Preparation method and application of cationic liposome loaded with S58 nucleic acid aptamer
Through the preparation method of cationic liposomes loaded with S58 nucleic acid aptamers, the problems of transient biological activity and poor stability of S58 nucleic acid aptamers were solved, and long-term inhibition of conjunctival fibroblast proliferation and migration were achieved, effectively inhibiting scar formation.
Patent Information
- Application Number
- CN202510271961.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-06-03
AI Technical Summary
The prior art is difficult to effectively prolong the biological activity of S58 nucleic acid aptamer, and has poor stability in serum, so it cannot effectively inhibit scar formation.
The preparation method of cationic liposomes loaded with S58 nucleic acid aptamer was adopted. The cationic liposomes loaded with S58 were prepared by mixing DOTAP and DSPE-PEG2000 and adding trichloromethane for ultrasonic resolving. After rotary evaporation, hydrating in PBS, and then adding S58 nucleic acid aptamer and 1-bromoheptafluorooctane for ultrasonic oscillation was performed.
The biological activity of S58 nucleic acid aptamer is extended, its decomposition in the serum is avoided, and the proliferation and migration of conjunctival fibroblasts is achieved for a long time, which effectively inhibits scar formation.
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Figure CN120078723A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of liposomes, and particularly relates to a preparation method and application of a cationic liposome loaded with S58 nucleic acid aptamer. Background Art
[0002] The nucleic acid aptamer S58 can effectively inhibit the expression of α-SMA in human fibroblasts and has an inhibitory effect on the transdifferentiation of fibroblasts. However, S58 has a short existence time in serum. Even when using chitosan nano-thermosensitive slow-release agar particles in the presence of serum, it can only extend the biological activity of S58 to about 48 hours, and cannot achieve the ideal effect of inhibiting scar formation. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a preparation method and application of a cationic liposome loaded with S58 nucleic acid aptamer in view of the deficiencies of the above-mentioned prior art. The prepared cationic liposome loaded with S58 nucleic acid aptamer can extend the biological activity of the nucleic acid aptamer S58, has no biological toxicity, can inhibit the proliferation and migration of human conjunctival fibroblasts, and can be applied to the preparation of drugs for inhibiting scar formation after conjunctival filtration surgery.
[0004] To solve the above technical problem, the technical solution adopted by the present invention is: a preparation method of a cationic liposome loaded with S58 nucleic acid aptamer, and the method is as follows:
[0005] After mixing DOTAP and DSPE-PEG2000, add chloroform, assist dissolution by water bath ultrasound, and then rotary evaporation to obtain a cationic liposome film. Add PBS and perform water bath ultrasound until the cationic liposome film is completely hydrated. Then add S58 nucleic acid aptamer and 1-bromooctadecafluorooctane to the hydrated liposome, and perform ultrasonic oscillation under the condition of ice bath to obtain a cationic liposome loaded with S58 nucleic acid aptamer;
[0006] The nucleotide sequence of the S58 nucleic acid aptamer is as shown in SEQ ID NO:1.
[0007] Preferably, the dosage ratio of DOTAP, DSPE-PEG2000, PBS, S58 nucleic acid aptamer and 1-bromooctadecafluorooctane is 12 mg: 4 mg: 2 mL: 20 nmol: 200 μL.
[0008] Preferably, the conditions of the rotary evaporation are: under the conditions of a temperature of 45 °C and a pressure of -0.1 kPa, rotary evaporation for 1 h.
[0009] Preferably, the conditions of the ultrasonic oscillation are: a power of 60 W, a working time of 3 min to 6 min, and stopping oscillation for 5 s every 5 s during oscillation.
[0010] The present invention also provides an application of the cationic liposome loaded with the S58 nucleic acid aptamer prepared by the above preparation method, and the cationic liposome loaded with the S58 nucleic acid aptamer is used for preparing a drug for inhibiting scar formation after trabeculectomy.
