Nano-liposome with permeation enhancing effect as well as preparation method and application of nano-liposome

Abstract

The invention provides a nano-liposome with a penetration enhancing effect as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The nano-liposome disclosed by the invention comprises a lipid composition and a medicine, the lipid composition comprises an ionic lipid, cholesterol, an amphiphilic lipid and DOPE-LA, wherein the ionic lipid is a cationic lipid or an ionizable lipid. The nano-liposome provided by the invention forms a disulfide bond with sulfydryl on the cell surface to realize a permeation enhancing effect, and the disulfide bond on the cell membrane surface delivers a drug into the cell and then the drug can be recycled to the cell membrane surface through an oxidation-reduction process of the sulfydryl in the cell, so that the sulfydryl on the cell surface is not depleted; therefore, eye discomfort cannot be caused.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a nano liposome with penetration-promoting effect and a preparation method and application thereof. Background Art

[0002] Gene silencing and gene editing technologies have brought about a revolution in biomedical technology, and at the same time have provided a new treatment for difficult and complicated eye diseases. For example, retinitis pigmentosa, choroideremia, and Leber hereditary optic neuropathy. Because most of the gene mutations that cause diseases have been confirmed, and the eye is an immune-exempt organ, it is particularly suitable for gene editing or mRNA technology to treat eye diseases. However, mRNA has a large molecular weight, carries a large amount of negative charge, and is easily degraded, making the delivery of mRNA more difficult. The eye has a special barrier system against foreign bodies, including physiological barriers and anatomical barriers. Physiological barriers include tear turnover, nasolacrimal duct drainage, and blinking. Anatomical barriers include dynamic barriers and static barriers, among which dynamic barriers include conjunctival blood, lymphatic flow, and tear drainage, and static barriers include corneal epithelium, corneal epithelium, stroma, and blood-aqueous barrier. Although the above barrier system protects the eye from foreign bodies and pathogens, it also prevents drugs from entering the eye to exert their therapeutic effects.

[0003] At present, in order to promote the entry of drugs into the eye, commonly used penetration enhancers such as alcohols, surfactants, pyrrolidones and azones are used. The above-mentioned penetration enhancers are all through the structure of destroying the cell membrane, opening the tight connection between cells, realizing the penetration of drug molecules, and often causing discomfort in the eye. Therefore, it is urgent to provide a delivery system that improves the permeability of nucleic acid drugs to the cornea and enhances the intraocular delivery effect without destroying the cell membrane structure. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a nanoliposome with a penetration-enhancing effect, which adopts a penetration-enhancing mechanism that does not destroy the cell membrane structure, improves the permeability of nucleic acid drugs to the cornea, and enhances the intraocular delivery effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A nano liposome with penetration-promoting effect, comprising a lipid composition and a drug; the lipid composition comprises an ionic lipid, cholesterol, an amphipathic lipid and DOPE-LA, and the ionic lipid is a cationic lipid or an ionizable lipid.

[0007] Preferably, the molar ratio of ionic lipid, cholesterol, amphiphilic lipid and DOPE-LA is (20-70):(20-70):(1-10):(5-50).

[0008] Preferably, the ionic lipid is any one of ALC-0315, SM-102, D-Lin-MC3-DMA, CL1, LP01, C12-200, DLinDMA, DLin-MC2-DMA, DLin-KC2-DMA, DODMA, DOTAP, DC-Chol and DOTMA.

[0009] Preferably, the amphiphilic lipid is one or more of DMG-PEG2000, DSPE-PEG, DPPE-mPEG5000, mPEG-c-DMG, mPEG2000-DSG and ALC-0159.

[0010] Preferably, the drug is a nucleic acid molecule, and the nucleic acid molecule is any one or more of DNA, siRNA, mRNA, dsRNA, antisense nucleic acid, microRNA, antisense microRNA, antagomir, microRNA inhibitor, microRNA activator and immunostimulatory nucleic acid.

[0011] Preferably, the particle size of the nanoliposome is 70-120 nm, and the PDI is less than 0.15%.

[0012] Another object of the present invention is to provide a method for preparing the nanoliposome, comprising the following steps: dissolving the drug in a buffer solution to obtain a drug solution; using a microfluidic device to mix the lipid composition and the drug solution, wherein the volume ratio of the lipid composition to the drug mixture is 1:2 to 6.

[0013] Preferably, the buffer is any one of a citric acid buffer solution, an acetic acid buffer solution, a phosphoric acid buffer solution, a boric acid buffer solution, a HEPES buffer solution and a Tris buffer solution, and the concentration of the buffer is 5 mM to 50 mM, and the pH value is 3.5 to 4.5.

