Use of black phosphorus quantum dots in the preparation of a drug for treating glaucoma

Abstract

The application discloses application of black phosphorus quantum dots in inhibition of RGC ferroptosis in preparation of a drug for treating glaucoma. The black phosphorus quantum dots prepared from bulk black phosphorus have more uniform nanoscale particle size and a larger surface volume ratio than common black phosphorus materials, and can be more easily taken up by cells; vitreous cavity injection of a low dose of the black phosphorus quantum dots can effectively block RGC ferroptosis caused by glaucoma, and the mechanism is that the black phosphorus quantum dots reduce Fenton oxidative damage secondary to an increase in divalent iron ions under glaucoma conditions, reduce the level of lipid peroxides in RGCs, and finally block RGC ferroptosis. The black phosphorus quantum dots in the application have the most prominent feature that they are safe, effective and have strong compatibility compared with other ferroptosis inhibitors used in current basic research.

Description

Technical Field

[0001] This invention relates to the fields of medical polymer materials and biomedicine, specifically to the application of black phosphorus quantum dots in the preparation of drugs for treating glaucoma. Background Technology

[0002] Glaucoma is the leading cause of irreversible blindness worldwide. Its pathological basis is the selective death of retinal ganglion cells (RGCs), leading to permanent optic nerve atrophy and visual field defects. Lowering intraocular pressure (IOP) through medication and surgery is currently the only effective clinical treatment for glaucoma, but it doesn't benefit all patients. Clinically, we have observed that many glaucoma patients, even after their IOP is controlled to a normal level (<21 mmHg), still experience progressive RGC loss and vision decline. There are many reasons for this phenomenon, with abnormal iron metabolism after IOP recovery and the resulting secondary RGC ferroptosis being a very important contributing factor.

[0003] Ferroprelation is a novel iron-dependent programmed cell death mechanism. Unlike other cell death mechanisms (such as apoptosis and necrosis), ferroptosis is closely regulated by iron metabolism, with characteristic molecular biological alterations including elevated levels of free ferrous ions in the cytoplasm, redox imbalance, and accumulation of lethal lipid peroxides. Ferroprelation has been clearly identified as participating in the pathophysiological processes of numerous clinical diseases, such as tumors, neurodegenerative diseases, and glaucoma. Blocking RGC ferroptosis with ferroptosis inhibitors can effectively alleviate RGC damage caused by glaucoma and protect visual function, proving to be a viable new therapeutic target for glaucoma.

[0004] However, to date, there is no clinically marketed ferroptosis inhibitor drug used in the treatment of glaucoma. The reasons behind this are as follows: (1) Ferroptosis inhibitors (such as iron chelators) lack targeting. In addition to clearing abnormally elevated iron ions in the RGC, they also clear iron ions required for normal metabolism in the retina. Long-term use will disrupt the body's normal iron metabolism balance and lead to iron deficiency complications; (2) Other ferroptosis inhibitors (such as Ferrostatin-1 and Liproxstatin-1) are small molecule drugs that are easily bound and cleared by other non-targeted proteins in tissues and cells, resulting in short drug half-life and unstable efficacy; (3) The toxicity of ferroptosis inhibitors and their metabolic end products in the eye is still unclear. Especially in the relatively closed space of the eyeball, the safety requirements of the drug itself are extremely high, and many drugs cannot meet these requirements; (4) Due to the complexity of the pathogenesis of glaucoma, drugs that target ferroptosis alone cannot completely reverse RGC damage. They need to be combined with drugs that target other targets to achieve satisfactory efficacy, which puts forward higher requirements for drug compatibility. Based on the above situation, there is an urgent need to develop a safer, more effective, and more compatible ferroptosis inhibitor to meet the pressing clinical needs for future glaucoma treatment. Summary of the Invention

[0005] In view of the aforementioned shortcomings, the purpose of this invention is to provide a safer, more effective, and more compatible inhibitor of ferroptosis—black phosphorus quantum dots—and to apply it to the treatment of glaucoma in ophthalmology. Black phosphorus is the most stable allotrope of phosphorus, and its metabolic end products in the body are phosphates and water, which are completely non-toxic to the human body. It is a very safe bioactive material and has shown great application potential in biomedical research fields such as drug delivery, tumor immunotherapy, and photothermal therapy. The black phosphorus quantum dots prepared by this invention using bulk black phosphorus as raw material have a more uniform nanoscale particle size and a larger surface-to-volume ratio than ordinary black phosphorus materials, making them easier for cells to take up. Intravitreal injection of low doses of black phosphorus quantum dots can effectively block ferroptosis of RGCs caused by glaucoma. The mechanism is that black phosphorus quantum dots alleviate Fenton oxidative damage secondary to the increase of divalent iron ions under glaucoma conditions, reduce the level of lipid peroxides in RGCs, and ultimately block ferroptosis of RGCs.

