WO2023165583A1

WO2023165583A1 - Delivery system and method targeting ocular cell

Info

Publication number: WO2023165583A1
Application number: PCT/CN2023/079439
Authority: WO
Inventors: 周昌阳; 孙怡迪; 彭文博; 汤竣杰
Original assignee: 益杰立科(上海)生物科技有限公司
Priority date: 2022-03-04
Filing date: 2023-03-03
Publication date: 2023-09-07

Abstract

Provided is an exogenous substance delivery method targeting a specific ocular cell, comprising applying a lipid nanoparticle (LNP) to a vitreous body. On the basis of molar percentage, the lipid nanoparticle comprises the following components: 20%-70% ionizable lipid, 30%-60% cholesterol-based lipid, 25%-50% structural lipid, and 1%-30% PEGylated lipid, wherein the ionizable lipid comprises a pH-sensitive lipid and a permanent cationic lipid, and the permanent cationic lipid and the pH-sensitive lipid are in a molar ratio (C/P value) of 1:24 to 24:1. By adjusting the C/P value, the LNP is allowed to target different intraocular cells, and accurate delivery targeting the intraocular cells is achieved.

Description

Delivery systems and methods targeting ocular cells

technical field

This application relates to the field of biomedicine, in particular to a delivery system and method targeting eye cells.

Background technique

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (Cas)) technology can edit the genome in a precise, sequence-dependent manner to produce permanent changes. Because of the ability to target disease-causing mutations, it holds the incredible promise of a once-in-a-lifetime cure for genetic diseases. To date, successful editing has been largely mediated by viral vectors that need to be painstakingly tailored to each target and have been compromised due to immunogenicity, production of antibodies that prevent repeated administration, and resistance to rare but dangerous integration events. Attention, poses a challenge to clinical translation. Clearly there remains a need for CRISPR/Cas editing via synthetic lipid nanoparticles (LNPs) to expand safe and effective applications of gene editing.

The routes of administration commonly used in the treatment of ocular diseases are topical, systemic, periocular and intravitreal. Topical administration is the optimal route due to the highest patient compliance and least invasiveness. Following topical administration, drug absorption occurs via corneal (cornea, aqueous humor, intraocular tissues) or non-corneal routes (conjunctiva, sclera, choroid/retinal pigment epithelium (RPE)). Only a small fraction (typically less than 5%) of topically administered drugs reaches intraocular tissues (Mishra GP et al., J. of Drug Delivery (2011) 2011:863734).

Systemic administration requires high dose administration due to the blood-aqueous and blood-retinal barriers. Such high doses can cause serious side effects. In addition, intravitreal administration requires frequent dosing, which may predispose to vitreous hemorrhage, retinal detachment, and endophthalmitis. Therefore, there is a clear need for improved drug delivery to the eye.

The viscosity of human blood is about 3-4mPa*s (cP), the viscosity is low, and the particles generally flow in the form of turbulent flow (turbulent flow), concentrated in the center of blood vessels; the viscosity of vitreous humor is much higher than that of human blood (viscosity see below table, about 100-300 times that of blood). The way LNP moves in the high-viscosity liquid in the eye is mainly based on the stokes force, which presents a laminar flow and is affected by rotational viscous resistance (ζT and ζR). LNP tends to move at the eyeball endothelial cells (Donati, S., Caprani , S.M., Airaghi, G., Vinciguerra, R., Bartalena, L., Testa, F., Mariotti, C., Porta, G., Simonelli, F. and Azzolini, C., 2014. Vitreous substitutes: the present and the future. BioMed research international, 2014.).

Table 1 Physicochemical properties of human eyeball vitreous humor

The retinal and corneal endothelial cells in the eye are positively charged, and the vitreous solution is mainly hyaluronic acid hydrogel (negatively charged); there is a divergent micro-electric field from the injection site of the vitreous to the periphery of the eyeball (Zhao, Min, et al. "Electrical signaling in control of ocular cell behaviors."Progress in retina and eye research 31.1(2012):65-88.).

The high-viscosity liquid in the vitreous has a different trajectory to the movement of LNP than blood, and because of its movement in the vitreous in the eye, the research experience in serum and other parts is difficult to apply. The influence of the content of cationic lipids in the LNP formula and the ratio of pH-responsive lipids (C/P value) on sub-ocular tendency is mainly realized from two aspects of competition and synergy: ① The ionization charge of LNP, with the increase of cationic lipid , the overall charge of LNP will increase, which will tend to migrate to low-charge areas; ②The adsorption of corona by hyaluronic acid: As the content of cationic lipids increases, although the ionization efficiency will be improved (positive charge), but it will also Adsorbing more hyaluronic acid on its surface not only increases the particle size of LNP, but also shields positive charges. Competing bidirectional effects lead to its movement in the vitreous body, and the research experience in serum and other parts is difficult to apply.

