Nucleic acid medicine eye drops and preparation method thereof

Abstract

The invention relates to nucleic acid medicine eye drops and a preparation method thereof. The nucleic acid drug eye drops comprise nucleic acid drug-loaded lipid nanoparticles and cell-penetrating peptides modified on the surfaces of the lipid nanoparticles, the lipid nanoparticle comprises a core and a surface layer, and the core comprises ionizable lipid and a phospholipid layer which are coated on the surface of the nucleic acid drug; the surface layer comprises ionizable lipid, phospholipid, cholesterol and modified polylysine; the modified polylysine is grafted with the cell-penetrating peptide. The nucleic acid drug eye drops have high stability, can realize noninvasive delivery of nucleic acid drugs, and complete treatment of posterior segment diseases of eyes.

Description

Technical Field

[0001] The present application relates to the field of nanomedicine technology, and in particular to a nucleic acid drug eye drop and a preparation method thereof. Background Art

[0002] Due to the physiological characteristics of cell membranes, negatively charged nucleic acid drugs are difficult to enter cells to exert their regulatory effects. There is a lack of good carriers for sustained and controlled release of drugs. Therefore, multiple intravitreal injections are required to treat eye blinding diseases.

[0003] At present, ocular trans-barrier drug delivery is an urgent development direction for the treatment of ocular diseases. The drugs involve small molecule compounds, protein drugs and gene drugs, and the targeted area is mainly for chronic blinding diseases in the posterior segment of the eye. The huge potential of lipid nanoparticles (LNP) in biomedicine, such as biocompatibility, low toxicity, and stretchability, has attracted extensive attention from researchers. The key to LNP using positive and negative electrical adsorption to form nanostructures to deliver nucleic acid drugs lies in the protonation of ionizable lipids under appropriate acidic pH conditions. However, changes in pH constitute structural defects in the stability of drug-loaded complexes. The stability of existing drug-loaded systems is relatively poor and cannot meet actual needs. Summary of the invention

[0004] Based on this, it is necessary to provide a non-invasive and highly effective nucleic acid drug eye drops with high stability, which can achieve non-invasive delivery of nucleic acid drugs and complete the treatment of posterior segment diseases of the eye.

[0005] The specific technical solutions are as follows:

[0006] A nucleic acid drug eye drop, comprising nucleic acid drug-loaded lipid nanoparticles and a membrane-penetrating peptide modified on the surface of the lipid nanoparticles;

[0007] The lipid nanoparticle comprises a core and a surface layer, wherein

[0008] The core includes an ionizable lipid and phospholipid layer coated on the surface of the nucleic acid drug;

[0009] The surface layer includes ionizable lipids, phospholipids, cholesterol and modified polylysine;

[0010] The modified polylysine is grafted with the cell-penetrating peptide.

[0011] In some embodiments, the modified polylysine comprises a polyethylene glycol modifier and polylysine.

[0012] In some embodiments, the structural formula of the polyethylene glycol modifier is .

[0013] In some embodiments, the average molecular weight of the polyethylene glycol modifier is above 800;

[0014] And / or, n is greater than or equal to 8.

[0015] In some embodiments, the structural formula of the ionizable lipid is selected from , and At least one of .

[0016] In some embodiments, the cell-penetrating peptide comprises at least one of a Pep peptide, a TAT peptide and a PENE peptide; and / or

[0017] The nucleic acid drug includes mRNA or siRNA.

[0018] In some embodiments, the mass ratio of the total ionizable lipids of the core and the surface layer, the total phospholipids of the core and the surface layer, the cholesterol, and the modified polylysine is (1-8): (1-4): (0.5-2): 1.

[0019] In some of the embodiments, the mass ratio of the lipid nanoparticles to the nucleic acid drug is (10~90):1.

[0020] The nucleic acid drug eye drops of the present application encapsulate nucleic acid drugs through the coordinated action of ionizable lipids, phospholipids, cholesterol and modified polylysine, and can self-assemble to form a stable lipid nanoparticle structure; the membrane-penetrating peptide is grafted with the modified polylysine, which increases the loading capacity of the ionizable lipids for nucleic acid drugs under neutral environmental conditions, improves the stability of the ionizable lipids, can efficiently participate in delivery and complete degradation in the body, has good biocompatibility, and further enables the nucleic acid drug eye drops to efficiently achieve non-invasive delivery of nucleic acid drugs and complete the treatment of posterior segment diseases of the eye.

