Preparation method of chiral cadmium selenide quantum dots and application of chiral cadmium selenide quantum dots in retinal degeneration

Abstract

The invention relates to the technical field of biological medicines, and particularly provides a preparation method of chiral cadmium selenide quantum dots and application of the chiral cadmium selenide quantum dots in retinal degeneration. The chiral cadmium selenide quantum dot with high efficiency is successfully synthesized by accurately controlling reaction conditions. The chiral cadmium selenide quantum dot has a remarkable protection effect on retinal injury caused by intraocular hypertension or oxidative stress, and can effectively improve the survival rate of retinal ganglion cells and relieve pathological changes of retina. In a rat model, a local point-to-eye administration mode shows a good treatment effect and biological safety. The invention provides a new thought and means for treatment of retinal degeneration, and has a wide clinical application prospect. In addition, the preparation method of the quantum dot and the compound thereof is simple and convenient, the cost is relatively low, and a new possibility is provided for expanding the application of the quantum dot in the field of biomedicine.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing chiral cadmium selenide quantum dots and an application thereof in retinal degeneration. Background Art

[0002] Retinal degeneration is a complex class of inherited and acquired ophthalmic diseases characterized by the gradual degeneration of retinal cells, which eventually leads to vision loss. Current treatments are limited and have varying effects. For example, the current treatment of retinal degeneration mainly relies on drugs, surgery, and gene therapy, but the effects of these methods vary from individual to individual and are not effective for all patients; and drug therapy can usually only relieve symptoms and cannot reverse the degeneration of retinal cells. With the continuous development of nanotechnology, quantum dots are increasingly used in the biomedical field. In particular, chiral quantum dots, due to their unique physicochemical properties and biocompatibility, have shown great potential in neuroprotection. However, their biocompatibility and toxicity issues have always been the focus of research. Ordinary quantum dots may have potential toxicity, and the preparation methods of chiral quantum dots in the existing technology are complex and costly, and their application effect in neuroprotection still needs to be improved. Summary of the invention

[0003] Based on the above problems, the present invention proposes a method for preparing chiral cadmium selenide quantum dots and its application in retinal degeneration.

[0004] The first aspect of the present invention provides a method for preparing chiral cadmium selenide quantum dots, the method comprising the following key steps:

[0005] Preparation of sodium selenate solution: dissolve selenium powder and sodium hydroxide in deionized water, heat to boiling and keep 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: Cadmium chloride is dissolved in deionized water, and then a chiral ligand D-cysteine ​​(Cys) is added, and the pH value of the solution is adjusted to a suitable range to form a Cd-D-Cys complex solution.

[0007] Formation of CdSe quantum dots: Sodium selenate solution is slowly added to the Cd-D-Cys complex solution, and vigorously stirred during the addition. After the addition is completed, the mixed solution is microwave-heated to a suitable temperature and maintained for a period of time to promote the formation of CdSe quantum dots.

[0008] Purification of quantum dots: The reaction solution is centrifuged using a centrifuge to separate the quantum dot precipitate. Subsequently, the precipitate is redispersed in deionized water and the centrifugation-dispersion step is 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. In addition, parameters such as the time of heating to boiling and holding, the heating temperature and time of the mixed solution, and the speed and time of the centrifuge are also optimized to obtain the best quantum dot preparation effect.

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

[0011] Compared with the prior art, the chiral cadmium selenide quantum dots of the present invention have good biocompatibility, providing new possibilities for expanding the application of quantum dots in the biomedical field. The chiral cadmium selenide quantum dots of the present invention have a significant protective effect on retinal damage caused by high intraocular pressure or oxidative stress, can effectively increase the survival rate of retinal ganglion cells, reduce retinal pathological changes, and thus improve the patient's vision. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0013] Figure 2 The chiral CdSe quantum dot pair consists of H 2 O 2 Induced oxidative stress damage results.

[0014] Figure 3 This is the result of the scavenging ability of chiral cadmium selenide quantum dots on the free radical DPPH.

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

[0016] Figure 5 Pathological changes of cardiac cells in rats with intraocular hypertension treated with chiral cadmium selenide quantum dots. Scale bar: 10μm. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand and implement the present invention, but 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, heated to boiling and maintained for 1 hour until the selenium powder was completely dissolved to obtain sodium selenate (Na 2 SeO 3 ) solution. 0.1366 g (0.64 mmol) of cadmium chloride (CdCl 2 ) was dissolved in 50 mL of deionized water, 0.124 g (1.28 mmol) of chiral ligand D-Cys was added, and the pH value 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 completed, microwave heating was performed to 120°C for 20 min to promote the formation of chiral cadmium selenide quantum dots. After the reaction was completed, centrifugation was performed at 8000 rpm for 10 minutes using a centrifuge, and the supernatant was discarded to obtain a 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 is different from that in Example 1 in that no chiral ligand D-Cys needs to be added, and the remaining preparation steps are the same as those in Example 1.

