A method for preparing chiral cadmium selenide quantum dots and their application in retinal degeneration

Abstract

This invention relates to the field of biomedical technology, specifically proposing a method for preparing chiral cadmium selenide quantum dots and their application in retinal degeneration. By precisely controlling the reaction conditions, highly efficient chiral cadmium selenide quantum dots were successfully synthesized. The chiral cadmium selenide quantum dots of this invention exhibit significant protective effects against retinal damage caused by high intraocular pressure or oxidative stress, effectively improving the survival rate of retinal ganglion cells and alleviating retinal pathological changes. In a rat model, topical ocular administration demonstrated good therapeutic efficacy and biosafety. This invention provides new ideas and methods for the treatment of retinal degeneration and has broad clinical application prospects. Furthermore, the preparation method of these quantum dots and their complexes is simple and low-cost, providing new possibilities for expanding the application of quantum dots in the biomedical field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a preparation method of chiral cadmium selenide quantum dots and application thereof in retinal degeneration. BACKGROUND

[0002] Retinal degeneration is a complex group of inherited and acquired ophthalmic diseases characterized by the gradual degeneration of retinal cells, ultimately leading to vision loss. Current treatment options are limited and have varying effects. For example, current treatments for retinal degeneration mainly rely on drugs, surgery and gene therapy, but the effects of these methods vary from individual to individual and are not effective for all patients. Drug treatment can only alleviate symptoms and cannot reverse the degeneration process of retinal cells. With the continuous development of nanotechnology, quantum dots are increasingly widely used in the field of biomedicine. In particular, chiral quantum dots have shown great potential in neuroprotection due to their unique physical and chemical properties and biocompatibility. However, the biocompatibility and toxicity of chiral quantum dots have always been the focus of research. Ordinary quantum dots may have potential toxicity, and the preparation method of chiral quantum dots in the prior art is complex and costly, and the application effect of chiral quantum dots in neuroprotection still needs to be improved. SUMMARY

[0003] Based on the above problems, the present application provides a preparation method of chiral cadmium selenide quantum dots and application thereof in retinal degeneration.

[0004] The first aspect of the present application provides a preparation method of chiral cadmium selenide quantum dots, which comprises the following key steps:

[0005] Preparation of sodium selenate solution: Dissolve selenium powder and sodium hydroxide in deionized water, heat to boiling and maintain for a period of time until the selenium powder is completely dissolved, thereby obtaining a sodium selenate solution.

[0006] Preparation of cadmium-modified chiral ligand complex solution: Dissolve cadmium chloride in deionized water, then add chiral ligand D-cysteine (Cys), and adjust the pH value of the solution to an appropriate range to form a Cd-D-Cys complex solution.

[0007] Formation of cadmium selenide quantum dots: Slowly add the sodium selenate solution to the Cd-D-Cys complex solution and perform vigorous stirring during the addition process. After the addition is completed, the mixed solution is microwave-heated to an appropriate temperature and maintained for a period of time to promote the generation of cadmium selenide quantum dots.

[0008] Purification of quantum dots: Use a centrifuge to centrifuge the solution after the reaction to separate the quantum dot precipitate. Then, the precipitate is re-dispersed in deionized water and the centrifugation-dispersion steps are repeated to obtain a purified chiral cadmium selenide quantum dot solution.

[0009] In a preferred embodiment, the molar ratio of selenium powder, sodium hydroxide, cadmium chloride, and D-Cys is precisely controlled at 0.32:0.5:0.64:1.28 to ensure efficient synthesis of quantum dots. Furthermore, parameters such as the heating time to boiling and holding, the heating temperature and time of the mixed solution, and the centrifuge speed and time are also optimized to obtain the best quantum dot preparation results.

[0010] A second aspect of this invention relates to the use of chiral cadmium selenide quantum dots in the preparation of drugs for treating retinal degeneration. Experimental data show that these quantum dots have a significant protective effect against retinal damage caused by high intraocular pressure or oxidative stress. Therefore, the chiral cadmium selenide quantum dots of this invention can be used to prepare drugs for treating retinal degeneration.