[0006] To achieve the above objectives, the present invention provides the application of black phosphorus quantum dots in the preparation of drugs for treating glaucoma.

[0007] Based on the same inventive concept, this invention also provides the application of black phosphorus quantum dots in inhibiting RGC ferroptosis in the preparation of drugs for treating glaucoma.

[0008] Based on the same inventive concept, the present invention also provides a pharmaceutical composition for treating glaucoma, comprising black phosphorus quantum dots.

[0009] According to one aspect of the invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0010] According to one aspect of the invention, the pharmaceutical composition further includes a small molecule drug disposed within the black phosphorus quantum dots.

[0011] According to one aspect of the present invention, the method for preparing the black phosphorus quantum dots includes the following steps:

[0012] Step 1: Add black phosphorus powder to the solvent and perform a first ultrasonic treatment under sealed conditions to obtain a dispersion;

[0013] Step 2: Under ice bath conditions, the dispersion is subjected to a second ultrasonic treatment, and the supernatant is collected after centrifugation;

[0014] Step 3: Centrifuge the supernatant to obtain the precipitate; rinse the precipitate with pure water and resuspend it;

[0015] Step 4: Repeat step 3 and resuspend the final precipitate in blowing water to obtain a black phosphorus quantum dot dispersion.

[0016] According to one aspect of the present invention, in step 1, the solvent is N-methyl-2-pyrrolidone; the first ultrasonic treatment specifically includes: ultrasonic power of 1200W, ultrasonic time of 4 hours, ultrasonic frequency of 19-25KHZ, and the wave generating probe working for 2 seconds every 4 seconds.

[0017] According to one aspect of the present invention, in step 2, the temperature of the ice bath is below 4°C; the second ultrasonic treatment specifically involves an ultrasonic power of 300W and an ultrasonic time of 10 hours; the centrifugation specifically involves centrifugation at 7000 rpm for 20 minutes.

[0018] According to one aspect of the invention, in step 3, the centrifugation specifically refers to centrifugation at 12,000 rpm for 20 minutes.

[0019] According to one aspect of the invention, in step 4, repeating step 3 specifically means repeating it 3 times.

[0020] The beneficial effects of this invention are:

[0021] This invention discloses the application of black phosphorus quantum dots in inhibiting RGC ferroptosis in the preparation of drugs for treating glaucoma. The most prominent features of the black phosphorus quantum dots in this invention compared to other ferroptosis inhibitors currently used in basic research are their safety, efficacy, and strong compatibility.

[0022] Safety was demonstrated by the following: after intravitreal injection of black phosphorus quantum dots, no obvious morphological damage was observed to the eyeball, heart, liver, spleen, lungs, or kidneys; and there was no significant effect on the blood routine and liver and kidney function of rats.

[0023] The efficacy was demonstrated as follows: intravitreal injection of black phosphorus quantum dots reduced malondialdehyde content in the retina of glaucoma rats by approximately 46%; increased glutathione content in the retina by approximately 1.2 times; increased the expression level of GPX4, a negative regulator of ferroptosis, and decreased the expression of ACSL4, a positive regulator of ferroptosis, in the retinal ganglion cell complex; increased the thickness of the retinal ganglion cell complex by approximately 0.5 times; and improved the amplitude of the ab wave on electroretinogram by 2.1 times.