[0011] The present invention has the following advantages compared with the prior art:
[0012] The present invention can solve the disadvantage that the nucleic acid aptamer S58 is easily decomposed, prolong the half-life and biological activity of the nucleic acid aptamer S58 to achieve the effect of inhibiting scar formation after conjunctival surgery, and can be applied to prepare a drug for inhibiting scar formation after trabeculectomy.
[0013] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings
[0014] Figure 1 It is the particle size and zeta potential of the cationic liposome loaded with the S58 nucleic acid aptamer in Example 1 of the present invention.
[0015] Figure 2 It is the electron microscope detection image of the cationic liposome loaded with the S58 nucleic acid aptamer in Example 1 of the present invention.
[0016] Figure 3 It is the release effect diagram of the S58 encapsulated in the cationic liposome loaded with the S58 nucleic acid aptamer in Example 1 of the present invention.
[0017] Figure 4 It is the CCK8 toxicity test diagram in Example 2 of the present invention.
[0018] Figure 5 It is the CCK8 proliferation test diagram in Example 2 of the present invention.
[0019] Figure 6 It is the Transwell cell invasion and migration test diagram in Example 2 of the present invention.
[0020] Figure 7 It is the Transwell statistical chart in Example 2 of the present invention.
[0021] Figure 8 It is the cell immunofluorescence image in Example 2 of the present invention. Detailed Embodiments
[0022] Example 1
[0023] The preparation method of the cationic liposome loaded with the S58 nucleic acid aptamer in this example is as follows:
[0024] After mixing 12 mg of DOTAP and 4 mg of DSPE-PEG2000, 5 mL of chloroform was added, and ultrasonic dissolution was assisted by a water bath. Then, under the conditions of a temperature of 45 °C and a pressure of -0.1 kPa, rotary evaporation was carried out for 1 h to obtain a cationic liposome film. 2 mL of PBS (phosphate buffered saline) was added, and ultrasonic treatment was carried out in a water bath until the cationic liposome film was completely hydrated. Then, 20 nmol of S58 aptamer and 200 μL of 1-bromoheptadecafluorooctane were added to the hydrated liposomes, and ultrasonic oscillation was carried out under ice bath conditions. The conditions for ultrasonic oscillation were: power of 60 W, working time of 3 min to 6 min, and during this period, oscillation was stopped for 5 s after every 5 s of oscillation (5 s / 5 s, on / off), to obtain cationic liposomes loaded with S58 aptamer;
[0025] The nucleotide sequence of the S58 aptamer is shown as SEQ ID NO:1.
[0026] DOTAP: (2,3-dioleyloxypropyl) trimethylammonium chloride, purchased commercially;
[0027] DSPE-PEG2000: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, purchased commercially;
[0028] (I) Screening of S58 concentration
[0029] In this example, the addition amount of the S58 aptamer was optimized, and at the same time, cationic liposomes loaded with S58 aptamer with addition amounts of 5, 10, 15, and 20 nmol of the S58 aptamer were prepared. After centrifugation, the S58 concentration in the supernatant was measured, and each group was repeated five times.
[0030] In this example, when the addition amount of the S58 aptamer was 20 nmol, the encapsulation efficiency was the highest, which was 84.37% ± 1.51%.
[0031] When the addition amount of the S58 aptamer was 5 nmol, the encapsulation efficiency was: 57.85% ± 5.52%.
[0032] When the addition amount of the S58 aptamer was 10 nmol, the encapsulation efficiency was: 71.33% ± 1.54%
[0033] When the addition amount of the S58 aptamer was 15 nmol, the encapsulation efficiency was: 79.08% ± 5.45%.
[0034] (II) Characterization and detection (particle size, zeta potential, electron microscopy) of the cationic liposomes loaded with S58 aptamer prepared in this example
[0035] (1) Experiment 1: Detection of particle size and zeta potential
[0036] As Figure 1 shown, the particle size and zeta potential of the cationic liposomes loaded with S58 nucleic acid aptamer prepared in this example were measured using a Malvern particle size and zeta potential analyzer. The results showed that the particle size distribution was concentrated, the particle size was 194.97 ± 21.98 nm, and the zeta potential was 1.80 ± 0.25 mV.