[0021] A method for preparing the nucleic acid drug eye drops as described above, characterized in that it comprises the following steps:

[0022] Mixing the ionizable lipid, the phospholipid, the cholesterol and the modified polylysine with a first solvent to prepare a first mixture;

[0023] Mixing the nucleic acid drug with a second solvent to prepare a second mixture;

[0024] The first mixture and the second mixture are mixed, and nucleic acid drug-loaded lipid nanoparticles are prepared by using microfluidics technology;

[0025] The cell-penetrating peptide is mixed with a third solvent, and then mixed with the lipid nanoparticles to prepare nucleic acid drug eye drops.

[0026] In some of the embodiments, the method for preparing the modified polylysine includes: coupling a polyethylene glycol modifier and polylysine to covalently link the polyethylene glycol modifier to the polylysine to obtain the modified polylysine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the preparation of lipid nanoparticles for gene delivery to the posterior segment of the eye in Example 1 of the present application;

[0028] Figure 2 Schematic diagram of the synthesis of ionizable lipid 1 (LNP1) in Example 1 of the present application;

[0029] Figure 3 Schematic diagram of the synthesis of ionizable lipid 2 (LNP2) in Example 1 of the present application;

[0030] Figure 4 Schematic diagram of the synthesis of ionizable lipid 3 (LNP3) in Example 1 of the present application;

[0031] Figure 5 This is a result diagram of the H-NMR detection of the ionizable lipid corresponding to the synthetic product in Example 1 of the present application;

[0032] Figure 6 Schematic diagram of the synthesis of PEG-PLLZ in Example 1 of the present application;

[0033] Figure 7 Schematic diagram of the synthesis of PEG-PLL in Example 1 of the present application;

[0034] Figure 8 This is a transmission electron microscopy test result of the lipid nanoparticles prepared in Example 1 of the present application;

[0035] Fig. 9 This is a comparison result of the transfection efficiency of LNPs with different compositions after preparation of various lipid nanoparticles in Example 1 of the present application;

[0036] Fig.10 This is a graph comparing the transfection efficiency of LNPs with different PEG-PLL incorporation in Example 1 of the present application;

[0037] Fig.11 This is a comparison result of the transfection efficiency of LNPs composed of different ionizable lipids after each LNP / mRNA preparation in Example 1 of the present application;

[0038] Fig.12 This is a graph showing the results of LNP-GFP mRNA delivery and gene expression detection in ARPE-19 cells in Example 1 of the present application;

[0039] Fig.13Schematic diagram of the structure of three polypeptide sequences used to modify LNP in Example 1 of the present application;

[0040] Fig.14 Schematic diagram of the synthesis of PENE-PEG-PLL in Example 1 of the present application;

[0041] Fig.15 This is a diagram showing the biodistribution and semi-quantitative analysis results of the lipid nanodelivery system in the fundus under different penetrating peptide modification methods in Example 1 of the present application. DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are provided in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0044] The term "comprises", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements defined by the sentence "comprising one..." do not exclude the presence of other identical elements in the process, method, article or equipment including the elements. The indefinite articles "a kind of" and "one" before the elements or components of the present application are not restrictive to the quantity requirements (i.e., the number of occurrences) of the elements or components. Therefore, "one" or "a kind of" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the number obviously refers only to the singular form. The meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0045] Except as shown in the operating examples or otherwise indicated, all numbers used in the specification and claims to indicate the amount of ingredients, physicochemical properties, etc. are understood to be adjusted by the term "about" in all cases. For example, therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the attached claims are approximate values, and those skilled in the art can use the teachings disclosed herein to seek to obtain the desired properties and appropriately change these approximate values. The use of numerical ranges expressed as endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0046] As shown in the background technology, in traditional technology, ordinary LNP preparation needs to be prepared under acidic conditions, and the change in pH constitutes a structural defect in the stability of the drug-loaded complex; when LNP is used in the eye, there are no modification sites in the classic structure, and the eye drops cannot achieve the non-invasive targeting effect of pathological sites in the posterior segment of the eye, and the purpose of efficient, stable and targeted delivery of nucleic acid drugs cannot be achieved.

