Application of deep eutectic solvent in preparation of medicine for promoting adeno-associated virus anterior chamber transfection

Abstract

The invention provides application of a deep eutectic solvent in preparation of a medicine for promoting adeno-associated virus anterior chamber transfection, and belongs to the technical field of medicine preparation. The invention relates to an application of a deep eutectic solvent in preparation of drugs for promoting adeno-associated virus or genetically modified adeno-associated virus transfection. The deep eutectic solvent is prepared from choline chloride and fructose in a molar ratio of 2: 1. The deep eutectic solvent (DES) has moderate viscosity and approximately clear and transparent appearance, can stay in an anterior chamber for a certain time without blocking chamber corners, has the advantages of low toxicity, biodegradability, simplicity and convenience in synthesis and the like, and can be used as a delivery carrier of an adeno-associated virus (AAV). The DES is injected into the animal model in the anterior chamber, the DES cannot cause changes of corneal transparency, corneal thickness, corneal barrier function, pump function and corneal endothelial cell morphology, the safety is good, the anterior chamber AAV transfection efficiency can be improved, and outflow of AAV along with aqueous humor circulation and immunogenicity are reduced.

Description

Technical Field

[0001] This application belongs to the field of drug preparation technology, specifically relating to the application of deep eutectic solvents (DES) in the preparation of drugs that promote the anterior chamber transfection of adeno-associated virus. Background Technology

[0002] Currently, adeno-associated virus (AAV) gene therapy is a gene therapy technology that uses AAV as a vector to introduce normal or therapeutic genes into the body to repair, replace, or regulate abnormal genes, thereby achieving the treatment of diseases. Due to the location of the corneal endothelium, we can only achieve gene overexpression in corneal endothelial cells through anterior chamber injection of AAV. However, unlike injection methods that can remain in solid organs for a long time, some of the AAV injected into the anterior chamber is lost through the needle tract, and it is also rapidly metabolized and cleared by the aqueous humor circulation, resulting in a limited local retention time. Therefore, the desired transfection efficiency can only be achieved by increasing the injection dose; previous literature reported an anterior chamber injection viral load of 1 × 10⁻⁶. 9 vg~5×10 9 Therefore, although the low immunogenicity of AAV can be utilized, in order to avoid excessive viral entry into the systemic circulation, it is still necessary to minimize the viral load while ensuring therapeutic efficacy, so as to achieve the best therapeutic effect with the lowest dose.

[0003] Studies show that hydrogels, when applied as AAV carriers to wound surfaces, can effectively improve the AAV transfection efficiency of local tissues, while also enhancing the carrier's targeting and reprogramming efficiency, and significantly promoting endothelial cell proliferation, migration, and angiogenesis. However, the degradation process and physical properties of hydrogels in the anterior chamber environment may obstruct the angle, not only failing to treat corneal endothelial lesions but also potentially inducing complications such as glaucoma. Furthermore, the literature has also reported the possibility of using metal nanoparticles as AAV carriers; however, these materials have potential toxicity, and their safety requires systematic verification. Moreover, even after encapsulating AAV, their particle size remains small, making them easily cleared by aqueous humor circulation, which is insufficient to meet the needs of intra-anterior chamber delivery. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a deep eutectic solvent in the preparation of drugs that promote the transfection of adeno-associated virus in the anterior chamber.

[0005] This invention provides the application of a deep eutectic solvent in the preparation of drugs that promote transfection of adeno-associated virus or genetically modified adeno-associated virus, wherein the deep eutectic solvent is made of choline chloride and fructose in a molar ratio of 2:1.

[0006] Preferably, the adeno-associated virus transfection includes adeno-associated virus anterior chamber transfection.

[0007] Preferably, the drug comprises an injectable solution.

[0008] Preferably, the genetically modified adeno-associated virus contains genes for the treatment of eye diseases.

[0009] Preferably, the therapeutic gene includes Foxo1 Gene.

[0010] Preferably, the method for preparing the deep eutectic solvent involves mixing choline chloride and fructose, stirring at 80°C for 60 minutes, and then filtering through a filter membrane after cooling.