[0024] The strong compatibility is manifested in the fact that black phosphorus quantum dots can physically adsorb other small molecule drugs, and deliver the small molecule drugs into the eye in a coordinated manner without destroying their physicochemical properties; this characteristic is not possessed by other ferroptosis inhibitor drugs. Attached Figure Description

[0025] Figure 1 A represents black phosphorus quantum dots detected by transmission electron microscopy and high-resolution transmission electron microscopy as described in Embodiment 1 of the present invention; Figure 1 B represents the black phosphorus quantum dots observed under an atomic force microscope as described in Example 1 of this invention; Figure 1 C represents the thickness analysis of black phosphorus quantum dots observed under an atomic force microscope as described in Example 1 of this invention;

[0026] Figure 2 This is a schematic diagram of the rat glaucoma injury model described in Example 2 of the present invention;

[0027] Figure 3 A is a quantitative graph showing the malondialdehyde content in the retina of glaucoma rats treated with black phosphorus quantum dots according to Example 3 of the present invention. Figure 3 B is a quantitative graph of glutathione content in the retina of glaucoma rats treated with black phosphorus quantum dots as described in Example 3 of this invention. Figure 3 C represents the expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthase long chain family member 4 (ACSL4) in the RGC of glaucoma rats treated with black phosphorus quantum dots as described in Example 3 of this invention.

[0028] Figure 4 A shows the tissue morphology staining of rats after black phosphorus quantum dot treatment as described in Example 4 of this invention; Figure 4 B represents the liver and kidney function test results of rats after phosphorus quantum dot treatment as described in Example 4 of this invention; Figure 4 C represents the blood routine results of rats after phosphorus quantum dot treatment as described in Example 4 of this invention;

[0029] Figure 5 A represents the thickness of the retinal ganglion cell complex in rats after treatment with black phosphorus quantum dots as described in Example 5 of this invention. Figure 5B represents the electroretinogram results of rats after phosphorus quantum dot treatment as described in Example 5 of this invention;

[0030] Figure 6 This study analyzes the fluorescence intensity of Cy5.5 in the eyeball within 24 hours after intravitreal injection of black phosphorus quantum dots containing Cy5.5 fluorescent molecules, as described in Example 6 of this invention. Detailed Implementation

[0031] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0032] Example 1

[0033] Preparation method of black phosphorus quantum dots:

[0034] Add 25 mg of black phosphorus powder to 25 ml of N-methyl-2-pyrrolidone and place the mixture in a 50 ml sealed conical tube. Sonicate the mixture for 3 hours at 1200 W using an ultrasonic probe. The ultrasonic frequency was 19 to 25 kHz, with the probe working for 2 seconds every 4 seconds. Continue sonicating the dispersion for 10 hours at 300 W using an ice bath below 4°C. Centrifuge the resulting dispersion at 7000 rpm for 20 minutes and collect the supernatant. Centrifuge the supernatant at 12000 rpm for 20 minutes, rinse the precipitate with pure water, and resuspend. Repeat step 4 three times. Resuspend the final precipitate in pure water; the black phosphorus quantum dot dispersion is now successfully prepared.

[0035] Identification of black phosphorus quantum dots:

[0036] The particle size and lattice structure of black phosphorus quantum dots in the prepared black phosphorus quantum dot dispersion were detected using transmission electron microscopy and high-resolution transmission electron microscopy. Figure 1 A). Surface roughness analysis of black phosphorus quantum dots in a black phosphorus quantum dot dispersion was performed using atomic force microscopy. Figure 1 BC). Figure 1 It can be seen that the black phosphorus quantum dots prepared by the present invention are uniform nanoparticles with a diameter of less than 10 nm and a lattice stripe size of 0.22 nm, indicating that the black phosphorus quantum dots have good crystallinity.

[0037] Example 2

[0038] Establishment of a rat model of glaucoma loss:

[0039] Eight-week-old female SD rats were selected and anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a dose of 80 mg / kg body weight. After complete anesthesia, compound tropicamide eye drops were used to dilate the pupils, and oxybuprofen hydrochloride eye drops were used to anesthetize the ocular surface. The ocular surface was then disinfected with 0.1% povidone-iodine. The rats were fixed on a control board and placed under a stereomicroscope. A 31G needle with saline solution attached to its end was inserted into the anterior chamber approximately 1 mm inside the limbus. The pressure in the anterior chamber was increased to 110 mmHg by adjusting the height of the infusion bottle and maintained for 60 minutes. Figure 2 After the anterior chamber is pressurized, the puncture needle is quickly removed, antibiotic eye drops are applied to the ocular surface to prevent infection, the rat is marked with an ear tag and then transferred to a cage to recover from anesthesia; the operated eye is given levofloxacin eye drops once a day for 3 consecutive days.