[0047] After a large number of creative experimental studies, the applicant found that by introducing chemical modification sites through amphiphilic block polymers and utilizing PEG-polylysine-doped lipid nanoparticle structures, the chemical modification of lipid nanoparticles by transmembrane peptides can be increased, thereby increasing the non-invasive delivery of nucleic acid drug eye drops to the posterior segment of the eye.

[0048] Nucleic acid is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and is a biological macromolecule composed of multiple nucleotide monomers; nucleic acid is composed of nucleotides, and nucleotide monomers are composed of pentose, phosphate, nitrogenous base, or any modified group. If the pentose is ribose, the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.

[0049] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA of 20 to 25 nucleotides in length. It induces the formation of gene silencing complexes in cells through RNA, sequence-specifically degrades mRNA, and thus downregulates the expression of the target protein.

[0050] mRNA, messenger RNA, Chinese translation: messenger ribonucleic acid, is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis. mRNA can be monocistronic mRNA or polycistronic mRNA. mRNA can also contain one or more functional nucleotide analogs, examples of which include: pseudouridine, 1-methyl-pseudouridine or 5-methylcytosine. The examples here are not exhaustive, and any modified mRNA or its derivatives can be applied to this application.

[0051] One embodiment of the present application provides a nucleic acid drug eye drop, which includes paper nanoparticles carrying nucleic acid drugs and a membrane-penetrating peptide modified on the surface of lipid nanoparticles;

[0052] Lipid nanoparticles include a core and a surface layer, wherein

[0053] The core includes an ionizable lipid and phospholipid layer coated on the surface of the nucleic acid drug;

[0054] The surface layer includes ionizable lipids, phospholipids, cholesterol, and modified polylysine;

[0055] Modified poly-lysine is grafted with cell-penetrating peptide.

[0056] In some embodiments, the modified polylysine comprises a polyethylene glycol modifier and polylysine.

[0057] It is understandable that polylysine itself does not have inherent cleavage properties, but the amino groups on its side chains can provide a large number of connection platforms as chemical attachment sites, supplying pH-sensitive characteristic groups. Secondly, polyamino acids have good biodegradability, can efficiently participate in delivery and complete degradation in the body, and have good biocompatibility.

[0058] In some embodiments, the polyethylene glycol modifier contains a thiol group and an amino group.

[0059] In one embodiment, the structural formula of the polyethylene glycol modifier is The polyethylene glycol modifier forms an -NH-CO- structure with polylysine to covalently link the polyethylene glycol modifier to polylysine. Further, the amino group of the polyethylene glycol modifier forms an -NH-CO- structure with the ester group of polylysine. It should be noted that the connection between the polyethylene glycol modifier and polylysine can also be connected by the NC bond required by the click chemistry reaction.

[0060] In some embodiments, the average molecular weight of the polyethylene glycol modifier is 800-4000. This arrangement prevents the polyethylene glycol modifier from blocking the chemical binding sites of polylysine in space, and the polyethylene glycol modifier has little effect on the activity of polylysine after modification. It should be noted that the use of a polyethylene glycol modifier with a molecular weight that is too large is likely to reduce the biological activity of polylysine, while the use of a polyethylene glycol modifier with a molecular weight that is too small has a weak effect on maintaining the stability of the modified polylysine.

[0061] In some embodiments, the above n is 8-45.

[0062] In a specific example, the structural formula of the polylysine is .

[0063] In some embodiments, the structural formula of the ionizable lipid is selected from , and At least one of .

[0064] In some embodiments, the above-mentioned cell-penetrating peptide includes at least one of Pep peptide, TAT peptide and PENE peptide.

[0065] In a specific example, one end of the cell-penetrating peptide is connected to a maleimide group.

