Delivery preparation containing isorhamnetin and CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 plasmid as well as preparation method and application of delivery preparation

Abstract

The invention relates to the technical field of medicines, in particular to a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmids as well as a preparation method and application of the delivery preparation. According to the isorhamnetin delivery preparation, the polylactic acid-glycolic acid copolymer is used as an inner shell material of the delivery preparation, a hydrophobic drug isorhamnetin is wrapped, a fat-soluble drug isorhamnetin is prepared into a hydrophilic preparation, and the problem that isorhamnetin is difficult to prepare into a medicine is solved; polyethyleneimine is used as a middle layer material of the delivery preparation, CRISPR Cas9 plasmid is loaded, efficient gene loading is achieved, ACSL4 gene is specifically knocked out, and ferroptosis is inhibited; 2, 3-dimethyl maleic anhydride functionalized poly-L-lysine is used as a middle layer material of the delivery preparation, and the charge reversal characteristic is introduced; by using the hypoxic retinal precursor cell membrane as the shell material of the delivery preparation, the delivery preparation has good biocompatibility and retina targeting effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid, and a preparation method and application thereof. Background Art

[0002] Retinal ischemia-reperfusion (RIR) injury is a common pathophysiological process in ophthalmology, implicated in the pathogenesis of numerous eye diseases, including acute angle-closure glaucoma, retinal vascular occlusion, and diabetic retinopathy. According to the World Health Organization, over 10 million people worldwide suffer from ischemic retinal diseases. RIR injury, a pathological phenomenon in which retinal damage worsens rather than alleviates after blood supply is restored to ischemic retinal tissue, is a common cause of many retinal diseases, including acute glaucoma, retinopathy of prematurity, age-related macular degeneration, and diabetic retinopathy. It leads to the death of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve, resulting in vision loss and permanent blindness.

[0003] The process of RIR injury leads to the death, morphological degeneration, functional loss of retinal ganglion cells (RGCs), and ultimately leads to vision loss. The mechanism of RIR injury is related to apoptosis factors, oxygen free radical damage, excitatory amino acids, calcium overload and inflammatory cytokines.

[0004] Isorhamnetin, a plant flavonoid, is one of the most important active ingredients in seabuckthorn fruit and ginkgo biloba leaves. In ischemic studies, isorhamnetin protects against myocardial injury induced by ischemic stroke by attenuating apoptosis and oxidative stress. Treatment of experimental stroke mice with isorhamnetin reduces infarct volume and caspase-3 activity (a biomarker of apoptosis) and improves neurological recovery.

[0005] Acyl-CoA synthase long-chain family member 4 (ACSL4) is a key enzyme for the esterification of fatty acids with a carbon chain length of 12-20. Studies have shown that ACSL4 may play an important role in ferroptosis. Other studies have shown that inhibiting ACSL4 can effectively prevent the progression of fibrosis and provide protection against the inflammatory environment. There is a positive regulatory relationship between lipopolysaccharide (LPS)-induced macrophage activity and ferroptosis sensitivity. Jia B et al. used specific shRNA to knock down ACSL4 and inhibited erastin-induced ferroptosis in HepG2 and HL60 cells (ferroptosis-sensitive cells). Therefore, it is necessary to provide a delivery formulation to treat retinal ischemia-reperfusion (RIR) injury. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid, as well as a preparation method and application thereof. The delivery preparation provided by the present invention has good biocompatibility and retinal targeting effect by using hypoxic retinal progenitor cell membrane as the shell material of the delivery preparation.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a delivery formulation containing isorhamnetin and a CRISPR Cas9 plasmid, comprising the following steps:

[0009] 1) mixing polyethyleneimine with acetone to obtain a polyethyleneimine solution;

[0010] mixing isorhamnetin with ethanol and ethyl acetate to obtain an isorhamnetin solution;

[0011] mixing polylactic acid-glycolic acid copolymer with acetone and dichloromethane to obtain a copolymer solution;

[0012] mixing the polyethyleneimine solution, the isorhamnetin solution and the copolymer solution to obtain an oil phase;

[0013] 2) mixing poloxamer 188 with DEPC water to obtain an aqueous phase;

[0014] The mass ratio of poloxamer 188 to isorhamnetin is 50:1;

[0015] 3) ultrasonically mixing the oil phase obtained in step 1) with the water phase obtained in step 2) to obtain a mixed solution;

[0016] 4) subjecting the mixed solution obtained in step 3) to rotary evaporation, ultrasonic treatment, and centrifugation in sequence to obtain a supernatant that is the encapsulated isorhamnetin delivery preparation;

[0017] 5) mixing the encapsulated isorhamnetin delivery formulation obtained in step 4) with the CRISPR Cas9 plasmid and incubating the mixture to obtain an incubation product;

[0018] 6) mixing the incubation product obtained in step 5) with 2,3-dimethylmaleic anhydride functionalized poly-L-lysine, and incubating again to obtain a secondary incubation product;

[0019] 7) ultrasonically treating the re-incubated product obtained in step 6) and the hypoxia-pretreated retinal progenitor cell membrane with an ice-bath probe to obtain an ultrasonicated product;

[0020] The sonicated product was sequentially extruded through polycarbonate porous membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm, and the obtained solution was then sonicated to obtain a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid.