[0040] Treatment options using black phosphorus quantum dots:

[0041] After the rat glaucoma model was established, a 31G puncture needle was removed from the anterior chamber. 2 μL of 10 μg / mL black phosphorus quantum dot dispersion (prepared in Example 1) was drawn using a Hamilton syringe. The needle was then inserted perpendicularly to the sclera at a point 2 mm posterior to the limbus at the 12 o'clock position on the rat's eyeball. 2 μL of the dispersion was slowly injected into the vitreous cavity using a Hamilton syringe. An equal volume of physiological saline was injected into the eyes of the control group.

[0042] Example 3

[0043] Inhibitory effect of black phosphorus quantum dots on ferroptosis:

[0044] The malondialdehyde (MDA) content in the retinas of glaucoma rats treated with black phosphorus quantum dots for 24 hours and control rats was detected using a malondialdehyde (MDA) detection kit. Figure 3 A); The glutathione content in the retinas of glaucoma rats treated with black phosphorus quantum dots for 24 hours and control rats was detected using a glutathione assay kit. Figure 3 B); Immunofluorescence staining was used to detect the expression of ferroptosis markers glutathione peroxidase 4 (GPX4) and acyl-CoA synthase long chain family member 4 (ACSL4) in the retinal cells of glaucoma rats and control rats treated with black phosphorus quantum dots for 24 hours. Figure 3 C). By Figure 3It is evident that the black phosphorus quantum dots prepared in this invention exhibit excellent inhibitory effects on ferroptosis. In animal experiments, intravitreal injection of black phosphorus quantum dots for 24 hours significantly reduced the malondialdehyde content in the retina of glaucoma rats and increased the retinal glutathione content. Simultaneously, it increased the expression of GPX4, a negative regulator of ferroptosis, and decreased the expression of ACSL4, a positive regulator of ferroptosis, in the retinal glucocorticoid retinopathy (RGC).

[0045] Example 4

[0046] Safety of black phosphorus quantum dots:

[0047] Twenty-four hours after intravitreal injection of 2 μL of 10 μg / ml black phosphorus quantum dot dispersion into rats, the rats' eyeballs, heart, liver, spleen, lungs, and kidneys were stained with hematoxylin and eosin to observe the histomorphological changes compared with the control group. Figure 4 A); 24 hours after intravitreal injection of 2 μL of 10 μg / ml black phosphorus quantum dot dispersion into rats, 1 ml of fresh blood was drawn from the rats for routine blood analysis. Figure 4 B) and liver and kidney function tests ( Figure 4 C), and compared with the control group. Figure 4 It was found that 24 hours after intravitreal injection of 2ul of 10ug / ml black phosphorus quantum dot dispersion, no obvious morphological damage was observed in the eyeball, heart, liver, spleen, lung, or kidney; it also had no significant effect on the blood routine and liver and kidney function of rats, indicating that black phosphorus quantum dots have good safety.

[0048] Example 5

[0049] The effectiveness of black phosphorus quantum dot therapy:

[0050] Seven days after intravitreal injection of 2 μL of 10 μg / ml black phosphorus quantum dot dispersion into glaucoma rats, the rat eyeballs were harvested for hematoxylin and eosin staining. The thickness of the ganglion cell complex (vertical distance from the nuclear layer to the nerve fiber layer) at ±800, ±1600, ±2400, ±3200, ±4000, and ±4800 μm distances from the optic disc was measured. The average thickness of each group was calculated and compared with the data of the control group rats. Figure 5 A) Seven days after intravitreal injection of 2 μL of 10 μg / ml black phosphorus quantum dot dispersion into glaucoma rats, electroretinography (ERG) was performed on the rats, and the ERG was recorded at 3.0 cd.s / m. 2 The changes in the difference between the a-wave and b-wave in electroretinography under white light stimulation were compared with data from the control group rats. Figure 5 B). By Figure 5It is evident that the black phosphorus quantum dots prepared in this invention can effectively alleviate retinal ganglion cell damage in glaucoma rats and improve visual function in glaucoma rats. Seven days after intravitreal injection of 2 μL of 10 μg / ml black phosphorus quantum dot dispersion, the thickness of the retinal ganglion cell complex in glaucoma rats increased compared to untreated rats. Furthermore, the amplitude of the ab wave shown on electroretinography also increased compared to untreated rats.