[0014] Preferably, the flow rate of the lipid composition is 1-6 mL / min, the flow rate of the drug solution is 3-30 mL / min, and the flow rate ratio of the lipid composition to the drug solution is 1:2-5.

[0015] Another object of the present invention is to provide the use of the nanoliposome or the preparation method in preparing drugs for treating eye diseases.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a nanoliposome with a penetration-promoting effect. The present invention uses DOPE-LA as a functional lipid excipient for promoting penetration, and provides a penetration-promoting LA-LNP delivery system. The nanoliposome of the present invention adopts a penetration-promoting mechanism that does not destroy the cell membrane structure, and achieves a penetration-promoting effect by forming a disulfide bond with the thiol group on the cell surface. After the disulfide bond on the cell membrane surface delivers the drug into the cell, it can be recirculated to the cell membrane surface through the redox process of the thiol group inside the cell. The present invention will not deplete the thiol group on the cell surface, and thus will not cause eye discomfort.

[0018] The present invention can quickly and efficiently deliver therapeutic nucleic acids into the eye and realize the therapeutic function of nucleic acids in cells. It can perform gene silencing, gene editing, and mRNA protein expression replacement for rare hereditary ophthalmic diseases, and has great development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the particle size distribution diagram of LA-LNP after mRNA is encapsulated by ionizable lipid ALC-0315 in Example 1 of the present invention;

[0020] Figure 2 This is the particle size distribution diagram of LA-LNP after mRNA is encapsulated using ionizable lipid SM-102 in Example 2 of the present invention;

[0021] Figure 3 This is the particle size distribution diagram of LA-LNP after mRNA is encapsulated by cationic lipid DOTAP in Example 3 of the present invention;

[0022] Figure 4 This is the particle size distribution diagram of LA-LNP after eGFP-DNA is encapsulated using ionizable lipid SM-102 in Example 4 of the present invention;

[0023] Figure 5 This is the particle size distribution diagram of LA-LNP prepared using ionizable lipid D-Lin-MC3-DMA in Example 5 of the present invention;

[0024] Figure 6 This is the particle size distribution diagram of LA-LNP labeled with Cy3 in Example 6 of the present invention;

[0025] Figure 7 This is a diagram of LA-LNP cell endocytosis labeled with Cy3 in Example 6 of the present invention;

[0026] Figure 8 The distribution diagram of LA-LNP labeled with Cy3 in the mouse cornea according to Example 7 of the present invention, wherein Cy3 is the distribution diagram of LA-LNP labeled with Cy3 in the mouse cornea, and the BF (Bright Field) detection result;

[0027] Fig. 9 This is the corneal fluorescence image of the mouse eye after eye drops were made using SM-102 as the ionizable lipid to prepare LA-LNP encapsulating eGFP-mRNA in Example 8 of the present invention, wherein the left column is the BF detection results of the mouse cornea; the right column is the fluorescence image after the green fluorescent protein is expressed in the cornea; the numbers 1, 2, and 3 represent three repeated experiments. DETAILED DESCRIPTION

[0028] The invention provides a nano liposome with penetration-promoting effect. The nano liposome comprises a lipid composition and a drug. The lipid composition comprises an ionic lipid, cholesterol, an amphiphilic lipid and DOPE-LA. The ionic lipid is a cationic lipid or an ionizable lipid.

[0029] The lipid composition of the present invention includes DOPE-LA (1,2-bis(9Z-oleoyl)-sn-glycerol-3-phosphoethanolamine lipoic acid conjugate). The thiol groups on the surface of corneal epithelial cells can undergo a ring-opening reaction with the dithiolane ring on the surface of the nanoliposomes of the present invention to form new disulfide bonds, thereby promoting the nanoliposomes of the present invention to enter the corneal epithelium, improving the permeability of nucleic acid drugs to the cornea, and enhancing the intraocular delivery effect. The DOPE-LA in the present invention is prepared by the method in CN118497281A.

[0030] In the present invention, the molar ratio of ionic lipids, cholesterol, amphiphilic lipids and DOPE-LA is (20-70):(20-70):(1-10):(5-50); preferably, the molar ratio of ionic lipids, cholesterol, amphiphilic lipids and DOPE-LA is (40-60):(40-60):(2-5):(10-30).