[0066] The cell-penetrating peptide is subjected to Michael addition reaction with the thiol group of the modified polylysine, thereby grafting the cell-penetrating peptide onto the modified polylysine. It should be noted that the cell-penetrating peptide is not limited to being directly connected to the modified polylysine through an active group, and the cell-penetrating peptide can also be connected to the modified polylysine through an indirect connection, for example, the modified polylysine uses a biotin-avidin system to connect polylysine to the cell-penetrating peptide. It should be noted that the indirect connection method is not limited to the biotin-avidin system, and can also be other intermediate connection systems.

[0067] In some embodiments, the nucleic acid drug comprises mRNA or siRNA.

[0068] In some embodiments, the mass ratio of the total ionizable lipids in the core and the surface layer, the total phospholipids in the core and the surface layer, the cholesterol, and the modified polylysine is (1-8):(1-4):(0.5-2):1.

[0069] In some of the embodiments, the mass ratio of the total ionizable lipids of the core and the surface layer, the total phospholipids of the core and the surface layer, the cholesterol, and the modified polylysine is (1-4): (1-2): (0.5-1.5): 1.

[0070] In some of the embodiments, the mass ratio of the lipid nanoparticles to the nucleic acid drug is (10~90):1.

[0071] It can be understood that the mass ratio of the above-mentioned lipid nanoparticles and the above-mentioned nucleic acid drug includes but is not limited to 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, and 90:1.

[0072] The above-mentioned nucleic acid drug eye drops encapsulate nucleic acid drugs through the coordinated action of ionizable lipids, phospholipids, cholesterol and modified polylysine, and can self-assemble to form a stable lipid nanoparticle structure; the membrane-penetrating peptide is grafted with modified polylysine, which increases the loading capacity of ionizable lipids for nucleic acid drugs under neutral environmental conditions, improves the stability of ionizable lipids, can efficiently participate in delivery and complete degradation in the body, has good biocompatibility, and further enables the nucleic acid drug eye drops to efficiently achieve non-invasive delivery of nucleic acid drugs and complete the treatment of posterior segment diseases of the eye.

[0073] Combination Figure 1 As shown, the present application also provides a method for preparing the above-mentioned nucleic acid drug eye drops, including steps S100 to S400.

[0074] Step S100: mixing the ionizable lipid, the phospholipid, the cholesterol and the modified polylysine with a first solvent to prepare a first mixture.

[0075] In some embodiments, the preparation method of the modified polylysine comprises: coupling reaction of a polyethylene glycol modifier and polylysine to covalently link the polyethylene glycol modifier and the polylysine to obtain the modified polylysine. Specifically, the reaction formula of the polyethylene glycol modifier and polylysine is as follows:

[0076]

[0077] In some embodiments, m is greater than or equal to 6, and further, m is 6 to 20. It should be noted that a larger degree of polymerization of the modified polylysine leads to a larger molecular weight, and the use of modified polylysine with an excessively large molecular weight is likely to reduce the biological activity of the cell-penetrating peptide.

[0078] In some embodiments, the first solvent includes one or more of anhydrous ethanol, acetone and methanol.

[0079] Step S200: Mixing the nucleic acid drug and a second solvent to prepare a second mixture.

[0080] In some embodiments, the second solvent comprises acetic acid-sodium acetate buffer with a concentration of 20 mM to 50 mM.

[0081] Step S300: Mix the first mixture and the second mixture, and prepare nucleic acid drug-carrying lipid nanoparticles using microfluidic technology.

[0082] Step S400: mixing the cell-penetrating peptide with a third solvent, and then mixing with the lipid nanoparticles to prepare nucleic acid drug eye drops.

[0083] In some embodiments, the third solvent includes water.

[0084] The above-mentioned nucleic acid drug eye drops encapsulate nucleic acid drugs through the ionizable lipids, phospholipids, cholesterol and modified polylysine, and can self-assemble to form a stable lipid nanoparticle structure. The lipid nanoparticle structure doped with PEG-polylysine can increase the chemical modification of lipid nanoparticles by transmembrane peptides. On the one hand, the introduction of polylysine increases the mRNA loading capacity of lipid nanoparticles under neutral buffer conditions, effectively improves the stability of lipid nanoparticles carrying nucleic acid drugs, prolongs the blood circulation time of lipid nanoparticles carrying nucleic acid drugs, makes the lipid nanoparticles carrying nucleic acid drugs have a better half-life in the blood flow, increases the effective accumulation of lipid nanoparticles carrying nucleic acid drugs in the target site, and provides convenience for the subsequent non-invasive delivery of nucleic acid drugs to target cells / tissues; on the other hand, through the modification of transmembrane peptides with tissue penetration, the drug is well enriched in the posterior segment of the eye.