[0021] Preferably, in step 1), the volume ratio of the polyethyleneimine to acetone is 3 mg:0.25 mL;

[0022] The mass ratio of the isorhamnetin to the volume of ethanol and the volume ratio of ethyl acetate is 3 mg:0.9 mL:0.6 mL;

[0023] The ratio of the mass of the polylactic acid-glycolic acid copolymer to the volume of acetone and the volume of dichloromethane is 30 mg:0.5625 mL:0.1875 mL.

[0024] Preferably, the mass ratio of the poloxamer 188 to the volume ratio of DEPC water is 150 mg:3 mL;

[0025] The DEPC water is ultrapure water treated with diethyl pyrocarbonate and then sterilized at high temperature and high pressure.

[0026] Preferably, the ultrasonic mixing conditions in step 1) include: ultrasonication at a power of 195 W for 30 minutes.

[0027] Preferably, the conditions of the rotary evaporation hydration in step 4) include: rotary evaporation at a temperature of 37° C. and a rotation speed of 70 rpm for 5 minutes;

[0028] The ultrasonic treatment conditions include: ultrasonic treatment at a power of 195W for 5 minutes, with a pause of 5 seconds after every 5 seconds of ultrasonic treatment;

[0029] The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 10 min.

[0030] Preferably, in step 5), the mass ratio of CRISPR Cas9 plasmid to isorhamnetin is 0.375:3;

[0031] The incubation temperature is 20-30° C. and the incubation time is 30 minutes.

[0032] Preferably, in step 6), the mass ratio of 2,3-dimethylmaleic anhydride functionalized poly-L-lysine to isorhamnetin is 1.64:3;

[0033] The temperature of the second incubation is 20-30° C., and the time is 30 minutes.

[0034] Preferably, the mass ratio of the retinal progenitor cell membrane pretreated with hypoxia in step 7) to the poly(lactic-co-glycolic acid) copolymer is 1:1;

[0035] The conditions for the ice bath probe ultrasonic treatment include: ultrasonication at a power of 195W for 30 minutes;

[0036] The ultrasonic treatment conditions include: ultrasonic treatment at a power of 195 W for 5 minutes, with a pause of 5 seconds after every 5 seconds of ultrasonic treatment.

[0037] The present invention also provides a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid prepared by the preparation method described in the above technical solution.

[0038] The present invention also provides the use of the delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury.

[0039] The beneficial effects of the present invention are:

[0040] (1) Using polylactic acid-co-glycolic acid copolymer as the inner shell material of the delivery formulation to encapsulate the hydrophobic drug isorhamnetin, the fat-soluble drug isorhamnetin was made into a hydrophilic formulation, solving the problem of isorhamnetin's difficulty in drug development;

[0041] (2) Polyethyleneimine is used as the middle layer material of the delivery preparation to load the CRISPR Cas9 plasmid. Polyethyleneimine has a strong DNA condensation ability, which can achieve efficient gene loading and has a certain proton sponge effect;

[0042] (3) 2,3-dimethylmaleic anhydride functionalized poly-L-lysine was used as the middle layer material of the delivery formulation to introduce charge reversal properties;

[0043] (4) Using hypoxic R28 cell membrane as the shell material of the delivery formulation has good biocompatibility and retinal targeting effect;

[0044] (5) The biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention can be used for inflammation caused by RIR damage, providing a new method for the treatment of retinal inflammation, and can also be used for ferroptosis and excessive reactive oxygen species caused by RIR damage, providing a new method for the treatment of RIR diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0046] Figure 1 This is a transmission electron micrograph of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared in the present invention;

[0047] Figure 2This is the particle size distribution diagram of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention;

[0048] Figure 3 This is the Zeta potential distribution diagram of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention;

[0049] Figure 4 The figure shows the detection results of the uptake of Cy5.5 delivery formulation by R28 cells;

[0050] Figure 5 The figure shows the test results of the secretion of inflammatory factor NO in each group of cells;

[0051] Figure 6 The graph shows the results of measuring the intracellular ATP content in each group;

[0052] Figure 7 Figure 2 is the fluorescence detection result of intracellular reactive oxygen species in each group;

[0053] Figure 8 The statistical results of intracellular reactive oxygen species fluorescence intensity in each group are shown;

[0054] Figure 9 Figure 2 shows the HE staining results of the retina of SD rats in each group;

[0055] Figure 10 The graph shows the immunofluorescence detection results of retinal neural tubulin β-III-Tubulin in each group of SD rats;

[0056] Figure 11 The statistical results of immunofluorescence intensity of retinal neural tubulin β-III-Tubulin in each group of SD rats are shown in FIG.