[0031] In the present invention, the ionic lipids are ALC-0315 (CAS No: 2036272-55-4), SM-102 (CAS No: 2089251-47-6), D-Lin-MC3-DMA (CAS No: 1224606-06-7), CL1 (CAS No: 1450888-71-7), LP01 (CAS No: 1799316-64-5), C12-200 (CAS No: 1220890-25-4), DLinDMA (CAS No: 871258-12-7), DLin-MC2-DMA (CAS No: 1221271-55-1), DLin-KC2-DMA (CAS No: 1190197-97-7), DODMA (CAS No: 104162-47-2), DOTAP (CAS Any one of DOTMA (CAS No: 104872-42-6) and DC-Chol (CAS No: 166023-21-8).

[0032] In the present invention, the amphiphilic lipid is one or more of DMG-PEG2000 (CAS No: 160743-62-4), DSPE-PEG (CAS No: 147867-65-0), DPPE-mPEG5000 (CAS No: 205494-72-0), mPEG-c-DMG (CAS No: 1019000-64-6), mPEG2000-DSG (CAS No: 308805-39-2) and ALC-0159 (CAS No: 1849616-42-7). When the amphiphilic lipid is a combination of two or more, they can be mixed in any proportion.

[0033] In the present invention, the drug is a nucleic acid molecule, and the nucleic acid molecule is any one or more of DNA, siRNA, mRNA, dsRNA, antisense nucleic acid, microRNA, antisense microRNA, antagomir, microRNA inhibitor, microRNA activator and immunostimulatory nucleic acid. When the nucleic acid molecule in the present invention is a combination of several, each nucleic acid molecule can be mixed in any proportion.

[0034] In the present invention, the particle size of the nanoliposome is 70-120 nm, and the PDI is less than 0.15%.

[0035] Another object of the present invention is to provide a method for preparing the nanoliposome, comprising the following steps: dissolving the drug in a buffer solution to obtain a drug solution; using a microfluidic device to mix the lipid composition and the drug solution, wherein the volume ratio of the lipid composition to the drug mixture is 1:2 to 6, preferably 1:3 to 5. After the lipid composition and the drug solution are mixed, the mixed solution is collected, and a dialysis bag with a molecular weight cutoff of 3.5 KDa is used to dialyze to obtain the nanoliposome; the present invention preferably uses PBS as the dialysis medium; the dialysis time is preferably 1.5 to 2.5 hours, and more preferably 2 hours.

[0036] In the preparation method provided by the present invention, the buffer solution is any one of a citric acid buffer solution, an acetate buffer solution, a phosphoric acid buffer solution, a boric acid buffer solution, a HEPES buffer solution and a Tris buffer solution, the concentration of the buffer solution is 5mM to 50mM, and the pH value is 3.5 to 4.5.

[0037] In the present invention, the flow rate of the lipid composition is 1 to 6 mL / min, the flow rate of the drug solution is 3 to 30 mL / min, and the flow rate ratio of the lipid composition to the drug solution is 1:2 to 5. The flow rate of the lipid composition is preferably 2 to 3 mL / min, and the flow rate of the drug solution is preferably 3 to 9 mL / min.

[0038] Another object of the present invention is to provide the use of the nanoliposome or the preparation method in the preparation of drugs for treating eye diseases. The present invention has found that due to the barrier effects such as blinking, tear secretion, and corneal barrier, the bioavailability of drugs in the eye is low. A nucleic acid drug has a large molecular weight and carries a large amount of negative charge, which makes it more difficult to enter the eye cells. The nanoliposome provided by the present invention can effectively improve the permeability of nucleic acid drugs to the cornea and improve the bioavailability of nucleic acid drugs in the eye.

[0039] The medicine prepared by the present invention can be used for gene silencing therapy, mRNA protein replacement therapy or gene editing therapy of eye diseases.

[0040] In the specific examples of the present invention, the ionic lipids, cholesterol, and amphiphilic lipids were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] Weigh 5 mg of ALC-0315, 1 mg of cholesterol, 2 mg of DOPE-LA and 0.2 mg of DSPE-PEG respectively, and dissolve them in 1 mL of anhydrous ethanol to obtain a lipid composition. Dissolve 400 μg of eGFP-mRNA in 3 mL of citric acid buffer solution (10 mM, pH 4.0) to obtain a drug solution. Use a microfluidic device to set the flow rate of the lipid composition to 1 mL / min and the flow rate of the drug solution to 3 mL / min, and mix the two solutions. Collect the mixed solution, use a dialysis bag with a molecular weight cutoff of 3.5 KDa, and dialyze with PBS as the dialysis medium for 2 hours to obtain nanoliposomes (LA-LNP) with a particle size of about 110 nm and a PDI of 0.13%.

[0044] The particle size distribution of LA-LNP after mRNA encapsulation using ionizable lipid ALC-0315 is as follows Figure 1 shown.