[0051] Example 6

[0052] Drug compatibility of black phosphorus quantum dots:

[0053] Black phosphorus quantum dots possess a loose, layered microstructure, allowing them to adsorb other small-molecule drugs through van der Waals forces, thus achieving drug delivery. Since this process is physical, black phosphorus quantum dots do not affect the structure of the loaded drug molecules or alter its efficacy, exhibiting excellent compatibility. This characteristic offers significant advantages for future applications of combined drug therapy for glaucoma. In this implementation plan, we simulate the fluorescent agent Cy5.5 as a small-molecule drug to be loaded within the black phosphorus quantum dots to verify their compatibility.

[0054] Mix an equal volume of 10 mg / ml black phosphorus quantum dot dispersion with 10 mg / ml Cy5.5 solution and incubate at 25°C for 8 hours. Centrifuge the mixture at 12000 rpm for 20 minutes, discard the supernatant, and resuspend the precipitate in ultrapure water. Repeat step 2 at least 3 times until no obvious Cy5.5 color residue remains in the supernatant. Adjust the final dispersion to 10 mg / ml. Inject 2 μL of 10 μg / ml dispersion into the vitreous cavity of rats after anesthesia, and detect Cy5.5 fluorescence in the rat eyes using a small animal in vivo imaging system at 5 minutes, 6 hours, and 24 hours after injection. Figure 6 This demonstrates that black phosphorus quantum dots incorporate Cy5.5 and exhibit good compatibility. Figure 6 It is known that the black phosphorus quantum dots prepared by this invention can bind small molecule drugs through physical adsorption while maintaining the properties of the adsorbed drugs, thus exhibiting good drug compatibility. After co-incubating Cy5.5 fluorescent molecules with black phosphorus quantum dot dispersion, unadsorbed Cy5.5 was removed, and the final black phosphorus quantum dot dispersion was injected into the vitreous cavity of rats. Fluorescent expression of Cy5.5 in the rat eyeballs was observed at different time points.

[0055] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Application of black phosphorus quantum dots in the preparation of drugs for treating glaucoma.

2. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition comprising black phosphorus quantum dots, which are the active ingredient of the pharmaceutical composition for treating glaucoma and treat glaucoma by inhibiting RGG ferroptosis.

3. The application according to claim 2, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

4. The application according to claim 2, characterized in that, The pharmaceutical composition also includes a small molecule drug carried within the black phosphorus quantum dots.

5. The application according to claim 2, characterized in that, The preparation method of the black phosphorus quantum dots includes the following steps: Step 1: Add black phosphorus powder to the solvent and perform a first ultrasonic treatment under sealed conditions to obtain a dispersion; Step 2: Under ice bath conditions, the dispersion is subjected to a second ultrasonic treatment, and the supernatant is collected after centrifugation; Step 3: Centrifuge the supernatant to obtain the precipitate; rinse the precipitate with pure water and resuspend it; Step 4: Repeat step 3 and resuspend the final precipitate in blowing water to obtain a black phosphorus quantum dot dispersion.

6. The application according to claim 5, characterized in that, In step 1, the solvent is N-methyl-2-pyrrolidone; the first ultrasonic treatment specifically involves: an ultrasonic power of 1200 W, an ultrasonic time of 4 hours, an ultrasonic frequency of 25 kHz, and the ultrasonic probe operating for 2 seconds every 4 seconds.

7. The application according to claim 5, characterized in that, In step 2, the temperature of the ice bath is below 4 ℃; the second ultrasonic treatment specifically involves an ultrasonic power of 300 W and an ultrasonic time of 10 hours; the centrifugation specifically involves centrifugation at 7000 rpm for 20 minutes.

8. The application according to claim 5, characterized in that, In step 3, the centrifugation specifically refers to centrifugation at 12,000 rpm for 20 minutes.

9. The application according to claim 5, characterized in that, In step 4, repeating step 3 specifically means repeating it 3 times.

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