[0011] This invention provides a viral preparation using a deep eutectic solvent as a solvent, and further includes adeno-associated virus or genetically modified adeno-associated virus; The deep eutectic solvent is made of choline chloride and fructose in a molar ratio of 2:1.

[0012] Preferably, the concentration of the adeno-associated virus or the genetically modified adeno-associated virus is 2.5 × 10⁻⁶. 11 Above vg / mL.

[0013] The present invention provides an ocular therapeutic drug comprising the aforementioned viral preparation.

[0014] Preferably, the dosage form of the ocular therapeutic agent includes an injection.

[0015] This invention provides the application of a deep eutectic solvent in the preparation of drugs that promote the transfection of adeno-associated virus (AAV) or genetically modified AAV. The deep eutectic solvent is composed of choline chloride and fructose in a molar ratio of 2:1. The deep eutectic solvent has moderate viscosity, a near-clear and transparent appearance, and can remain in the anterior chamber for a certain period without clogging the angle. The DES also possesses advantages such as low toxicity, biodegradability, and ease of synthesis, and has shown application potential in multiple fields including extraction and separation, inorganic and organic synthesis, ionogels, drug sustained release, gas absorption, and nanomaterial preparation. Therefore, it was selected as the delivery carrier for AAV in this study. To evaluate its in vivo safety, DES was injected into the anterior chamber of an animal model. The results showed that anterior chamber injection of DES did not cause changes in corneal transparency, corneal thickness, corneal barrier function and pump function, or corneal endothelial cell morphology; at the same time, it could improve the transfection efficiency of AAV in the anterior chamber, reduce the outflow of AAV with aqueous humor circulation, and reduce immunogenicity. Therefore, the application provided by this invention offers a new strategy for the treatment of corneal injury-related lesions. Attached Figure Description

[0016] Figure 1The results show the characterization of deep eutectic solvent (DES), where A represents the raw materials for DES synthesis; B represents the appearance of the DES sample; and C represents the viscosity measurement results of DES. Figure 2 The safety assessment results for DES are as follows: A) Slit-lamp radiography showing corneal transparency in mice in the PBS and DES groups (n=6); B) OCT detection of central corneal thickness in mice (n=6); ns, with no statistically significant difference; C) Immunofluorescence detection of Na in CEnCs on mouse corneal slices. + -K + The expression level of the pump (n=4); D is the expression level of ZO-1 in CEnCs detected by immunofluorescence on mouse corneal slices (n=4). Figure 3 The effect of DES on AAV transfection is shown in Figure A, where A represents the effect of DES on AAV transfection efficiency in corneal endothelial cells on mouse corneal flaps (n = 6); and B represents the results of statistical analysis of AAV transfection efficiency. P <0.01); C represents the morphology of the DES-AAV sample; D represents the structural characterization results of the AAV virus particles encapsulated in DES. Figure 4 For AAV- Foxo1 With DES-AAV- Foxo1 The effect of the delivery system on improving UVA corneal endothelial damage, where A is the change in corneal transparency before and after UVA irradiation in mice as shown by slit-lamp photography (n=6); B is the change in corneal thickness before and after UVA irradiation in mice as shown by OCT images (n=6); C is the central corneal thickness measured by OCT at different time points before and after UVA irradiation. p <0.05; D represents the Na content in CEnCs detected by mouse corneal flap assay. + -K + The pump's level of expression. Detailed Implementation

[0017] This invention provides the application of a deep eutectic solvent in the preparation of drugs that promote transfection of adeno-associated virus or genetically modified adeno-associated virus, wherein the deep eutectic solvent is made of choline chloride and fructose in a molar ratio of 2:1.

[0018] In this invention, the adeno-associated virus (AAV) transfection preferably includes anterior chamber transfection with AAV. The anterior chamber transfection is preferably performed by injecting AAV into the anterior chamber. The AAV preferably includes AAV6 or AAV2 AAV. The drug preferably comprises an injection solution. The drug preferably comprises a deep eutectic solvent. In an embodiment of this invention, the concentration of the AAV or genetically modified AAV is 2.5 × 10⁻⁶. 11Above vg / mL.