Therefore, there remains a need to develop new lipid nanoparticles for preferential delivery to the eye.

Contents of the invention

This application provides a method to achieve precise enrichment in different parts of the eyeball by regulating the ratio of cationic lipids content and pH-responsive lipids. By regulating the surface charge of LNP in the specific environment of the vitreous body and the interaction with hyaluronic acid, its enrichment to different eye regions is achieved.

In one aspect, the present application provides a method for delivering exogenous substances targeting specific cells in the eye, comprising administering lipid nanoparticles (LNP) to the vitreous, calculated by molar percentage, the lipid nanoparticles comprising the following components : Ionizable lipid 20%-70%, cholesterol-based lipid 30%-60%, structured lipid 25%-50%, pegylated lipid 1%-30%, wherein the ionizable lipid It includes pH-sensitive lipids and permanent cationic lipids, and the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is 1:24 to 24:1.

In some embodiments, the eye-specific cells include corneal endothelial cells or choroidal cells.

In certain embodiments, wherein the LNP is targeted to different cells of the eye by adjusting the C/P value in the LNP.

In certain embodiments, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 1:24 to 2:1.

In certain embodiments, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 8:42 to 32:18.

In certain embodiments, wherein said LNP targets choroidal cells when said C/P value is not less than 12:1.

In certain embodiments, wherein said LNP targets choroidal cells when said C/P value is not less than 24:1.

In certain embodiments, wherein said LNP targets choroidal cells when said C/P value is in the range of 12:1 to 24:1.

In some embodiments, the content of the ionizable lipid is about 40%-60% by mole percentage.

In certain embodiments, the ionizable lipid is present in an amount of no more than about 50% by mole percent.

In some embodiments, the content of the ionizable lipid is about 40%-50% by mole percentage.

In certain embodiments, the content of the ionizable lipid is about 45%-55% by mole percentage.

In some embodiments, the content of the ionizable lipid is about 50% by mole percentage.

In certain embodiments, the content of the ionizable lipid is about 40-60%, and the C/P value is in the range of 8:42 to 32:18, the LNP Targets corneal endothelial cells.

In certain embodiments, wherein said ionizable lipid is present in an amount of about 40-60% on a molar basis and said C/P ratio is not less than 12:1, said LNP targets choroidal cells.

In certain embodiments, wherein the permanent cationic lipid is selected from the group consisting of: DOTAP, DDAB, DOTMA, DC-Chol, and combinations thereof.

In certain embodiments, wherein the pH-sensitive lipid is selected from MC-3, LP-01, and combinations thereof.

In certain embodiments, wherein the structural lipid is selected from DPPC, DSPC, DOPE and combinations thereof.

In certain embodiments, wherein the PEGylated lipid is selected from DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG-PEG, ceramide PEG, and combinations thereof.

In certain embodiments, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.

In some embodiments, the exogenous substances include compounds, nucleic acids, antibodies or active fragments thereof, peptides, lipids, protein drugs, protein conjugate drugs, enzymes, oligonucleotides or ribozymes.

In certain embodiments, wherein the nucleic acid is selected from siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-related nucleic acid, single guide RNA (sgRNA) ), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single One or more of stranded DNA (ssDNA), single stranded RNA (ssRNA) and double stranded RNA (dsRNA).

In certain embodiments, wherein the nucleic acid comprises mRNA and/or sgRNA.

In certain embodiments, wherein the mRNA comprises a nucleic acid sequence encoding an enzyme.

In some embodiments, wherein the mRNA comprises a nucleic acid sequence encoding a Cas protein.

In certain embodiments, wherein the lipid nanoparticle is a nucleic acid lipoplex, wherein the molar ratio of element N to element P is 1:1 to 9:1.

In another aspect, the present application provides an LNP delivery system targeting specific cells in the eye, wherein the LNP comprises the following components: ionizable lipid 20%-70%, cholesterol-based lipid 30%-60%, Structured lipids 25%-50%, PEGylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the permanent cationic lipids and pH-responsive lipids The molar ratio of mass (C/P value) is between 1:24 and 24:1.