[0037] Experiment 2: Electron microscopy detection
[0038] As Figure 2 shown, the morphology of the cationic liposomes loaded with S58 nucleic acid aptamer was detected using a low-voltage transmission electron microscope. Under the electron microscope, it was a spherical structure with a smooth surface.
[0039] (3) In vitro release determination of cationic liposomes
[0040] Figure 3 The figure shows the release effect diagram of the S58 encapsulated in the cationic liposomes loaded with S58 nucleic acid aptamer of Example 1. The dialysis method was used: the prepared cationic liposomes were placed in a dialysis bag, and the dialysis bag was placed in water and continuously stirred. The amount of S58 released from the dialysis bag was measured daily, and the release rate for 7 days with 3 repeated measurements was 85.53% ± 2.30%.
[0041] The cationic liposomes prepared by the present invention prolong the biological activity of S58, prevent it from being decomposed, and can continuously and slowly release the nucleic acid aptamer S58, thereby being able to inhibit scar formation for a long time.
[0042] Example 2
[0043] This example is the application of the cationic liposomes loaded with S58 nucleic acid aptamer prepared in Example 1. The cationic liposomes loaded with S58 nucleic acid aptamer are used to evaluate the biological toxicity and the inhibitory effect on human conjunctival fibroblasts.
[0044] (1) Experiment 1: CCK8 toxicity experiment
[0045] This experiment was to test the cytotoxicity of the cationic liposomes loaded with S58 nucleic acid aptamer prepared in Example 1 for evaluating the biological toxicity.
[0046] Experimental steps:
[0047] 1. Cell culture:
[0048] Human conjunctival fibroblasts were inoculated in a 96-well plate, with 6000 cells inoculated in each well, and cultured in a cell culture incubator for 24 h.
[0049] 2. Sample treatment:
[0050] After the cells adhered and grew to an appropriate density, the control group (Conrrol group) was added with the complete medium for human conjunctival fibroblasts;
[0051] The experimental group was divided into four groups, and TGF-β 2 (human conjunctival fibroblasts), S58, blank liposomes, and cationic liposomes loaded with S58 aptamer were added to the complete medium for human conjunctival fibroblasts for treatment.
[0052] Among them, the blank liposomes (LP) were the hydrated liposomes prepared by the preparation method of Example 1 (that is, the hydrated liposomes obtained from the liposome film + PBS);
[0053] The specific grouping is as follows:
[0054] Control group (Conrrol group): Complete medium for human conjunctival fibroblasts.
[0055] Experimental group:
[0056] TGF-β 2 Group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL.
[0057] LP group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL, blank liposomes with a final concentration of 200 μg / mL.
[0058] S58 group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL, S58 with a final concentration of 10 nmol / mL.
[0059] LP-S58 group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL, cationic liposomes loaded with S58 aptamer with a final concentration of 200 μg / mL.
[0060] 3. Continue culturing:
[0061] The cells were continuously cultured for 48 h after treatment.
[0062] 4. Add CCK-8 reagent:
[0063] 10 μL of CCK-8 reagent was added to each well at 0 h, 24 h, and 48 h after adding the drug. The 96-well plate was gently shaken to ensure that the reagent was fully mixed with the cells.
[0064] 5. Incubate:
[0065] The 96-well plate was placed back in the incubator at 37 °C and incubated for 2 hours.
[0066] 6. Measure the absorbance:
[0067] Use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.
[0068] The results are as Figure 4 shown. In the figure, ns indicates no difference between groups, and * indicates a difference with statistical significance. Figure 4 On the 1st day, there was no statistical difference in the absorbance values between groups, indicating that there was no difference in the number of cells between groups before drug treatment, avoiding experimental errors caused by different cell numbers. On the 2nd and 3rd days, the lack of statistical difference in the absorbance values between groups indicated that there was no difference in the number of cells between groups after drug treatment, that is, the drugs used in the experiment had no toxic effect on the cells.