[0057] Figure 12 The graph shows the immunofluorescence detection results of glial cell-specific protein IBA1 in SD rats of each group;

[0058] Figure 13 The statistical results of immunofluorescence intensity of glial cell-specific protein IBA1 in each group of SD rats are shown in the figure;

[0059] Figure 14 The graph shows the immunofluorescence detection results of glial cell-specific protein GFAP in each group of SD rats;

[0060] Figure 15 The statistical results of immunofluorescence intensity of glial cell-specific protein GFAP in each group of SD rats are shown in the figure;

[0061] Figure 16The retinal fluorescence detection results of SD rats without modeling and SD rats with modeling on the 7th day;

[0062] Figure 17 The graph shows the retinal fluorescence detection results of SD rats without modeling and SD rats with modeling on the 28th day. DETAILED DESCRIPTION

[0063] The present invention provides a method for preparing a delivery formulation containing isorhamnetin and a CRISPR Cas9 plasmid, comprising the following steps:

[0064] 1) mixing polyethyleneimine with acetone to obtain a polyethyleneimine solution;

[0065] mixing isorhamnetin with ethanol and ethyl acetate to obtain an isorhamnetin solution;

[0066] mixing polylactic acid-glycolic acid copolymer with acetone and dichloromethane to obtain a copolymer solution;

[0067] mixing the polyethyleneimine solution, the isorhamnetin solution and the copolymer solution to obtain an oil phase;

[0068] 2) mixing poloxamer 188 with DEPC water to obtain an aqueous phase;

[0069] The mass ratio of poloxamer 188 to isorhamnetin is 50:1;

[0070] 3) ultrasonically mixing the oil phase obtained in step 1) with the water phase obtained in step 2) to obtain a mixed solution;

[0071] 4) subjecting the mixed solution obtained in step 3) to rotary evaporation, ultrasonic treatment, and centrifugation in sequence to obtain a supernatant that is the encapsulated isorhamnetin delivery preparation;

[0072] 5) mixing the encapsulated isorhamnetin delivery formulation obtained in step 4) with the CRISPR Cas9 plasmid and incubating the mixture to obtain an incubation product;

[0073] 6) mixing the incubation product obtained in step 5) with 2,3-dimethylmaleic anhydride functionalized poly-L-lysine, and incubating again to obtain a secondary incubation product;

[0074] 7) ultrasonically treating the re-incubated product obtained in step 6) and the hypoxia-pretreated retinal progenitor cell membrane with an ice-bath probe to obtain an ultrasonicated product;

[0075] The sonicated product was sequentially extruded through polycarbonate porous membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm, and the resulting solution was further sonicated to obtain a delivery formulation containing isorhamnetin and CRISPR Cas9 plasmid. In a specific embodiment of the present invention, the CRISPR Cas9 plasmid was purchased from Suzhou Genetron Health Co., Ltd.

[0076] The present invention mixes polyethyleneimine with acetone to obtain a polyethyleneimine solution; mixes isorhamnetin with ethanol and ethyl acetate to obtain an isorhamnetin solution; mixes poly(lactic acid-glycolic acid) copolymer with acetone and dichloromethane to obtain a copolymer solution; and mixes the polyethyleneimine solution, isorhamnetin solution, and copolymer solution to obtain an oil phase. In the present invention, the weight ratio of the polyethyleneimine to the volume of acetone is preferably 3 mg:0.25 mL. In the present invention, the weight ratio of the isorhamnetin to the volume of ethanol and ethyl acetate is preferably 3 mg:0.9 mL:0.6 mL. In the present invention, the weight ratio of the poly(lactic acid-glycolic acid) copolymer to the volume of acetone and dichloromethane is preferably 30 mg:0.5625 mL:0.1875 mL. In a specific embodiment of the present invention, the poly(lactic acid-glycolic acid) copolymer is preferably purchased from Shanghai Macklin Biochemical Co., Ltd., with a lactide:glycolide ratio of 50:50 and a molecular weight of 38,000-54,000.

[0077] The present invention mixes poloxamer 188 with DEPC water to obtain an aqueous phase; the mass ratio of poloxamer 188 to isorhamnetin is 50:1. In the present invention, the volume ratio of the mass of poloxamer 188 to DEPC water is 150 mg:3 mL. In the present invention, the DEPC water is preferably ultrapure water treated with diethyl pyrocarbonate and then sterilized at high temperature and high pressure. The present invention does not particularly limit the preparation of the DEPC water, and those skilled in the art can prepare it using conventional methods.