[0019] In this invention, the genetically modified adeno-associated virus preferably contains a gene for treating eye diseases. This invention does not impose any special limitation on the type of gene for treating eye diseases; any gene therapy drug known in the art capable of treating eye diseases can be used. In embodiments of this invention, overexpression... Foxo1 adeno-associated virus (AAV- Foxo1 For example, to illustrate Foxo1 Gene transfection efficiency. The expression... Foxo1 The recombinant AAV6 adeno-associated virus vector was used for viral packaging to obtain the final AAV6-Foxo1 packaged with a final titer of 6.91 × 10⁻⁶. 12 The vg / ml construction work was specifically commissioned to Yuan Biotechnology (Shanghai) Co., Ltd. Foxo1 Gene-selected mice Foxo1 Gene (Gene ID: 56458). Results showed that, compared to injection of AAV- Foxo1 Compared to DES-AAV- Foxo1 It can greatly improve Foxo1 The fluorescence immunoassay intensity indicates that DES can effectively promote... Foxo1 Transfection of anterior chamber endothelial cells.

[0020] In this invention, the preferred method for preparing the deep eutectic solvent involves mixing choline chloride and fructose, stirring at 80°C for 60 minutes, cooling, and then filtering through a filter membrane. The filter membrane... The present invention provides a viral preparation using a deep eutectic solvent as a solvent, and further includes adeno-associated virus or genetically modified adeno-associated virus; the deep eutectic solvent is preferably made of choline chloride and fructose in a molar ratio of 2:1.

[0021] In this invention, the concentration of the adeno-associated virus or the genetically modified adeno-associated virus is preferably 2.5 × 10⁻⁶. 11 The concentration of DES in mice is above vg / mL. The deep eutectic solvent has moderate viscosity, a near-clear and transparent appearance, and can remain in the anterior chamber for a certain period without clogging the anterior chamber angles. DES also possesses advantages such as low toxicity, biodegradability, and ease of synthesis, and has shown application potential in multiple fields including extraction and separation, inorganic and organic synthesis, ionogels, drug sustained release, gas absorption, and nanomaterial preparation. Therefore, it can be used as a delivery carrier for AAV.

[0022] The present invention provides an ocular therapeutic drug comprising the aforementioned viral preparation.

[0023] In this invention, the dosage form of the ocular therapeutic drug preferably includes an injection. This invention does not impose any particular limitation on the preparation method of the drug; any preparation method for injection-type drugs well-known in the art can be used.

[0024] The following examples illustrate the application of the deep eutectic solvent provided by the present invention in the preparation of drugs that promote anterior chamber transfection of adeno-associated virus. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 For the preparation method of deep eutectic solvent (DES), see [link to documentation]. Figure 1 To solve problem A, the specific steps are as follows: Choline chloride and fructose were mixed in a 2:1 molar ratio in a round-bottom flask, sealed, and stirred at 80°C for 60 min. After the reaction solution cooled to room temperature, a homogeneous liquid was obtained, which was then filtered through a 0.22 μm filter membrane and stored at room temperature for later use.

[0026] The prepared DES appeared as a clear, nearly transparent, and homogeneous liquid. The viscosity of the DES was measured using a rheometer, and the results are shown below. Figure 1 The viscosity of C and DES is uniform.

[0027] Example 2 Toxicity testing of DES (1) After one week of acclimatization feeding, female C57BL / 6J mice were injected with 1 μL of PBS and DES in the anterior chamber.

[0028] (2) Observe its toxicity after two weeks, including observing the corneal transparency of mice with a slit lamp, detecting the central corneal thickness of mice with OCT, and detecting Na in mouse corneal endothelial cells (CEnCs) with immunofluorescence on mouse corneal smears. + -K + Expression levels of pump and ZO-1.

[0029] See results Figure 2 Slit-lamp radiography showed no statistically significant difference in corneal transparency between the PBS and DES groups, and OCT analysis also revealed no statistically significant difference in central corneal thickness. Immunofluorescence assays of mouse corneal slices showed that Na+ in CEnCs... + -K + The expression levels of pump and ZO-1 showed no significant changes. This indicates that DES is a safe and non-toxic AAV delivery vector.