[0069] The results showed that none of the 4 groups in the experimental group had cytotoxicity, indicating that the liposomes prepared by the present invention would not cause harm to normal conjunctival tissue and could be used in human and animal experiments.
[0070] (2) Experiment 2: CCK8 proliferation experiment
[0071] This experiment was a cell proliferation experiment to test the cationic liposomes loaded with S58 nucleic acid aptamer prepared in Example 1, and was used to evaluate the inhibitory effect of cationic liposomes loaded with nucleic acid aptamer S58 on the proliferation of human conjunctival fibroblasts.
[0072] The experimental steps were the same as those in Experiment 1.
[0073] The results are as Figure 5 shown. In the figure, ns indicates no difference between groups, * indicates a result with statistical significance, and the more * there are, the more obvious the difference is.
[0074] Figure 5 On the 1st day, there was no statistical difference in the absorbance values between groups, indicating that there was no difference in the number of cells between groups before drug treatment, avoiding experimental errors caused by different cell numbers. On the 2nd and 3rd days, there was a statistical difference in the absorbance values between groups. Among them, the OD values of the TGF-β 2 and TGF-β 2 +LP group were significantly higher than those of the other three groups, and the LP-S58 group was lower than the S58 group.
[0075] The results showed that TGF-β2 could stimulate the proliferation of human conjunctival fibroblasts. Both S58 and LP-S58 (i.e., the cationic liposome loaded with the S58 nucleic acid aptamer prepared in Example 1) could inhibit the proliferative effect of TGF-β2. The blank liposome (LP) had no inhibitory effect on TGF-β2. At the same time, the inhibitory effect of LP-S58 on TGF-β2 was stronger than that of pure S58. The cationic liposome loaded with the S58 nucleic acid aptamer prepared in this experiment prolonged the biological activity of S58 and could inhibit the proliferation of fibroblasts for a long time to achieve the effect of inhibiting scar formation. Therefore, it could be used to prepare drugs for inhibiting scar formation after conjunctival filtration surgery.
[0076] (III) Experiment 3: Transwell invasion experiment
[0077] This experiment was a Transwell invasion experiment to test the cationic liposome loaded with the S58 nucleic acid aptamer prepared in Example 1, and was used to detect the inhibitory effect of the cationic liposome loaded with the S58 nucleic acid aptamer on the invasion and migration of human conjunctival fibroblasts.
[0078] 1. Cell pretreatment: Starve human conjunctival fibroblasts 24 hours in advance.
[0079] 2. Cell seeding: Seed human conjunctival fibroblasts into Transwell chambers, 105 cells per well. Add 200 μL of serum-free blank medium to the chambers, place the chambers in a 24-well plate, and add complete medium to the chambers for drug treatment.
[0080] The specific experimental groups were as follows:
[0081] Control group (Conrrol group): Complete medium for human conjunctival fibroblasts.
[0082] Experimental groups:
[0083] TGF-β 2 group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL.
[0084] TGF-β 2 +LP group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL, blank liposome with a final concentration of 200 μg / mL.
[0085] TGF-β 2 +S58 group: Complete medium for human conjunctival fibroblasts, TGF-β2 with a final concentration of 10 ng / mL, S58 with a final concentration of 10 nmol / mL.
[0086] TGF-β 2+LP-S58 group: Complete medium for human conjunctival fibroblasts, TGF-β2 at a final concentration of 10 ng / mL, and cationic liposomes loaded with S58 nucleic acid aptamer at a final concentration of 200 μg / mL.
[0087] The blank medium is DMEM medium, commercially available, brand gibco.
[0088] The complete medium is Hconf complete medium for human conjunctival fibroblasts, commercially available, brand Huatuo Biotechnology.