[0078] The present invention ultrasonically mixes the obtained oil phase with the aqueous phase to obtain a mixed solution. In the present invention, the ultrasonic mixing conditions preferably include: ultrasonication at a power of 195W for 30 minutes.

[0079] The present invention sequentially subjectes the obtained mixed solution to rotary evaporation, ultrasonic treatment, and centrifugation, and the resulting supernatant is the encapsulated isorhamnetin delivery formulation. In the present invention, the rotary evaporation conditions preferably include: rotary evaporation at a temperature of 37°C and a rotation speed of 70 rpm for 5 minutes. In the present invention, the ultrasonic treatment conditions preferably include: ultrasonication at a power of 195W for 5 minutes, with a 5-second pause every 5 seconds of ultrasonication. In the present invention, the centrifugation conditions preferably include: a rotation speed of 5000 rpm for 10 minutes.

[0080] The present invention mixes the encapsulated isorhamnetin delivery formulation obtained with a CRISPR Cas9 plasmid and incubates the mixture to obtain an incubation product. In the present invention, the mass ratio of the CRISPR Cas9 plasmid to isorhamnetin is preferably 0.375:3. In the present invention, the incubation temperature is preferably 20-30°C, and the incubation time is preferably 30 minutes.

[0081] The present invention mixes the obtained incubation material with 2,3-dimethylmaleic anhydride functionalized poly-L-lysine and incubates again to obtain a re-incubated material. In the present invention, the mass ratio of the 2,3-dimethylmaleic anhydride functionalized poly-L-lysine (LiX, Xu Q, Zhang P, Zhao X, Wang Y. Cutaneous microenvironment responsive microneedle patch for rapid gene release to treat subdermal tumor. J Control Release. 2019; 314: 72-80. doi: 10.1016 / j.jconrel.2019.10.016) to isorhamnetin is preferably 1.64: 3. In the present invention, the temperature of the re-incubation is preferably 20 to 30 ° C, and the time is preferably 30 min.

[0082] In the present invention, the mass ratio of the hypoxia-pretreated retinal progenitor cell membrane to the poly(lactic-co-glycolic acid) copolymer is preferably 1:1. In the present invention, the conditions for the ice-bath probe ultrasonic treatment preferably include ultrasonication at a power of 195 W for 30 minutes. In the present invention, the conditions for the ultrasonication preferably include ultrasonication at a power of 195 W for 5 minutes, with a 5-second pause after every 5-second ultrasonication.

[0083] The present invention provides a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid prepared by the preparation method described in the above technical solution.

[0084] The present invention also provides the use of the delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury.

[0085] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0086] Example 1

[0087] Preparation of biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulations

[0088] Step 1: Prepare the oil phase

[0089] Accurately weigh 3 mg of polyethyleneimine and dissolve it in 0.25 mL of acetone, accurately weigh 3 mg of isorhamnetin and dissolve it in 1.5 mL of ethanol / ethyl acetate (3:2, v / v) mixed solvent, accurately weigh 30 mg of polylactic acid-glycolic acid copolymer and dissolve it in 0.75 mL of acetone / dichloromethane (3:1, v / v) mixed solvent, and mix the above three solutions together to obtain a transparent and clear oil phase.

[0090] Step 2: Prepare the aqueous phase

[0091] 150 mg of poloxamer 188 (50 times the mass of isorhamnetin) was accurately weighed and dissolved in 3 mL of DEPC water (ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure) to obtain an aqueous phase.

[0092] Step 3: Mix the oil phase and water phase

[0093] The oil phase prepared in step 1 was slowly added to the water phase prepared in step 2, and ultrasonicated with an ice bath probe (195W) for 30 min to obtain a mixed solution.

[0094] Step 4: Preparation of a delivery formulation containing isorhamnetin

[0095] The mixed solution obtained in step 3 was placed on a rotary evaporator for 5 minutes (hydration) at a temperature of 37° C. and a speed of 70 r / min. The mixed solution was then ultrasonically treated using a cell disruptor, with each ultrasonic treatment lasting 5 seconds and a pause of 5 seconds for a total of 5 minutes to obtain a clear, transparent liquid with a yellow opalescence. The clear, transparent liquid with a yellow opalescence was divided into 1.5 mL centrifuge tubes and centrifuged at 5000 r / min for 10 minutes to separate the free isorhamnetin (precipitate), thereby obtaining a delivery formulation (supernatant solution) fully encapsulated with isorhamnetin.