[0085] The following is a detailed description in conjunction with specific examples. The following examples do not include other components except for inevitable impurities unless otherwise specified. The reagents and instruments used in the examples are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are implemented according to conventional conditions, such as the conditions described in the literature, books, or the methods recommended by the manufacturer. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0086] Example 1

[0087] 1. Preparation of ionizable lipids

[0088] 1. Saturated aliphatic hydrocarbons without double bonds are used for the synthesis of ionizable lipids and LNP preparation (LNP1)

[0089] Take 0.5 mM 4-amino-1-butanol and 1.1 mM octadecyl acrylate and react in an oil bath at 70°C for 48 h. The obtained product is dialyzed with a dialysis bag (molecular weight cutoff: 300 Da) for 2 days. The product is freeze-dried and used for later use. Figure 2 shown.

[0090] 2. Unsaturated aliphatic hydrocarbons containing one double bond are used for the synthesis of ionizable lipids and LNP preparation (LNP2)

[0091] Take 0.5 mM 4-amino-1-butanol and 1.1 mM oleyl methacrylate and react in an oil bath at 70°C for 48 hours. The obtained product is dialyzed for 2 days using a dialysis bag (molecular weight cutoff: 300 Da). The product is freeze-dried and used for later use. Figure 3 shown.

[0092] 3. Unsaturated aliphatic hydrocarbons containing two double bonds are used for the synthesis of ionizable lipids and LNP preparation (LNP3)

[0093] Take 2 mM linoleyl alcohol and dissolve it in dichloromethane (DCM), slowly add the acid-binding agent triethylamine, and add 2.2 mM acryloyl chloride while stirring, and react for 3 hours at room temperature. After the reaction, acryloyl chloride is quenched in an ice bath, and linoleyl acrylate is obtained after rotary evaporation and concentration. The obtained product is dialyzed with a dialysis bag (molecular weight cutoff: 100 Da) for 2 days, and the product is freeze-dried for use.

[0094] Take 0.5 mM 4-amino-1-butanol and 1.1 mM linoleyl acrylate and react in an oil bath at 70°C for 48 h. The obtained product is dialyzed with a dialysis bag (molecular weight cutoff: 300 Da) for 2 days. The product is freeze-dried and used for later use. Figure 4 shown.

[0095] The ionizable lipid product prepared above was subjected to nuclear magnetic resonance hydrogen spectrum (H-NMR) detection, and the results were as follows Figure 5 shown.

[0096] 2. Polyethylene glycol-polylysine (PEG-PLL) synthesis

[0097] The synthesis steps are as follows: 0.1 mM SH-PEG-NH2 (MW 2000) and 0.8 mM N6-benzyloxycarbonyl-L-lysine cyclic anhydride (LLZ-NCA) are dissolved in 5 mL of anhydrous DMF, and a small amount of DTT is added to prevent sulfhydryl oxidation. The reaction is carried out under N2 protection for 72 hours. The solution after the reaction is precipitated with 40 times the volume of methyl tert-butyl ether, centrifuged at 8000 rpm for 5 min, and the supernatant is discarded to obtain SH-PEG-PLLZ. The product is freeze-dried and set aside. The synthesis diagram is shown in the figure. Figure 6~Figure 7 shown.