[0089] 3. Culturing cells:
[0090] Place the 24-well plate in an incubator at 37 °C and 5% CO 2 and incubate for 24 hours.
[0091] 4. Cell fixation and staining:
[0092] After culturing, take out the Transwell chamber, rinse the top well with PBS to remove non-penetrating cells.
[0093] Fix the cells with 4% paraformaldehyde for 10 minutes, and then rinse with PBS.
[0094] Stain the cells that have penetrated to the bottom with crystal violet.
[0095] 5. Cell counting:
[0096] Observe the bottom of the Transwell chamber under the microscope, and select multiple random fields to count the number of penetrating cells.
[0097] The results of cell staining are as Figure 6 shown. The purple part in the figure represents the conjunctival fibroblasts that have invaded through the Transwell chamber, that is, the cell number can represent the strength of cell migration and invasion ability. Compared with the control group ( Figure 6 a), the migration and invasion abilities of human conjunctival fibroblasts stimulated by TGF-β 2 ( Figure 6 b) are significantly enhanced (more in number), and there is no significant difference after adding blank liposomes ( Figure 6 c). After adding S58 ( Figure 6 d), the number of migrating cells decreases, indicating that S58 can inhibit the migration and invasion abilities of conjunctival fibroblasts treated with TGF-β 2 . After adding cationic liposomes loaded with S58 nucleic acid aptamer ( Figure 6 e), the cell number is even less, indicating that the cationic liposomes loaded with S58 nucleic acid aptamer have a stronger ability to inhibit migration and invasion than pure S58.
[0098] As Figure 7As shown, **** indicates that there are statistically significant differences in the number of cells between groups, where TGF-β 2 and TGF-β 2 The number of cells in the +LP group was significantly higher than that in the other three groups, and the number of cells in the LP-S58 group was lower than that in the S58 group.
[0099] The results showed that TGF-β2 could stimulate the invasion of human conjunctival fibroblasts, and both S58 and the cationic liposome loaded with S58 aptamer could inhibit the invasion of TGF-β2, while the blank liposome had no inhibitory effect. At the same time, the inhibitory effect of the cationic liposome loaded with S58 aptamer was stronger than that of pure S58. The cationic liposome loaded with S58 aptamer prepared in the present invention can more effectively inhibit the invasion and migration of fibroblasts, can be used to inhibit scar formation, and prepare drugs for inhibiting scar formation.
[0100] (IV) Experiment 4: Cellular immunofluorescence experiment
[0101] This experiment was a cellular immunofluorescence experiment to test the cationic liposome loaded with S58 aptamer prepared in Example 1, and was used to detect the expression of actin-α (α-SMA) and type I collagen (collagen 1) in cells.
[0102] Experimental steps:
[0103] 1. Cell culture:
[0104] Human conjunctival fibroblasts were seeded on cell culture slides and cultured to 50% confluence. The drug treatment was the same as in Experiment 3.
[0105] 2. Cell fixation:
[0106] Wash the cells twice with PBS to remove the culture medium.
[0107] Fix the cells with 4% paraformaldehyde for 20 minutes.
[0108] Wash the cells with PBS 2-3 times to remove the residual fixative.
[0109] 3. Blocking treatment:
[0110] Treat the cells with 10% normal goat serum and incubate at room temperature for 1 h.
[0111] 4. Primary antibody incubation:
[0112] Incubate with the primary antibody overnight. After incubation, wash the cells 3 times with PBS to remove the unbound primary antibody.
[0113] 5. Secondary antibody incubation:
[0114] Incubate the cells with the appropriately diluted fluorescently labeled secondary antibody for 1 hour in the dark.
[0115] Wash the cells three times and remove the unbound secondary antibody with PBS.
[0116] 6. Nuclear staining: Stain the cell nuclei with 4',6-diamidino-2-phenylindole (DAPI).