[0096] Step 5: Add CRISPR Cas9 plasmid to the isorhamnetin-encapsulated delivery formulation

[0097] Add 0.375 mg of CRISPR Cas9 plasmid (12.5% of the mass of polyethyleneimine) to the isorhamnetin-encapsulated delivery formulation prepared in step 4, vortex for 30 seconds, and incubate on a shaker at room temperature for 30 minutes.

[0098] Step 6: Add 2,3-dimethylmaleic anhydride functionalized poly-L-lysine

[0099] Continue to add 2,3-dimethylmaleic anhydride functionalized poly-L-lysine to the delivery formulation encapsulating isorhamnetin. The amount of 2,3-dimethylmaleic anhydride functionalized poly-L-lysine added is 1.64 mg (the carbon-nitrogen molar ratio of 2,3-dimethylmaleic anhydride functionalized poly-L-lysine to polyethyleneimine is 5:1), vortex for 30 seconds, and incubate on a shaker at room temperature for 30 minutes.

[0100] Step 7: Retinal progenitor cells (R28 cells) were cultured overnight. The next day, the cells were replaced with a sugar-free medium and transferred to a hypoxic chamber for culture for 8 hours. The medium was then replaced with complete medium for culture for 4 hours. The cells were collected and the cell membranes were extracted using a cell membrane protein and cytoplasm protein extraction kit to obtain hypoxia-pretreated retinal progenitor cell membranes. Adding retinal progenitor cell membranes: Continue to add retinal progenitor cell membranes (R28 cell membranes) that have been pretreated with hypoxia to the delivery preparation encapsulating isorhamnetin. The amount of R28 cell membranes pretreated with hypoxia is 30 mg (the mass ratio of R28 cell membrane protein to polylactic acid-glycolic acid copolymer is 1:1). Ultrasound is performed for 30 minutes using an ice bath probe (195W) to obtain a mixed solution, which is then extruded through polycarbonate porous membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm, respectively. The solution is ultrasonically treated using a cell disruptor, with a pause of 5 seconds for each 5-second ultrasonication, and a total treatment of 5 minutes to obtain a clear isorhamnetin and CRISPR Csa9 plasmid delivery preparation with yellow opalescence, which is stored at 4°C for later use.

[0101] Since the present invention modifies the natural R28 cell membrane on the surface of the synthetic nanoparticles, the prepared isorhamnetin and CRISPR Cas9 plasmid delivery preparation has excellent intraocular biocompatibility and is a biomimetic delivery preparation.

[0102] Example 2

[0103] Detecting the encapsulation efficiency of biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulations

[0104] The biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention was dissolved in methanol, and the concentration of isorhamnetin was measured at a wavelength of 376 nm using a Perkin Elmer Lambda 6 UV-visible spectrophotometer produced by Perkin Elmer, Boston, Massachusetts, USA. Finally, the encapsulation efficiency of isorhamnetin was calculated based on the measured concentration.

[0105] It was calculated that the isorhamnetin encapsulation efficiency of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation was 84.30%±1.31%.

[0106] Example 3

[0107] Characterization of biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulations

[0108] 1. Morphological characterization

[0109] The morphology of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared above was characterized by transmission electron microscopy. The obtained transmission electron micrographs are shown in Figure 1 .

[0110] 2. Particle size and Zeta potential characterization

[0111] The particle size and Zeta potential of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparations prepared above were characterized using the DelsaNano C nanoparticle size / Zeta potential distribution analyzer. The obtained particle size distribution and Zeta potential distribution are shown in Figure 2. Figure 2 and Figure 3 .

[0112] Depend on Figure 1 、 Figure 2 and Figure 3 It can be seen that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention is regular spherical, uniform in size, with a particle size of about 153.63 nm, a Zeta potential of -21.32 mV, good dispersibility and high stability.

[0113] Example 4

[0114] Exploring the therapeutic effects of biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulations on RIR damage at the cellular level

[0115] Prepare isorhamnetin solution: dissolve 3 mg of isorhamnetin in 1.5 mL of ethanol / ethyl acetate (3:2, v / v) mixed solvent to obtain isorhamnetin solution for later use.

[0116] Prepare a blank preparation: Prepare a mixed solution, i.e., a blank preparation, by following steps 1 (preparing an oil phase), 2 (preparing an aqueous phase), and 3 (mixing the oil phase and the aqueous phase) in sequence for later use.