[0098] 3. Microfluidic Preparation of LNP Drug-Loaded Nanoparticles

[0099] The lipid component consisted of ionizable lipids, cholesterol, DOPE, and SH-PEG-PLL (mass ratio of 4:2:1:1), and the lipids were dissolved in anhydrous ethanol. mRNA was dissolved in pH 5.6 acetic acid-sodium acetate buffer (50 mM). Lipid nanoparticles (LNPs) were prepared using a microfluidic device with a volume flow rate ratio of 3:1 between the aqueous phase and the organic phase and a total flow rate of 20 mL min -1 The LNP whose ionizable lipid is lonizable lipid 1 is named LNP 1, the LNP whose ionizable lipid is lonizable lipid 2 is named LNP 2, and the LNP whose ionizable lipid is lonizable lipid 3 is named LNP 3. The schematic diagram of lipid nanoparticle preparation is shown in FIG. Figure 1 As shown; Microfluidic device means microfluidic device, Lipid nanoparticle means lipid nanoparticle, Lonizable lipid means ionizable lipid, High transfection efficiency means high transfection efficiency, Low cytotoxicity means low cytotoxicity, Cholesterol means cholesterol, DOPE means phospholipid, SH-PEG-PLL means modified polylysine, and mRNA means nucleic acid drug.

[0100] The lipid nanoparticles prepared above were examined by transmission electron microscopy (TEM). Figure 8 As shown, the results showed that LNP 1, LNP 2, and LNP 3 all had spherical nanoparticle morphology.

[0101] 1. Detection of the effect of poly-lysine introduced into LNP on transfection of ocular cells

[0102] 1) 100 μL ARPE-19 cell suspension (1×10 4 Cells were seeded in 96-well plates and incubated overnight in a cell culture incubator.

[0103] 2) 2 h before transfection, the culture medium was replaced with serum-free and antibody-free culture medium and cultured for another two hours to keep the cells in a "starved" state.

[0104] 3) Prepare mRNA-LNP complexes according to a certain ratio in step 3 above (total lipids: mRNA = 20:1 / 40:1 / 60:1 / 80:1, wt), with an mRNA mass of 0.4 μg per well. Discard the original culture medium and add 100 μL / well of LNPs solution diluted in serum-free and double-antibody-free culture medium. Use cationic liposome transfection reagent Lipofectamine 2000 (Lipo) as a positive control.

[0105] 4) After culturing in a cell culture incubator for 4 h, add 100 μL / well of culture medium containing 10% fetal bovine serum and continue to culture in the cell culture incubator.

[0106] After 20 h, the expression of EGFP was observed under an inverted fluorescence microscope, and ARPE-19 cells were collected by trypsin digestion. The expression efficiency of EGFP-mRNA in ARPE-19 cells was quantitatively analyzed by flow cytometry.

[0107] The results are as follows Fig. 9 As shown, the results showed that the introduction of polylysine increased the mRNA loading capacity of LNP under neutral buffer conditions, improved the stability of LNP, and the introduction of polylysine increased the transfection ability of LNP to eye cells.

[0108] 2. Transfection efficiency of LNPs with different PEG-PLL incorporation

[0109] The lipid components were composed of ionizable lipid, cholesterol, DOPE, SH-PEG-PLL in a mass ratio of 8:4:1:2, ionizable lipid, cholesterol, DOPE, SH-PEG-PLL in a mass ratio of 8:4:2:2, and ionizable lipid, cholesterol, DOPE, SH-PEG-PLL in a mass ratio of 8:4:3:2, respectively. The lipids were dissolved in anhydrous ethanol. mRNA was dissolved in pH 5.6 acetic acid-sodium acetate buffer (50mM) (total lipids: mRNA = 20:1 / 40:1 / 60:1 / 80:1, wt). Lipid nanoparticles (LNPs) were prepared using a microfluidic device, with a volume flow rate ratio of 3:1 between the aqueous phase and the organic phase, and a total flow rate of 20 mL min -1 . Ionizable lipids are lonizable lipid 1.

[0110] The lipid nanoparticles prepared above were transfected into cells according to the operation in step (1). The results are as follows: Fig.10 shown.

[0111] 3. Transfection efficiency of LNPs composed of different ionizable lipids after preparation of each lipid nanoparticle

[0112] The lipid component consisted of ionizable lipids, cholesterol, DOPE, and SH-PEG-PLL (mass ratio of 4:2:1:1), and the lipids were dissolved in anhydrous ethanol. mRNA was dissolved in pH 5.6 acetic acid-sodium acetate buffer (50 mM) (total lipids: mRNA = 20:1 / 40:1 / 60:1 / 80:1, wt). Lipid nanoparticles (LNPs) were prepared using a microfluidic device, with a volume flow rate ratio of 3:1 between the aqueous phase and the organic phase, and a total flow rate of 20 mL min -1 The LNP whose ionizable lipid is lonizable lipid 1 is named LNP1, the LNP whose ionizable lipid is lonizable lipid 2 is named LNP 2, and the LNP whose ionizable lipid is lonizable lipid 3 is named LNP 3.