[0022] Example 3

[0023] The cell model used in the present invention is an in vitro pressurized culture model of retinal ganglion cells (RGCs) apoptosis, that is, purified rat primary RGCs are cultured in an incubator with the setting parameters of 37°C and 5% CO 2 After the cells were completely attached to the wall, they were cultured in a pressure incubator and randomly divided into 5 groups: blank group, model group, 5μM treatment group, 10μM treatment group and 20μM treatment group. The pressure values ​​of the four groups except the blank group were set to 60 mmHg, 37°C, 5% CO 2 After culturing for 24 h, the treatment group was added with a culture medium containing chiral cadmium selenide quantum dots at a final concentration of the corresponding concentration, and the blank group was placed in a carbon dioxide incubator; the model group and the treatment group were placed in a carbon dioxide pressure incubator, and the pressure was set to 60 mmHg; the other parameters of all groups were maintained at 37°C and 5% CO 2 , CCK8 detection was performed after 24h.

[0024] Depend on Figure 1It can be seen that compared with the blank, the survival rate of RGCs in the model group was significantly reduced, indicating that the in vitro model of RGCs apoptosis was successfully established; compared with the model group, the survival rate of RGCs in the chiral cadmium selenide quantum dot treatment groups with different concentrations was significantly increased, confirming the inhibitory effect of chiral cadmium selenide quantum dots on RGCs apoptosis in vitro, and also preliminarily revealing its feasibility as a potential neuroprotective agent. Among them, the survival rate of the 10μM treatment group was the best, and this concentration was selected in the subsequent examples of the present invention.

[0025] Example 4

[0026] Following the same cell culture process as in Example 3, RGCs were first cultured in vitro under pressure. After the cells were completely attached, the purified rat primary 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 carbon dioxide incubator, while the model group and the two treatment groups were cultured in a carbon dioxide pressure incubator set at a pressure of 60 mm Hg. All groups were maintained at 37°C and 5% CO 2 After the cell culture was stable, the oxidative stress inducer H was added to the retinal cell cultures of the model group and the treatment group. 2 O 2 , to simulate the oxidative stress environment in vivo. Subsequently, a solution containing chiral CdSe quantum dots was added to the culture system of the CdSe quantum dot treatment group, and a solution containing CdSe quantum dots was added to the culture system of the CdSe quantum dot treatment group, while only an equal amount of solvent was added to the model group as a control to detect the scavenging ability of the chiral CdSe quantum dots on the free radical DPPH.

[0027] like Figure 2 As shown in Figure 2, compared with the blank control group, the model group showed a significant increase in the number of oxidative stress inducers after addition of H 2 O 2 After H 2 O 2 The results successfully induced cellular oxidative stress and caused damage. Although the CdSe quantum dot treatment group also showed a certain protective effect under the same conditions, the improvement was very limited. Compared with the model group, the survival rate of RGCs in the chiral CdSe quantum dot treatment group was significantly improved. Chiral CdSe quantum dots may interact with specific biological molecules in cells in a more matching and efficient way, thereby more effectively alleviating cell damage caused by oxidative stress and promoting cell survival and recovery. This indicates that chiral CdSe quantum dots have a protective effect on H 2 O 2 It has a certain protective effect against oxidative stress damage induced by

[0028] like Figure 3As shown, the present invention further detected the ability of quantum dots to scavenge the free radical DPPH. The experimental results showed that compared with the control group without chiral cadmium selenide quantum dots, the solution containing chiral cadmium selenide quantum dots showed a significant scavenging effect on DPPH free radicals, and was much higher than the cadmium selenide quantum dot treatment group, which shows that chiral cadmium selenide quantum dots have strong antioxidant activity and can effectively scavenge free radicals, thereby reducing the damage of oxidative stress to cells.

[0029] In summary, chiral CdSe quantum dots have a significant effect on the H 2 O 2 It has a significant protective effect against induced oxidative stress damage and has a strong free radical scavenging ability.

[0030] Example 5

[0031] Fifteen SPF SD rats were randomly divided into three groups: sham operation group, high intraocular pressure model group and treatment group. Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (dose of 45 mg / kg), and the left eye was selected as the surgical eye. Before surgery, 0.5% proparacaine eye drops were instilled into the left eye for surface anesthesia. Then, the bulbar conjunctiva was cut from 6:00 to 14:00 in a clockwise direction 1 mm behind the corneal limbus, and the fascia and muscle under the conjunctiva were carefully separated. Subsequently, three "Y"-shaped suprascleral veins were found on both sides of the superior rectus muscle and near the bottom of the lateral rectus muscle. The common branches of the veins were freed and lifted, clamped with clips, and the main trunk of the vein on the far side of the cornea and sclera was cauterized with a heated pin to close the vein. After successful cauterization, the bulbar conjunctiva was reset and sutured. In the sham operation group, the bulbar conjunctiva of the left eye was cut at the same position, but the suprascleral vein was not cauterized. After the operation, levofloxacin eye ointment was applied to the conjunctival sac of the left eye, and the rats were placed on a warming blanket until they woke up naturally, and then returned to the cage. Within 5 days after the operation, levofloxacin eye ointment was applied to the conjunctival sac once a day. Rats in the treatment group received local eye drops after surgery, with a volume of 0.2mL / kg (containing 2mg / mL chiral cadmium selenide quantum dots), once a day for two weeks. Rats in the sham operation group and the high intraocular pressure model group were given the same volume of distilled water eye drops, once a day, for two weeks. After the experimental period, the eyeball tissue and heart tissue of the rats were taken for HE staining observation.