[0117] Preparation of fluorescently labeled biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation: Cy5.5 was used to label the delivery formulation. The red fluorescent-labeled isorhamnetin and CRISPR Csa9 plasmid delivery formulation was prepared according to step 1 (preparing the oil phase) by adding Cy5.51 mg, step 2 (preparing the aqueous phase), step 3 (mixing the oil phase and the aqueous phase), step 4 (preparing the delivery formulation encapsulating isorhamnetin), step 5 (adding the CRISPR Cas9 plasmid to the delivery formulation encapsulating isorhamnetin), step 6 (adding 2,3-dimethylmaleic anhydride functionalized poly-L-lysine) and step 7 (adding retinal progenitor cell membrane), which was recorded as Cy5.5 delivery formulation.

[0118] 1. Cellular uptake experiment

[0119] Retinal progenitor cells (R28 cells) were seeded in a 2 cm confocal microdish and incubated overnight to allow the cells to adhere to the wall. 100 nmol of the previously prepared Cy5.5 delivery agent was added and the culture medium containing the Cy5.5 delivery agent was washed away after 0 h, 4 h, 8 h, 12 h, and 24 h. After cell fixation, permeabilization, and DAPI labeling of the cell nuclei, a laser confocal microscope was used to observe the uptake of the Cy5.5 delivery agent by R28 cells over time.

[0120] The results of the detection of the uptake of Cy5.5 delivery formulation by R28 cells are shown in Figure 4 .Depend on Figure 4 As can be seen, the Cy5.5 delivery formulation was successfully taken up by R28 cells over time, and the uptake was time-dependent. This indicates that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention has good biocompatibility and cellular uptake ability.

[0121] 2. Anti-inflammatory activity research

[0122] Primary microglia extracted from mouse brains were inoculated into 24-well plates and divided into control group, model group, blank preparation group and biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group. After incubation overnight, the blank preparation group was pretreated with the blank preparation (final concentration of 10 μmol / L) prepared above for 2 h, the isorhamnetin-loaded preparation group was pretreated with the isorhamnetin-loaded preparation (final concentration of 10 μmol / L) prepared above for 2 h, the CRISPR Cas9 plasmid preparation group was pretreated with the CRISPR Cas9 plasmid preparation (final concentration of 20 μmol / L) prepared above for 2 h, and the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group were pretreated with the biomimetic isorhamnetin and CRISPR The cells were pretreated with the Cas9 plasmid delivery preparation (final concentration 10 μmol / L) for 2 h, and then lipopolysaccharide (LPS, final concentration 100 ng / mL) was added to the other groups except the control group to establish an inflammatory model. The control group did not receive any treatment. After 24 h, the content of the inflammatory factor NO secreted by the cells in the cell supernatant was detected using a total nitric oxide (NO) detection kit.

[0123] The results of the test on the secretion of inflammatory factor NO in each group are shown in Figure 5 .Depend on Figure 5 It can be seen that compared with the model group, the addition of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation significantly inhibited the production of the inflammatory factor NO. This shows that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention has good anti-inflammatory efficacy.

[0124] 3. Research on retinal cell protection ability

[0125] R28 cells were seeded in 96-well plates and divided into a blank preparation group, an isorhamnetin group, and a biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group. After incubation overnight, the blank preparation group was replaced with a full culture medium containing different concentrations of the previously prepared blank preparation for pretreatment for 24 h. The biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group was replaced with a full culture medium containing different concentrations of the previously prepared biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation for pretreatment for 24 h. After the pretreatment, each group was replaced with a sugar-free culture medium and the 96-well plate was placed in a hypoxic chamber (simulating an ischemic and hypoxic environment to establish an RIR injury model). After incubation at 37°C for 12 h, the 96-well plate was removed and the intracellular ATP content of each group was detected.

[0126] The results of the determination of ATP content in each group are shown in Figure 6 .Depend on Figure 6It can be seen that the presence of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation significantly inhibited the decrease in intracellular ATP content after establishing the RIR injury model, and this effect was concentration-dependent. This shows that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention exhibits good retinal cell protection ability in vitro.

[0127] 4. Research on Retinal Cell Reactive Oxygen Species Generation

[0128] R28 cells were seeded in 2 cm confocal microplates and divided into control group, model group, blank preparation group, and biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group. After incubation overnight, the blank preparation group was replaced with full culture medium containing the previously prepared blank preparation (final concentration of 10 μmol / L) for pretreatment for 24 h, and the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group was replaced with full culture medium containing the previously prepared biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation (final concentration of 10 μmol / L) for pretreatment for 24 h. After the pretreatment, all groups except the control group were replaced with sugar-free culture medium and the microplates were placed in a hypoxic chamber. The control group did not receive any treatment. After incubation at 37°C for 12 h, the microplates were removed, the sugar-free culture medium was discarded, and the cells were washed with phosphate buffer. The generation of intracellular reactive oxygen species in each group was detected using a reactive oxygen species detection kit, and the generation of reactive oxygen species was photographed using a confocal microscope.