[0045] Example 2

[0046] Weigh 5 mg of SM-102, 1 mg of cholesterol, 2 mg of DOPE-LA and 0.2 mg of DPPE-mPEG5000 respectively, and dissolve them in 1 mL of anhydrous ethanol to obtain a lipid composition. Dissolve 400 μg of eGFP-mRNA in 3 mL of acetate buffer solution (25 mM, pH 4.0) to obtain a drug solution. Use a microfluidic device to set the flow rate of the lipid composition to 1 mL / min and the flow rate of the drug solution to 3 mL / min, and mix the two solutions. Collect the mixed solution, use a dialysis bag with a molecular weight cutoff of 3.5 KDa, and dialyze with PBS as the dialysis medium for 2 hours to obtain nanoliposomes (LA-LNP) with a particle size of about 120 nm and a PDI of 0.14%.

[0047] The particle size of LA-LNP after mRNA encapsulation using ionizable lipid SM-102 is as follows Figure 2 shown.

[0048] Example 3

[0049] Weigh 4 mg of DOTAP, 1.5 mg of cholesterol, 2.5 mg of DOPE-LA and 0.2 mg of DSPE-PEG respectively, and dissolve them in 1 mL of anhydrous ethanol to obtain a lipid composition. Dissolve 400 μg of eGFP-mRNA in 3 mL of HEPES buffer solution (10 mM, pH 4.0) to obtain a drug solution. Use a microfluidic device to set the flow rate of the lipid composition to 2 mL / min and the flow rate of the drug solution to 8 mL / min, and mix the two solutions. Collect the mixed solution, use a dialysis bag with a molecular weight cutoff of 3.5 KDa, and dialyze with PBS as the dialysis medium for 2 hours to obtain nanoliposomes (LA-LNP) with a particle size of about 100 nm and a PDI of 0.11%.

[0050] The particle size of LA-LNP after mRNA encapsulation using cationic lipid DOTAP is as follows Figure 3 shown.

[0051] Example 4

[0052] Weigh 6 mg of SM-102, 2 mg of cholesterol, 3 mg of DOPE-LA and 0.2 mg of DMG-PEG2000 respectively, and dissolve them in 1 mL of anhydrous ethanol to obtain a lipid composition. Dissolve 400 μg of eGFP-DNA in 3 mL of phosphate buffer solution (20 mM, pH 4.0) to obtain a drug solution. Use a microfluidic device to set the flow rate of the lipid composition to 1 mL / min and the flow rate of the drug solution to 4 mL / min, and mix the two solutions. Collect the mixed solution, use a dialysis bag with a molecular weight cutoff of 3.5 KDa, and dialyze with PBS as the dialysis medium for 2 hours to obtain nanoliposomes (LA-LNP) with a particle size of about 100 nm and a PDI of 0.14%.

[0053] The particle size of LA-LNPs after eGFP-DNA was encapsulated by ionizable lipid SM-102 is as follows: Figure 4 shown.

[0054] Example 5

[0055] Weigh 3 mg of D-Lin-MC3-DMA, 5 mg of cholesterol, 3 mg of DOPE-LA and 0.5 mg of DMG-PEG2000 respectively, and dissolve them in 1 mL of anhydrous ethanol to obtain a lipid composition. Use a microfluidic device to set the flow rate of the lipid composition to 1 mL / min, and the flow rate of the boric acid buffer solution (20 mM, pH 4.0) to 4 mL / min, and mix the two solutions. Collect the mixed solution, use a dialysis bag with a molecular weight cutoff of 3.5 KDa, and dialyze with PBS as the dialysis medium for 2 hours to obtain nanoliposomes (LA-LNP) with a particle size of about 120 nm and a PDI of 0.11%.

[0056] The particle size of LA-LNP prepared using ionizable lipid MC3 is as follows: Figure 5 shown.

[0057] Example 6

[0058] Preparation of Cy3-labeled LA-LNP: The DSPE-PEG in Example 1 was replaced with Cy3-DSPE-PEG to obtain Cy3-labeled LA-LNP with a particle size of about 100 nm and a PDI of 0.12% (eg Figure 6 Hela cells were cultured at 5×10 4 / well were inoculated in a 24-well plate. After 24 hours, the culture medium was discarded, and the cells were washed three times with PBS. 100 μL of Cy3-labeled LA-LNP and 400 μL of PBS were added to each well and incubated with the cells for 5, 15, 30, and 60 min, respectively. After the incubation, the cells were washed three times with PBS, and the cell membrane was stained with DiO, and then the cells were fixed with paraformaldehyde, and finally the cell nucleus was stained with DAPI. The internalization of LA-LNP by cells was observed using a fluorescence microscope.