[0032] The results of HE staining of eyeball tissue sections of rats in each group are as follows Figure 4As shown in the figure, the retina of rats in the sham operation group had a clear structure, distinct layers, neatly arranged cells in each layer, and normal morphology. The retinal nerve fiber layer (NFL) was continuous and complete, the number of cells in the retinal ganglion cell (RGCs) layer was sufficient, the morphology was full, and the nuclei were clearly visible, indicating that the sham operation did not cause obvious 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 layer of the retina were disordered, and there were varying degrees of cell loss and morphological abnormalities. In particular, the RGCs layer had a significant decrease in the number of cells, atrophy of the cell morphology, and condensation or fragmentation of the cell nuclei, indicating that the high intraocular pressure model was successfully established. Compared with the high intraocular pressure model group, the number of cells in the RGCs layer of the retina of rats in the treatment group was more, and the cell morphology was relatively complete, indicating that the chiral cadmium selenide quantum dots administered topically by eyedrop had a certain protective effect on retinal damage induced by high intraocular pressure. At the same time, the degree of edema, vascular morphology, and inflammatory cell infiltration of the retina in the treatment group were also improved, further verifying the therapeutic effect of chiral cadmium selenide quantum dots.

[0033] HE staining results of cardiac tissue sections of rats in each group are shown in Figure 5 As shown, the cardiomyocytes in all groups were stained evenly, arranged regularly, without edema and necrosis, and without inflammatory cell infiltration, indicating that chiral cadmium selenide quantum dots did not produce obvious toxic effects on myocardial tissue during the treatment of rats with high intraocular pressure, indicating preliminary confirmation of the biosafety of this nanomaterial.

[0034] In summary, through HE staining observation, we can clearly see the pathological changes in the retina of rats in the sham operation group, high intraocular pressure model group and treatment group. Chiral cadmium selenide quantum dots have a significant protective effect and therapeutic effect on retinal damage induced by high intraocular pressure, and no obvious toxic effect on myocardial tissue was produced during the treatment process. These results provide a strong experimental basis 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 of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work 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 following steps are involved: S1: dissolving selenium powder and sodium hydroxide in deionized water, heating to boiling and maintaining for 1 hour until the selenium powder is completely dissolved to obtain a sodium selenate solution; S2: dissolving cadmium chloride in deionized water, adding chiral ligand D-Cys, and adjusting the pH value to a suitable range to obtain a Cd-D-Cys complex solution; S3 slowly add the sodium selenate solution dropwise to the Cd-D-Cys complex solution while stirring vigorously; After the addition of S4 is completed, the mixed solution is heated to a suitable temperature and maintained for a period of time to promote the formation of cadmium selenide quantum dots; S5 uses a centrifuge to centrifuge the solution after the reaction, discards the supernatant, and obtains quantum dot precipitation; S6: Re-dispersing the quantum dot precipitate in deionized water, repeating the centrifugation-dispersion steps, and obtaining a purified chiral cadmium selenide quantum dot solution.

2. The preparation method according to claim 1, characterized in that: The molar ratio of the selenium powder, sodium hydroxide, cadmium chloride and D-Cys is 0.32:0.5:0.64:1.

28.

3. The preparation method according to claim 1, characterized in that: The pH value of S2 is 8.

6.

4. The preparation method according to claim 1, characterized in that: S4 The mixed solution was heated to 120°C by microwave and maintained for 20 minutes.

5. The preparation method according to claim 1, characterized in that: The rotation speed of the centrifuge in S5 is 8000 rpm, and the centrifugation time is 10 minutes.

6. Use of the chiral cadmium selenide quantum dots according to any one of claims 1 to 5 in the preparation of drugs for treating retinal degeneration.

7. The use according to claim 6, characterized in that: The drug can act in vitro, and by co-incubating with retinal ganglion cells RGCs cultured in vitro under pressure at a drug concentration of 5 to 20 μM, the survival rate of the RGCs can be increased.

8. The use according to claim 6, characterized in that: The drug can act in vivo to treat retinal damage caused by high intraocular pressure or oxidative stress.

9. The use according to claim 6, characterized in that: The drug can act on the body and be administered by local eye drops.

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