[0129] The results of intracellular reactive oxygen species fluorescence detection in each group are shown in Figure 7 , the fluorescence intensity statistics are shown in Figure 8 .Depend on Figure 7 and Figure 8 It can be seen that compared with the control group, the model group had significantly increased intracellular reactive oxygen species levels, while after treatment with the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation, the reactive oxygen species levels decreased. This shows that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention can significantly inhibit intracellular reactive oxygen species levels.

[0130] Example 5

[0131] Exploring the therapeutic effects of biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulations on RIR damage at the animal level

[0132] The RIR injury model in SD rats was established using the anterior chamber pressurization method.

[0133] 1. Retinal HE staining

[0134] The modeled SD rats were divided into a model group, a blank preparation group, and a biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group.

[0135] The blank preparation prepared above was injected into the vitreous of the SD rats in the blank preparation group, and the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared above were injected into the vitreous of the SD rats in the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group, with an injection dose of 4 μL. The SD rats in the model group did not receive any treatment. After 7 days, the SD rats in each group were killed, and the eyeballs were removed. After fixation, paraffin embedding, and hematoxylin-eosin staining, the stained sections were placed under a microscope to observe the retinal structure and morphology.

[0136] The results of HE staining of rat retina are shown in Figure 9 .Depend on Figure 9 It can be seen that the retinal layers of the SD rats in the model group were thinned and retinal ganglion cells were lost. Compared with the model group, the retinal morphology and thickness of the SD rats in the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation group were significantly restored after vitreous injection of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention. This indicates that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention successfully inhibited retinal cell apoptosis after ischemia-reperfusion and improved retinal function.

[0137] 2. Optic nerve cell protection experiment

[0138] The modeled SD rats were divided into a model group, a blank preparation group, and a biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group.

[0139] The blank preparation prepared above was injected into the vitreous of SD rats in the blank preparation group, and the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared above were injected into the vitreous of SD rats in the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation group, with an injection dose of 4 μL. The SD rats in the model group did not receive any treatment. After 7 days, the SD rats in each group were killed, and the eyeballs were removed. After sucrose dehydration, 4% paraformaldehyde fixation, and cryoembedding, frozen sections were made with a thickness of 8 μm. The retinal neural tubulin β-III-Tubulin was fluorescently stained, and the cell nuclei were labeled with DAPI. The fluorescence intensity was observed using a laser confocal microscope.

[0140] The results of immunofluorescence detection of retinal tubulin β-III-Tubulin in each group of SD rats are shown in Figure 10 , the fluorescence intensity statistics are shown in Figure 11 . Figure 10 and Figure 11 It can be seen that compared with the model group, the fluorescence intensity of retinal tubulin β-III-Tubulin in SD rats was significantly increased after administration of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention. This shows that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention has a good protective effect on retinal nerve cells after RIR occurs.

[0141] 3. Retinal inflammation inhibition experiment

[0142] Retinal frozen sections obtained in the previous optic nerve cell protection experiment were taken, and the glial cell-specific proteins IBA1 and GFAP were fluorescently stained, and the cell nuclei were labeled with DAPI. The fluorescence intensity was observed using a laser confocal microscope.

[0143] The results of immunofluorescence detection of glial cell-specific protein IBA1 in each group of SD rats are shown in Figure 12 , the fluorescence intensity statistics are shown in Figure 13 The results of immunofluorescence detection of glial cell specific protein GFAP in each group of SD rats are shown in Figure 14 , the fluorescence intensity statistics are shown in Figure 15 .Depend on Figure 12 、 Figure 13 、 Figure 14 and Figure 15 It can be seen that the fluorescence intensity of glial cell-specific proteins IBA1 and GFAP in SD rats in the model group increased significantly, indicating that glial cells were activated under ischemia-reperfusion conditions. Compared with the model group, the fluorescence of glial cell-specific proteins IBA1 and GFAP in SD rats after administration of the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention decreased significantly, indicating that the activation and proliferation of glial cells and the inflammatory response were suppressed. This shows that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery formulation prepared by the present invention has a good ability to inhibit retinal inflammation in vivo.

[0144] 4. Retinal drug uptake experiment

[0145] The Cy5.5 delivery preparation prepared above was injected into the vitreous of unmodeled SD rats (sham operation group), and the Cy5.5 delivery preparation prepared above was also injected into the vitreous of modeled SD rats (model group). The injection dose was 4 μL. The eyeballs were removed on the 7th and 28th days, respectively. After fixation, the retinas were flattened. After the retinas were soaked in methanol, the fluorescence of the retinal pigment epithelium was observed using a laser confocal microscope.