[0059] The endocytosis process of Cy3-labeled LA-LNP in cells is as follows Figure 7 As shown, it can be seen that LA-LNP has entered the cells after 5 minutes of incubation.

[0060] Example 7

[0061] The Cy3-labeled LA-LNPs in Example 6 were used to conduct a mouse corneal distribution study.

[0062] The mice were KM mice purchased from Charles River Company and were raised and tested in the SFP animal room. The present invention randomly divided the mice into three groups after 7 days of adaptive culture. 50 μL Cy3-labeled LA-LNP was dripped into the eyes of the mice in each group. The mice in the three groups were killed 30, 60, and 90 minutes after the Cy3-labeled LA-LNP was dripped, and the eyeballs were removed and fixed with paraformaldehyde. The cornea was peeled off and observed under a fluorescence microscope.

[0063] The distribution of Cy3-labeled LA-LNP in the mouse cornea is shown in Figure 2. Figure 8 As shown, it can be seen that the LA-LNP of the present invention can be distributed in the corneal tissue in a short time.

[0064] Example 8

[0065] The LA-LNP in Example 1 was used to study the expression of eGFP mRNA in the mouse cornea.

[0066] The mice were KM mice purchased from Charles River Company and were raised and tested in the SFP animal room. After the mice were adaptively cultured for 7 days, 50 μL of LA-LNPs loaded with eGFP-mRNA were dripped into each eye of the mice, and the mice were killed 30 minutes later. The eyeballs were removed and fixed with paraformaldehyde, and the corneas were peeled off and observed under a fluorescence microscope. The experiment was repeated 3 times.

[0067] The expression of eGFP mRNA in mouse cornea is shown in Fig. 9 shown. Fig. 9 In the figure, the left column is the corneal BF image, the right column is the fluorescence image after the green fluorescent protein is expressed in the cornea, and 1, 2, and 3 represent the results of testing one eyeball of a mouse in three experiments. Fig. 9 The results showed that LA-LNP could rapidly deliver mRNA to the cornea via eye drops.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanoliposome having a penetration-enhancing effect, characterized in that: The nanoliposome comprises a lipid composition and a drug; the lipid composition comprises an ionic lipid, cholesterol, an amphipathic lipid and DOPE-LA, and the ionic lipid is a cationic lipid or an ionizable lipid.

2. The nanoliposome according to claim 1, characterized in that The molar ratio of ionic lipid, cholesterol, amphiphilic lipid and DOPE-LA is (20-70):(20-70):(1-10):(5-50).

3. The nanoliposome according to claim 1, characterized in that The ionic lipid is any one of ALC-0315, SM-102, D-Lin-MC3-DMA, CL1, LP01, C12-200, DLinDMA, DLin-MC2-DMA, DLin-KC2-DMA, DODMA, DOTAP, DC-Chol and DOTMA.

4. The nanoliposome according to claim 1, characterized in that The amphiphilic lipid is one or more of DMG-PEG2000, DSPE-PEG, DPPE-mPEG5000, mPEG-c-DMG, mPEG2000-DSG and ALC-0159.

5. The nanoliposome according to claim 1, characterized in that The drug is a nucleic acid molecule, and the nucleic acid molecule is any one or more of DNA, siRNA, mRNA, dsRNA, antisense nucleic acid, microRNA, antisense microRNA, antagomir, microRNA inhibitor, microRNA activator and immunostimulatory nucleic acid.

6. The nanoliposome according to claim 1, characterized in that The particle size of the nanoliposome is 70-120 nm, and the PDI is less than 0.15%.

7. The method for preparing nanoliposomes according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: dissolving the drug in a buffer solution to obtain a drug solution; and mixing the lipid composition and the drug solution using a microfluidic device, wherein the volume ratio of the lipid composition to the drug mixture is 1:2-6.

8. The preparation method according to claim 7, characterized in that: The buffer solution is any one of a citric acid buffer solution, an acetic acid buffer solution, a phosphoric acid buffer solution, a boric acid buffer solution, a HEPES buffer solution and a Tris buffer solution. The concentration of the buffer solution is 5 mM to 50 mM, and the pH value is 3.5 to 4.

5.

9. The preparation method according to claim 7, characterized in that: The flow rate of the lipid composition is 1-6 mL / min, the flow rate of the drug solution is 3-30 mL / min, and the flow rate ratio of the lipid composition to the drug solution is 1:2-5.

10. Use of the nanoliposome according to any one of claims 1 to 6 or the preparation method according to any one of claims 7 to 9 in the preparation of drugs for treating eye diseases.

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