[0113] The lipid nanoparticles prepared above were transfected into cells according to the operation in step (1). The results are as follows: Fig.11 shown.

[0114] 4. ARPE-19 cell detection of LNP-GFP mRNA delivery and gene expression detection

[0115] The lipid component consisted of ionizable lipids, cholesterol, DOPE, and SH-PEG-PLL (mass ratio of 4:2:1:1), and the lipids were dissolved in anhydrous ethanol. mRNA was dissolved in pH 5.6 acetic acid-sodium acetate buffer (50 mM) (total lipids: mRNA = 20:1 / 40:1 / 60:1 / 80:1, wt). Lipid nanoparticles (LNPs) were prepared using a microfluidic device, with a volume flow rate ratio of 3:1 between the aqueous phase and the organic phase, and a total flow rate of 20 mL min -1 The LNP whose ionizable lipid is lonizable lipid 1 is named LNP1, the LNP whose ionizable lipid is lonizable lipid 2 is named LNP 2, and the LNP whose ionizable lipid is lonizable lipid 3 is named LNP 3.

[0116] The lipid nanoparticles prepared above were transfected into cells according to the operation in step (1), and the microscopic detection results were as follows: Fig.12 shown.

[0117] 4. Peptide Modification

[0118] In order to increase the biological barrier penetration of non-invasive eye drops, functional peptides were introduced with the help of the reaction sites on PEG to increase the biodistribution of eye drops in the posterior segment of the eye.

[0119] The three peptide sequences are: Pep peptide (Mal-SPALHFL (SEQ ID NO: 1)), TAT peptide (Mal-STPESANL (SEQ ID NO: 2)) and PENE peptide (Mal-RQIKIWFQNRRMKWKK (SEQ ID NO: 3)), and the structural schematic diagram is shown in Fig.13 shown.

[0120] The specific steps of peptide modification are:

[0121] 1) Solid phase preparation: Weigh 1 g of dichlororesin (1 g of resin with a substitution rate of 1.0-1.1 mmol) into a solid phase synthesis tube and add 10 mL of DCM to soak for 15 min to swell the resin.

[0122] 2) Add the first amino acid: discard DCM, quickly add DCM (10 mL) solution containing 1.5 mmol amino acid AA1 and 3 mmol N, N-diisopropylethylamine (DIEA, 496 μL), and react for 1 h. (Note: DIEA is added later, 165 μL / mmol.)

[0123] 3) Capping: Discard the reaction solution, wash with DCM 5 times to remove the amino acids not connected to the solid phase, add 12 mL of blocking solution, and cap for 10 minutes.

[0124] Blocking solution: DIEA:DCM:MeOH = 1:17:2 (v / v), 0.6:10.2:1.2 (mL).

[0125] 4) Remove Fmoc: discard the blocking solution, wash with DCM 5 times, N, N-dimethylformamide (DMF)

[0126] Wash 5 times, add 50% diethylamine (DMF 50% Vol) and react for 30 minutes.

[0127] 5) Access to the second amino acid: Wash with DMF 5 times, and add a DMF solution containing 3 mmol amino acid AA2, 3 mmol HBTU (1.138 g), and 6 mmol DIEA (990 μL).

[0128] 6) Repeat steps 4) to 5) until fully extended.

[0129] 7) Add maleimidopropionic acid: wash with DMF 5 times, add DMF solution containing 3 mmol maleimidopropionic acid (0.507 g), 3 mmol HBTU (1.138 g), and 6 mmol DIEA (990 μL).

[0130] 7) Cutting: Discard the reaction solution, wash with DMF 5 times, wash with DCM 5 times, add the cutting reagent, react for 30 min, collect the filtrate, remove trifluoroacetic acid by rotary evaporation, precipitate with anhydrous ether, filter and discard the supernatant, and freeze-dry to obtain Mal-Peptide.