[0011] Compared with existing technologies, the chiral cadmium selenide quantum dots of this invention exhibit excellent biocompatibility, providing new possibilities for expanding the application of quantum dots in the biomedical field. The chiral cadmium selenide quantum dots of this invention have a significant protective effect against retinal damage caused by high intraocular pressure or oxidative stress, effectively improving the survival rate of retinal ganglion cells, alleviating pathological changes in the retina, and thus improving the patient's vision. Attached Figure Description

[0012] Figure 1 The results show the inhibitory effect of different concentrations of chiral cadmium selenide quantum dots on apoptosis of retinal ganglion cells (RGCs) in vitro.

[0013] Figure 2 The results show the effects of H2O2-induced oxidative stress damage on chiral cadmium selenide quantum dots.

[0014] Figure 3 The results show the scavenging ability of chiral cadmium selenide quantum dots against the free radical DPPH.

[0015] Figure 4 The results show the improvement of retinal damage induced by high intraocular pressure induced by chiral cadmium selenide quantum dots in vivo. Scale bar: 10 μm.

[0016] Figure 5 The image shows the pathological changes in cardiac cells of rats with high intraocular pressure treated with chiral cadmium selenide quantum dots. Scale bar: 10 μm. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention.

[0018] Example 1

[0019] Synthesis of chiral cadmium selenide quantum dots: 0.0254 g (0.32 mmol) of selenium powder and 0.02 g of sodium hydroxide (NaOH, 0.5 mmol) were dissolved in 10 mL of deionized water. The solution was heated to boiling and maintained for 1 hour until the selenium powder was completely dissolved, yielding a sodium selenate (Na2SeO3) solution. 0.1366 g (0.64 mmol) of cadmium chloride (CdCl2) was dissolved in 50 mL of deionized water. 0.124 g (1.28 mmol) of chiral ligand D-Cys was added, and the pH was adjusted to 8.6 to obtain a Cd-D-Cys complex solution. The sodium selenate solution was slowly added dropwise to the Cd-D-Cys complex solution while stirring vigorously. After the addition was complete, the mixture was microwaved to 120 °C and maintained for 20 min to promote the formation of chiral cadmium selenide quantum dots. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was discarded to obtain the quantum dot precipitate. The precipitate was redispersed in deionized water, and the centrifugation-dispersion steps were repeated 2-3 times to obtain a purified chiral cadmium selenide quantum dot solution.

[0020] Example 2

[0021] The method for preparing cadmium selenide quantum dots differs from that in Example 1 in that the chiral ligand D-Cys is not required, while the remaining preparation steps are the same as in Example 1.

[0022] Example 3

[0023] The cell model used in this invention is an in vitro pressure-cultured retinal ganglion cell (RGC) apoptosis model. Purified primary rat RGCs were cultured in an incubator at 37°C and 5% CO2. After complete cell adhesion, the cells were placed in a pressure incubator and randomly divided into five groups: a control group, a model group, a 5μM treatment group, a 10μM treatment group, and a 20μM treatment group. The four groups other than the control group were cultured at 60 mmHg, 37°C, and 5% CO2 for 24 hours. The treatment group was supplemented with culture medium containing chiral cadmium selenide quantum dots at the corresponding final concentration. The control group was placed in a carbon dioxide incubator. The model group and the treatment group were cultured in a carbon dioxide pressure incubator at 60 mmHg. All other parameters for all groups were maintained at 37°C and 5% CO2. CCK8 assays were performed after 24 hours.

[0024] Depend on Figure 1As can be seen, compared with the blank control, the survival rate of RGCs in the model group was significantly reduced, indicating that an in vitro model of RGC apoptosis was successfully established. Compared with the model group, the survival rate of RGCs in the treatment groups with different concentrations of chiral cadmium selenide quantum dots was significantly increased, confirming the inhibitory effect of chiral cadmium selenide quantum dots on RGC apoptosis in vitro, and also preliminarily revealing its feasibility as a potential neuroprotective agent. The 10 μM treatment group showed the best survival rate, and this concentration was used in subsequent embodiments of this invention.

