaged retinas of mice was carried out. First, experimental eye drops were prepared: Au 22 GSH 18 、Au 22 The drug concentration of the GSH-CR8 solution was configured to be 3 μg / μL; in order to construct a light-damaged retina model, a dark box equipped with a blue light source on the top and mirrors on the four sides was used. Six-week-old female BALB / c mice were subjected to dark adaptation for 16 h, and 0.5% tropicamide solution was topically applied to the corneas of the mice to ensure pupil dilation. Then they were exposed to strong blue light for 1 h. Subsequently, they were returned to the dark for another 16 h. The six-week-old female BALB / c mice with the constructed light-damaged retina model were randomly divided into 4 groups, and the above groups were treated with PBS, Au 22 GSH 18 、Au 22 GSH-CR8 and commercially available sodium hyaluronate eye drops, and were topically instilled into the light-damaged retina mouse model twice a day (3 μg / μL × 5 μL). Healthy mice were used as positive controls (four eyeballs in each group). On the 3rd and 7th days after administration, optical coherence tomography (OCT) was performed to evaluate the outer nuclear layer (ONL) and total retinal thickness of the mice from each treatment group. As Figure 14 and Figure 15 showed, the light-damaged retina mice after treatment showed similar ONL and total retinal thickness, but were significantly thinner than the 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, the mice treated with Au 22 GSH-CR8 showed a significant recovery of ONL and total retinal thickness, indicating that it could effectively reduce retinal degeneration by preventing photoreceptor apoptosis. To further confirm the anti-apoptotic efficacy of Au 22 GSH-CR8, the eyeballs were subjected to HE staining on the 3rd and 7th days after different treatments. AsFigure 16 As shown, in the retinas of light-damaged retinal mice treated with PBS, Au 22 GSH 18 and commercially available sodium hyaluronate eye drops, significant inflammatory cell infiltration and destruction of the standard structure of photoreceptor cells in the ONL were observed, characterized by morphological changes, disordered arrangement, and reduced thickness. In contrast, Au 22 GSH-CR8 effectively reduced inflammatory cell infiltration on both the 3rd and 7th days, prevented photoreceptor apoptosis, and greatly improved the structural arrangement of the ONL. Overall, these results demonstrated that Au 22 GSH-CR8 was effectively delivered to the posterior eye and significantly alleviated retinal degeneration by preventing apoptosis. Therefore, the ultra-small gold nanoparticles of the present invention can be applied in the preparation of drugs for treating and preventing light-damaged retinas.

[0091] In view of the numerous embodiments of the present invention, the raw materials and dosages involved can be selected according to actual needs within the limited range. The experimental data of each embodiment are huge and numerous, and it is not suitable to list them one by one here. However, the content to be verified and the final conclusions obtained in each embodiment are similar. Therefore, the verification content of each embodiment will not be described one by one here.

[0092] The above description only elaborates in detail on the preferred embodiments and principles of the present invention. For those of ordinary skill in the art, based on the idea provided by the present invention, there will be changes in the specific implementation manners, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A super-small gold nanoparticle capable of crossing the blood-eye barrier, characterized in that: The ultra-small gold nanoparticles contain atomically precise gold nanoclusters Au 22 cores; ligands are covalently modified on the surface of the Au 22 cores through Au-S bonds; the ligands are glutathione GSH, and the number of ligands is 10-16; cell-penetrating peptide CR8, whose sequence is CRRRRRRR, replaces part of the GSH ligands through Au-S bonds, and the modification amount is 2-8 CR8 molecules / Au 22 cluster.

2. The ultra-small gold nanoparticles capable of crossing the blood-eye barrier according to claim 1, characterized in that, The hydrated particle size of the ultra-small gold nanoparticles is 1.8 - 2.5 nm, and the Zeta potential of the ultra-small gold nanoparticles is -5 to -10 mV.

3. An eye drop containing the ultra-small gold nanoparticles capable of crossing the blood-eye barrier according to claim 1 or 2, characterized in that: It includes ultra-small gold nanoparticles, phosphate buffer solution and sodium hyaluronate; it is packaged in a single-dose sterile form; the final concentration of the ultra-small gold nanoparticles is 0.1 - 5 μg / μL; the pH value of the phosphate buffer solution is 7.2 - 7.6; the concentration of the sodium hyaluronate is 0.05% - 0.1% (w / v).