[0117] 7. Mounting:
[0118] Add an appropriate amount of anti-fluorescence quenching agent on the glass slide to prevent fluorescence quenching.
[0119] 8. Observation under fluorescence microscope:
[0120] Observe the cells using a fluorescence microscope and select an appropriate filter for fluorescence imaging.
[0121] Take fluorescence images as needed and record the data.
[0122] Figure 8 a is the staining map of actin-α (α-SMA), DAPI staining map, and the merged Merge map; Figure 8 b is the staining map of type I collagen (collagen 1), DAPI staining map, and the merged Merge map;
[0123] Actin-α (α-SMA), type I collagen (collagen 1), 4',6-diamidino-2-phenylindole (DAPI), and Merge are merged maps, which are produced by superimposing the previous two maps.
[0124] As Figure 8 shown, Figure 8 in a, the red fluorescence intensity represents the expression level of α-SMA. Compared with the Control group, the expression of αSMA in the TGF-β 2 and TGF-β 2 +LP groups increased significantly. The expression in the TGF-β 2 +S58 group and the TGF-β 2 +LP-S58 group decreased, and the expression in the TGF-β 2 +LP-S58 group was less than that in the S58 group. Figure 8 In b, the green fluorescence intensity represents the expression level of Collagen 1. For the Control group, the expression of αSMA in the TGF-β 2 and TGF-β 2 +LP groups increased significantly. The expression in the TGF-β 2 +S58 group and the TGF-β 2 +LP-S58 group decreased, and the expression in the TGF-β 2The expression of the +LP-S58 group is less than that of the S58 group. Immunofluorescence assay shows that the preparation of the present invention can reduce the expression of actin-α and type I collagen in human conjunctival fibroblasts after being stimulated by TGF-β 2 and can be used to reduce scar formation after trabeculectomy.
[0125] The cationic liposomes loaded with the S58 nucleic acid aptamer prepared by the present invention have a particle size range of less than 300 nm, are positively charged and are easily taken up by cells; the biological activity of the nucleic acid aptamer S58 is prolonged; it has no biological toxicity, and can inhibit the proliferation, invasion and migration of conjunctival fibroblasts, and can be used for scar formation after trabeculectomy and for preparing drugs for inhibiting scar formation after trabeculectomy.
[0126] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent changes made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.
Claims
1. A method for preparing a cationic liposome loaded with S58 nucleic acid aptamers, characterized in that: The method is: After mixing DOTAP and DSPE-PEG2000, chloroform was added, and solubilized by water bath ultrasound, and then rotary evaporated to obtain a cationic liposome film, PBS was added, and sonicated in a water bath until the cationic liposome film was completely hydrated, and then S58 nucleic acid aptamers and 1-bromoheptadecafluorooctane bromide were added to the hydrated liposomes, and sonicated in an ice bath to obtain cationic liposomes loaded with S58 nucleic acid aptamers; The nucleotide sequence of the S58 nucleic acid aptamer is shown in SEQ ID NO:
1.
2. The method for preparing a cationic liposome loaded with S58 nucleic acid aptamers according to claim 1, characterized in that: The dosage ratio of DOTAP, DSPE-PEG2000, PBS, S58 nucleic acid aptamer and 1-bromoheptadecafluorooctane bromide is 12 mg: 4 mg: 2 mL: 20 nmol: 200 μL.
3. The cation loaded with S58 nucleic acid aptamer according to claim 1 is rotary evaporated for 1 hour at a temperature of 45°C and a pressure of -0.1 kPa.
4. The method for preparing a cationic liposome loaded with S58 nucleic acid aptamers according to claim 1, characterized in that: The conditions of the ultrasonic oscillation are: power of 60W, working time of 3min to 6min, and stopping oscillation for 5s after oscillating for 5s.
5. An application of a cationic liposome loaded with S58 nucleic acid aptamer prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The cationic liposome loaded with S58 nucleic acid aptamer is used for preparing a drug for inhibiting scar formation after conjunctival filtration surgery.