[0025] Example 4

[0026] Following the same cell culture procedure as in Example 3, RGCs were first cultured under in vitro pressure. After complete cell adhesion, purified primary rat RGCs were randomly divided into four groups: a blank control group, a model group, a cadmium selenide quantum dot treatment group (10 μM), and a chiral cadmium selenide quantum dot treatment group (10 μM). The blank control group was cultured in a conventional CO2 incubator, while the model group and the two treatment groups were cultured in a CO2 pressure incubator with a pressure of 60 mm Hg. All groups were maintained at 37°C and 5% CO2. After cell culture stabilized, the oxidative stress inducer H2O2 was added to the retinal cell cultures of the model group and the treatment groups to simulate the in vivo oxidative stress environment. Subsequently, a solution containing chiral cadmium selenide quantum dots was added to the culture system of the chiral cadmium selenide quantum dot treatment group, and a solution containing cadmium selenide quantum dots was added to the culture system of the cadmium selenide quantum dot treatment group, while the model group only received an equal amount of solvent as a control. The scavenging ability of chiral cadmium selenide quantum dots against the free radical DPPH was then tested.

[0027] like Figure 2 As shown, compared with the blank control group, the survival rate of RGCs in the model group was significantly reduced after the addition of the oxidative stress inducer H2O2, indicating that H2O2 successfully induced cellular oxidative stress and caused damage. While the cadmium selenide quantum dot treatment group also showed some protective effect under the same conditions, this improvement was very limited. Compared with the model group, the chiral cadmium selenide quantum dot treatment group showed a significant increase in the survival rate of RGCs. Chiral cadmium selenide quantum dots may interact with specific intracellular biomolecules in a more matched and efficient manner, thereby more effectively alleviating cellular damage caused by oxidative stress and promoting cell survival and recovery. This suggests that chiral cadmium selenide quantum dots have a certain protective effect against H2O2-induced oxidative stress damage.

[0028] like Figure 3As shown, this invention further investigated the scavenging ability of quantum dots against the free radical DPPH. Experimental results showed that, compared to the control group without chiral cadmium selenide quantum dots, the solution containing chiral cadmium selenide quantum dots exhibited a significant scavenging effect on DPPH free radicals, far exceeding that of the cadmium selenide quantum dot treatment group. This indicates that chiral cadmium selenide quantum dots possess strong antioxidant activity, effectively scavenging free radicals and thus mitigating oxidative stress-induced cell damage.

[0029] In summary, chiral cadmium selenide quantum dots exhibit significant protective effects against H2O2-induced oxidative stress damage in an in vitro pressurized RGCs model, and also possess strong free radical scavenging capabilities.

[0030] Example 5

[0031] Fifteen SPF-grade SD rats were randomly divided into three groups: a sham-operated group, a high intraocular pressure model group, and a treatment group. Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (45 mg / kg), with the left eye selected as the surgical eye. Before surgery, the left eye was topically anesthetized with 0.5% promecaine eye drops. Next, the bulbar conjunctiva was incised clockwise from 6:00 to 14:00, 1 mm posterior to the corneal limbus, and the subconjunctival fascia and muscles were carefully dissected. Subsequently, three Y-shaped suprascleral veins were located on either side of the superior rectus muscle and near the inferior lateral rectus muscle. The main branches of these veins were freed and lifted, clamped, and the distal main vein of the cornea and sclera was cauterized with a heated pin to close the veins. After successful cauterization, the bulbar conjunctiva was repositioned and sutured. In the sham-operated group, the left bulbar conjunctiva was incised at the same location, but the suprascleral veins were not cauterized. Postoperatively, levofloxacin eye ointment was applied to the conjunctival sac of the left eye. The rats were placed on a warming blanket until they naturally awoke, and then returned to their cages. For the first 5 days postoperatively, levofloxacin eye ointment was applied to the conjunctival sac once daily. Rats in the treatment group received topical eye drops of 0.2 mL / kg (containing 2 mg / mL chiral cadmium selenide quantum dots) once daily for two weeks. Rats in the sham-operated group and the high intraocular pressure model group received the same volume of distilled water eye drops once daily for two weeks. At the end of the experimental period, ocular and cardiac tissues from the rats were collected for HE staining and observation.