[0131] Cleavage reagent: 95% trifluoroacetic acid (TFA): 2.5% triisopropylsilane (TIS): 2.5% H2O (v / v), i.e. 19 mL TFA: 0.5 mL TIS: 0.5 mL H2O.

[0132] The reaction diagram is as follows Fig.14 shown.

[0133] The distribution of lipid nanoparticles in the posterior segment of the eye was monitored by the lipid nanoparticles with different modification modes and lipid nanoparticles without membrane-penetrating peptide modification. The results of biological tissue distribution and semi-quantitative analysis in the in vivo experiment were as follows: Fig.15 shown.

[0134] The modification of the surface of drug-loaded lipid nanoparticles with membrane-penetrating peptides can significantly enhance the transmembrane effect of nanodrugs in the anterior segment of the eye. Once the drug molecules have passed through the corneal barrier, with the help of the good vascular network of the sclera and choroid of the eye, the drugs can be well enriched in the posterior segment of the eye, providing a new therapeutic approach for the non-invasive treatment of eye blinding diseases with mRNA drugs.

[0135] In summary, the above-mentioned embodiment can increase the chemical modification of LNP by the transmembrane peptide through the PEG-polylysine doped LNP nanostructure. The introduction of polylysine increases the loading capacity of LNP for mRNA under neutral buffer conditions and improves the stability of LNP, thereby achieving the purpose of efficient, stable and targeted delivery of mRNA to the posterior segment of the eye by the nano-delivery system eye drops.

[0136] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A nucleic acid drug eye drop, characterized in that: The nucleic acid drug eye drops include nucleic acid drug-loaded lipid nanoparticles and a membrane-penetrating peptide modified on the surface of the lipid nanoparticles; The lipid nanoparticle comprises a core and a surface layer, wherein The core includes an ionizable lipid and phospholipid layer coated on the surface of the nucleic acid drug; The surface layer includes ionizable lipids, phospholipids, cholesterol and modified polylysine; The modified polylysine is grafted with the cell-penetrating peptide.

2. The nucleic acid drug eye drops according to claim 1, characterized in that The modified polylysine comprises a polyethylene glycol modifier and polylysine.

3. The nucleic acid drug eye drops according to claim 2, characterized in that: The structural formula of the polyethylene glycol modifier is .

4. The nucleic acid drug eye drops according to claim 3, characterized in that: The average molecular weight of the polyethylene glycol modifier is above 800; And / or, n is greater than or equal to 8.

5. The nucleic acid drug eye drops according to any one of claims 1 to 4, characterized in that The structural formula of the ionizable lipid is selected from , and At least one of .

6. The nucleic acid drug eye drops according to any one of claims 1 to 4, characterized in that The cell-penetrating peptide comprises at least one of Pep peptide, TAT peptide and PENE peptide; and / or The nucleic acid drug includes mRNA or siRNA.

7. The nucleic acid drug eye drops according to any one of claims 1 to 4, characterized in that The mass ratio of the total ionizable lipids of the core and the surface layer, the total phospholipids of the core and the surface layer, the cholesterol, and the modified polylysine is (1-8):(1-4):(0.5-2):

1.

8. The nucleic acid drug eye drops according to any one of claims 1 to 4, characterized in that The mass ratio of the lipid nanoparticles to the nucleic acid drug is (10~90):

1.

9. A method for preparing the nucleic acid drug eye drops according to any one of claims 1 to 8, characterized in that: The following steps are involved: Mixing the ionizable lipid, the phospholipid, the cholesterol and the modified polylysine with a first solvent to prepare a first mixture; Mixing the nucleic acid drug with a second solvent to prepare a second mixture; The first mixture and the second mixture are mixed, and nucleic acid drug-loaded lipid nanoparticles are prepared by using microfluidics technology; The cell-penetrating peptide is mixed with a third solvent, and then mixed with the lipid nanoparticles to prepare nucleic acid drug eye drops.

10. The method for preparing the nucleic acid drug eye drops according to claim 9, characterized in that: The preparation method of the modified polylysine comprises: performing a coupling reaction on a polyethylene glycol modifier and polylysine so as to covalently connect the polyethylene glycol modifier and the polylysine to obtain the modified polylysine.

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