[0146] The retinal fluorescence detection results of SD rats without modeling and SD rats with modeling on the 7th and 28th days are shown in Figure 2. Figure 16 and Figure 17 .Depend on Figure 16 and Figure 17 It can be seen that: on the 7th day, the fluorescence intensity of the model group was the strongest, indicating that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention can accurately target the retina and can be absorbed by the retina for a long time. Until the 28th day, red fluorescence can still be detected in the model group, indicating that the biomimetic isorhamnetin and CRISPR Cas9 plasmid delivery preparation prepared by the present invention has good biocompatibility and long-term sustained release.

[0147] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a delivery formulation containing isorhamnetin and CRISPR Cas9 plasmid, characterized in that: The following steps are involved: 1) mixing polyethyleneimine with acetone to obtain a polyethyleneimine solution; mixing isorhamnetin with ethanol and ethyl acetate to obtain an isorhamnetin solution; mixing polylactic acid-glycolic acid copolymer with acetone and dichloromethane to obtain a copolymer solution; mixing the polyethyleneimine solution, the isorhamnetin solution and the copolymer solution to obtain an oil phase; 2) mixing poloxamer 188 with DEPC water to obtain an aqueous phase; The mass ratio of poloxamer 188 to isorhamnetin is 50:1; 3) ultrasonically mixing the oil phase obtained in step 1) with the water phase obtained in step 2) to obtain a mixed solution; 4) subjecting the mixed solution obtained in step 3) to rotary evaporation, ultrasonic treatment, and centrifugation in sequence to obtain a supernatant that is the encapsulated isorhamnetin delivery preparation; 5) mixing the encapsulated isorhamnetin delivery formulation obtained in step 4) with the CRISPR Cas9 plasmid and incubating the mixture to obtain an incubation product; 6) mixing the incubated product obtained in step 5) with 2,3-dimethylmaleic anhydride functionalized poly-L-lysine, and incubating again to obtain a secondary incubated product; 7) ultrasonically treating the re-incubated product obtained in step 6) and the hypoxia-pretreated retinal progenitor cell membrane with an ice-bath probe to obtain an ultrasonicated product; The sonicated product was sequentially extruded through polycarbonate porous membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm, and the obtained solution was then sonicated to obtain a delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid.

2. The preparation method according to claim 1, characterized in that In step 1), the volume ratio of polyethyleneimine to acetone is 3 mg:0.25 mL; The mass ratio of the isorhamnetin to the volume of ethanol and the volume ratio of ethyl acetate is 3 mg:0.9 mL:0.6 mL; The ratio of the mass of the polylactic acid-glycolic acid copolymer to the volume of acetone and the volume of dichloromethane is 30 mg:0.5625 mL:0.1875 mL.

3. The preparation method according to claim 1, characterized in that In step 2), the volume ratio of poloxamer 188 to DEPC water is 150 mg:3 mL; The DEPC water is ultrapure water treated with diethyl pyrocarbonate and then sterilized at high temperature and high pressure.

4. The preparation method according to claim 1, characterized in that The conditions of the ultrasonic mixing in step 3) include: ultrasonic mixing at a power of 195 W for 30 minutes.

5. The preparation method according to claim 1, characterized in that The conditions of the rotary evaporation hydration in step 4) include: rotary evaporation at a temperature of 37° C. and a rotation speed of 70 rpm for 5 minutes; The ultrasonic treatment conditions include: ultrasonic treatment at a power of 195W for 5 minutes, with a pause of 5 seconds after every 5 seconds of ultrasonic treatment; The centrifugal conditions include: a rotation speed of 5000 rpm and a time of 10 min.

6. The preparation method according to claim 1, characterized in that In step 5), the mass ratio of CRISPR Cas9 plasmid to isorhamnetin is 0.375:3; The incubation temperature is 20-30° C. and the incubation time is 30 minutes.

7. The preparation method according to claim 1, characterized in that In step 6), the mass ratio of 2,3-dimethylmaleic anhydride functionalized poly-L-lysine to isorhamnetin is 1.64:3; The temperature of the second incubation is 20-30° C., and the time is 30 minutes.

8. The preparation method according to claim 1, characterized in that In step 7), the mass ratio of the hypoxic pretreated retinal progenitor cell membrane to the poly(lactic-co-glycolic acid) copolymer is 1:1; The conditions for the ice bath probe ultrasonic treatment include: ultrasonication at a power of 195W for 30 minutes; The ultrasonic treatment conditions include: ultrasonic treatment at a power of 195 W for 5 minutes, with a pause of 5 seconds after every 5 seconds of ultrasonic treatment.

9. A delivery preparation containing isorhamnetin and CRISPR Cas9 plasmid prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the delivery formulation containing isorhamnetin and CRISPR Cas9 plasmid according to claim 9 in the preparation of a drug for treating retinal ischemia-reperfusion injury.

Images