4. The preparation method of the eye drops according to claim 3, characterized in that, It includes the following steps: (1)Synthesize atomically precise gold nanoclusters Au 22 GSH 18 ; (2) Ligand exchange modification of the cell-penetrating peptide CR8; (3) Adding excipients to prepare eye drops.

5. The preparation method according to claim 4, wherein The specific steps of step (1) include the following steps: (a) Prepare an aqueous solution of HAuCl4·3H2O with a concentration of 10 - 30 mM, an aqueous solution of GSH with a concentration of 40 - 60 mM, an aqueous solution of NaBH4 with a concentration of 5 - 15 mM, an aqueous solution of NaOH with a concentration of 1 - 2 mM and an aqueous solution of HCl; (b) Add the aqueous solution of HAuCl4·3H2O and the aqueous solution of GSH to deionized water successively, and then stir vigorously at a speed of 1000 r - 1200 r for 2 - 5 min; the addition amounts of the aqueous solution of HAuCl4·3H2O and the aqueous solution of GSH satisfy: the molar ratio of HAuCl4 to GSH is 1:1 - 1:2; (c) After the solution changes from light yellow to turbid, add the aqueous solution of NaOH to adjust the pH of the solution to 11.8 - 12.2, and the solution becomes bright yellow after adjusting the pH; (d) Slowly drip the aqueous solution of NaBH4 and adjust the speed to 450 - 500 r; the addition amount of the aqueous solution of NaBH4 satisfies: the molar ratio of NaBH4 to HAuCl4 is NaBH4:HAuCl4 = 1:35 - 1:45; (e) React for 10 - 20 min and the solution becomes orange, and after reacting for 30 - 40 min, add HCl to adjust the pH of the solution to 2.4 - 2.6; (f) Adjust the rotation speed to 150 - 200 r, and slowly react for 8 - 9 h. After purification, atomically precise gold nanoclusters Au are obtained 22 GSH 18 .

6. The preparation method according to claim 5, characterized in that, The purification method is: (a) After the reaction is completed, add NaOH to adjust the pH of the solution to 6.2 - 6.8; (b) Prepare weak alkaline water with a pH of 8.0 - 8.6; (c) Use weak alkaline water for ultrafiltration centrifugation to remove unreacted substrates and concentrate; the ultrafiltration centrifugation temperature is 4 - 30 °C, the ultrafiltration centrifugation speed is 2000 - 4000 rpm, the ultrafiltration centrifugation time is 5 - 30 min; the number of ultrafiltration times is 8 - 10 times; the membrane pore size of the ultrafiltration tube is 3 - 50 kDa.

7. The preparation method according to claim 4, characterized in that, The specific steps of step (2) include the following steps: (a) Dissolve CR8 with a NaOH solution with a concentration of 1 - 2 mM, and the concentration of CR8 is 4 - 5 mM; (b) Quantitatively determine the GSH concentration. Slowly add CR8 to the atomically precise gold nanocluster Au in an amount with a molar ratio of CR8:GSH = 1:5 - 1:

10. 22 GSH 18 Adjust the rotation speed to 400 - 600 rpm and react for 1 - 3 h. (c) Use weak alkaline water for ultrafiltration centrifugation to remove unreacted substrates and concentrate; the ultrafiltration centrifugation temperature is 4 - 30 °C, the ultrafiltration centrifugation speed is 2000 - 4000 rpm, the ultrafiltration centrifugation time is 5 - 30 min; the number of ultrafiltration times is 8 - 10 times; the membrane pore size of the ultrafiltration tube is 3 - 50 kDa, and after purification, it is freeze-dried.

8. The preparation method according to claim 4, characterized in that The specific steps of step (3) include the following steps: The freeze-dried complex was redissolved in phosphate buffer PBS to a concentration of 0.1-5 μg / μL, sonicated for 5-10 min to ensure no visible particles to the naked eye, slowly added 0.1% sodium hyaluronate in a ratio of 1:1-1:2, magnetically stirred at 200-300 r for 15-30 min, and the mixture was aseptically filtered through a 0.2-0.22 μm filter membrane and then aliquoted.

9. Use of the ultrasmall gold nanoparticles according to claim 1 or 2 in the preparation of a medicament for treating ROS-related diseases in the posterior segment of the eye, said diseases including: Photodamage retinopathy, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy (DR).

10. The application according to claim 9, wherein The ultra-small gold nanoparticles are administered 2-3 times a day, with a dose of 5-10 μL each time; the ultra-small gold nanoparticles penetrate the cornea and the blood-retinal barrier, greatly increasing the concentration of gold nanoclusters in the retinal tissue.

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