[0030] Example 3 DES-AAV- Foxo1 Solvent preparation method AAV- Foxo1The virus was mixed with DES, bringing the final concentration of AAV-Foxo1 virus to 2.5 × 10⁻⁶. 11 vg / mL, appearance morphology see Figure 3 C. Using transmission electron microscopy to examine DES-AAV- Foxo1 Structural characterization was performed using solvents. Results are shown below. Figure 3 The results showed that the viral capsid was intact and structurally complete.

[0031] Example 4 The Fuchs corneal endothelial dystrophy (FECD) model was constructed and its function was validated. (1) After one week of acclimatization feeding, female C57BL / 6J mice were injected with AAV- into the anterior chamber. Foxo1 and DES-AAV- Foxo1 AAV- Foxo1 The total amount is 2.5 × 10 8 VG was collected 2 weeks later to observe its transfection efficiency, and the AAV transfection status of corneal endothelial cells was evaluated by corneal flaps of mice.

[0032] (2) Mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (50 mg / kg) and topical anesthesia was performed by 0.5% promecaine hydrochloride eye drops. (3) Irradiate the cornea with UVA for 20 min 57 s, with an irradiation flux of 500 J / cm. 2 ; (4) Successful modeling was indicated by corneal swelling in mice. Anterior segment photography and corneal OCT were performed on days 1, 3, 5, and 7. Samples were collected on day 7 for subsequent experiments. Slit-lamp photography was used to determine changes in corneal transparency before and after UVA irradiation. Simultaneously, OCT was used to detect changes in corneal thickness and central corneal thickness before and after UVA irradiation. Furthermore, immunofluorescence assays were used to detect Na+ in CEnCs using mouse corneal flaps. + -K + The pump's level of expression.

[0033] Transfection efficiency results are shown in Figure 3 The results showed that, compared with simple injection of AAV- into the anterior chamber... Foxo1 Compared to mice injected with DES-AAV- Foxo1 The efficiency of corneal endothelial transfection in mice was increased by nearly 3 times.

[0034] The results of corneal morphology are shown in Figure 4 AAV- Foxo1 With DES-AAV- Foxo1A comparison of the effects of the delivery system on improving UVA corneal endothelial damage showed that slit-lamp radiography significantly improved corneal transparency in mice before and after UVA irradiation. Simultaneously, OCT images revealed a significant difference in corneal thickness changes before and after UVA irradiation, specifically a significant decrease in central corneal thickness on days 1 and 3. Furthermore, immunofluorescence assays of Na in CEnCs were performed on mouse corneal slices. + -K + The expression level of the pump was significantly increased, and the cell density was significantly elevated, indicating that DES, as a delivery system, facilitated the expression of AAV- Foxo1 towards Highly efficient intracorneal delivery effectively improves corneal function.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a deep eutectic solvent in the preparation of drugs that promote transfection of adeno-associated virus or genetically modified adeno-associated virus, wherein the deep eutectic solvent is made of choline chloride and fructose in a molar ratio of 2:

1.

2. The application according to claim 1, characterized in that, The adeno-associated virus transfection includes adeno-associated virus anterior chamber transfection.

3. The application according to claim 1, characterized in that, The drug includes an injectable solution.

4. The application according to claim 1, characterized in that, The genetically modified adeno-associated virus contains genes for the treatment of eye diseases.

5. The application according to claim 4, characterized in that, The therapeutic gene includes Foxo1 Gene.

6. The application according to any one of claims 1 to 5, characterized in that, The method for preparing the deep eutectic solvent involves mixing choline chloride and fructose, stirring at 80°C for 60 minutes, and then filtering through a filter membrane after cooling.

7. A viral preparation, characterized in that, Using deep eutectic solvents as solvents, it also includes adeno-associated virus or genetically modified adeno-associated virus; The deep eutectic solvent is made of choline chloride and fructose in a molar ratio of 2:

1.

8. The viral preparation according to claim 7, characterized in that, The concentration of the adeno-associated virus or genetically modified adeno-associated virus is 2.5 × 10⁻⁶. 11 Above vg / mL.

9. An ophthalmic therapeutic drug, characterized in that, Includes the viral preparation described in claim 7 or 8.

10. The ocular treatment drug according to claim 9, characterized in that, The dosage form of the ophthalmic treatment includes an injection.

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