[0032] HE staining results of rat ocular tissue sections in each group are as follows: Figure 4As shown, the retinal structure of rats in the sham-operated group was clear, with distinct layers, and cells in each layer were neatly arranged and morphologically normal. The retinal nerve fiber layer (NFL) was continuous and intact, and the retinal ganglion cell (RGC) layer had a sufficient number of cells, full morphology, and clearly visible nuclei, indicating that the sham-operated procedure did not cause significant damage or impact on the rat retina. In contrast, the retina of rats in the high intraocular pressure model group showed significant pathological changes. The cells in each retinal layer were disordered, with varying degrees of cell loss and morphological abnormalities. In particular, the RGC layer showed a significant reduction in cell number, cell atrophy, and pyknosis or fragmentation of cell nuclei, indicating the successful establishment of the high intraocular pressure model. Compared to the high intraocular pressure model group, the treatment group rats retained more RGC cells in the retinal layer, and the cell morphology was relatively intact, indicating that the chiral cadmium selenide quantum dots administered topically have a certain protective effect against high intraocular pressure-induced retinal damage. At the same time, the degree of retinal edema, vascular morphology, and inflammatory cell infiltration in the treatment group were also improved, further verifying the therapeutic effect of chiral cadmium selenide quantum dots.

[0033] HE staining results of heart tissue sections from each group of rats are as follows: Figure 5 As shown, the myocardial cells in all groups were uniformly stained, arranged regularly, and showed no edema, necrosis, or inflammatory cell infiltration. This indicates that the chiral cadmium selenide quantum dots did not produce significant toxic effects on myocardial tissue during the treatment of rats with high intraocular pressure, demonstrating the preliminary affirmation of the biosafety of this nanomaterial.

[0034] In summary, HE staining clearly revealed the pathological changes in the retina of rats in the sham-operated group, the high intraocular pressure model group, and the treatment group. Chiral cadmium selenide quantum dots exhibit significant protective and therapeutic effects against retinal damage induced by high intraocular pressure, and did not produce significant toxic effects on myocardial tissue during treatment. These results provide strong experimental evidence for further in-depth research on the application of chiral cadmium selenide quantum dots in the treatment of ophthalmic diseases.

[0035] The embodiments described above are only some embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. 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.

Claims

1. A method for preparing chiral cadmium selenide quantum dots, characterized in that, The method comprises the following steps: S1: Dissolve selenium powder and sodium hydroxide in deionized water, heat to boiling and keep for 1 h until the selenium powder is completely dissolved to obtain a sodium selenite solution; S2: Dissolve cadmium chloride in deionized water, add chiral ligand D-Cys, and adjust the pH value to 8.6 to obtain a Cd-D-Cys complex solution; S3: Slowly add the sodium selenite solution to the Cd-D-Cys complex solution while stirring vigorously; S4: After the addition is completed, microwave the mixed solution to 120℃ and keep for 20 min to promote the formation of cadmium selenide quantum dots; S5: Centrifuge the solution after the reaction using a centrifuge at a speed of 8000 rpm for 10 min, discard the supernatant, and obtain quantum dot precipitate; S6: Redisperse the quantum dot precipitate in deionized water, repeat the centrifugation-dispersion steps, and obtain a purified chiral cadmium selenide quantum dot solution; The molar ratio of the selenium powder, sodium hydroxide, cadmium chloride and D-Cys is 0.32:0.5:0.64:1.

28.

2. Use of the chiral cadmium selenide quantum dots prepared by the method of claim 1 in the preparation of a drug for treating retinal degeneration.

3. Use according to claim 2, characterized in that, The drug can act on the outside of the body, and by co-incubating 5-20 μM of the drug with retinal ganglion cells (RGCs) cultured in vitro under pressure, the survival rate of the RGCs can be improved.

4. Use according to claim 2, characterized in that, The drug can act on the inside of the body, and is used for treating retinal damage caused by high intraocular pressure or oxidative stress.

5. Use according to claim 2, characterized in that, The drug can act on the inside of the body, and is administered by the way of local eye drops. The drug can act on the outside of the body, and by co-incubating 5-20 μM of the drug with retinal ganglion cells (RGCs) cultured in vitro under pressure, the survival rate of the RGCs can be improved. The drug can act on the inside of the body, and is used for treating retinal damage caused by high intraocular pressure or oxidative stress. The drug can act on the inside of the body, and is administered by the way of local eye drops.

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