In certain embodiments, wherein the content of the ionizable lipid is about 50%, and the C/P value is in the range of 8:42 to 32:18, the LNP targeting Corneal endothelial cells.

In certain embodiments, wherein the ionizable lipid content is about 50% on a molar percentage basis and the C/P ratio is not less than 24:1, the LNP targets choroidal cells.

In certain embodiments, when the LNP is selected from #2, #3, #4 and #5 in Table 1, the LNP targets corneal endothelial cells.

In certain embodiments, when the LNP is selected from #6 in Table 1, the LNP targets choroidal cells.

In certain embodiments, the LNP delivery system is configured as a liquid for intravitreal administration.

In certain embodiments, the administering comprises intravitreal injection.

In another aspect, the present application provides an LNP delivery system for targeted delivery of specific cells in the eye, wherein the LNP comprises the following components: ionizable lipid 20%-70%, cholesterol-based lipid 30%-60%, structured lipids 25%-50%, PEGylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, and the permanent cationic lipids The molar ratio (C/P value) of lipids and pH-responsive lipids was between 1:24 and 24:1.

In another aspect, the present application provides the use of the LNP delivery system described in the present application in the preparation of a medicament for preventing and/or treating eye diseases or disorders.

In another aspect, the present application provides a pharmaceutical composition for treating ocular diseases or disorders, comprising lipid nanoparticles comprising a therapeutic agent, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70%, cholesterol-based lipids 30%-60%, structured lipids 25%-50%, pegylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is between 1:24 and 24:1.

In another aspect, the present application provides a pharmaceutical composition for treating corneal endothelial cell diseases or disorders, comprising lipid nanoparticles comprising a therapeutic agent, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70% lipids, 30%-60% cholesterol-based lipids, 25%-50% structured lipids, 1%-30% pegylated lipids, wherein the ionizable lipids include pH-sensitive lipids quality and permanent cationic lipids, the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is between 1:24 and 2:1.

In another aspect, the present application provides a pharmaceutical composition for treating choroidal cell diseases or disorders, comprising lipid nanoparticles comprising a therapeutic agent, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70%, cholesterol-based lipids 30%-60%, structured lipids 25%-50%, pegylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is not less than 12:1.

In certain embodiments, wherein the therapeutic agent is selected from the group consisting of:

i) a. a Cas protein or a polynucleotide encoding a Cas protein; and b. a guide RNA that hybridizes to a target site of a gene, wherein the gene encodes a protein that contributes to an eye disease or disorder;

ii) antisense oligonucleotides; and

iii) mRNA encoding a protein whose deficiency causes said eye disease disease or condition.

In another aspect, the present application provides a method of treating an ocular disease or condition in a subject, the method comprising administering to the subject's eye a lipid nanoparticle comprising a therapeutic agent, the lipid nanoparticle comprising The following components: 20%-70% ionizable lipids, 30%-60% cholesterol-based lipids, 25%-50% structured lipids, 1%-30% pegylated lipids, wherein the The ionized lipids include pH-sensitive lipids and permanent cationic lipids, and the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is between 1:24 and 24:1.

ii) antisense oligonucleotides; and

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows a TEM electron micrograph of the LNP described in this application.

Figure 2 shows the eye distribution of Ai9 mice.

Figure 3 shows that #2 is specifically enriched in eyeball corneal endothelial cells.

Figure 4 shows the transfection effect of LNP eyeball endothelial cells with different C/P values.

Figure 5 shows the transfection effect of LNP eyeball choroid with different C/P values of the present application.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Not intending to be limited by any theory, the following examples are only for explaining the lipid nanoparticles, preparation method and application of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1

Example 1.1 Preparation of Lipid Nanoparticles

By fully mixing the organic phase and the aqueous phase, LNP nanoparticles are prepared to achieve effective embedding of gene editing tools or other nucleic acid drugs.

Wherein, the organic phase: contains at least one ionizable lipid, at least one supporting lipid, at least one amphiphilic block copolymer and cholesterol, and is dissolved by an organic solvent that is miscible with water. The organic solvent is preferably selected from ethanol, acetonitrile, acetone and the like.

Aqueous phase: an aqueous solution of gene editing tools, wherein the content of nucleic acid substances (such as mRNA) is 0.5-50% (w/v), and the pH is 3.0-7.0. The aqueous salt solution is selected from: citrate buffer, phosphate buffer, Tris-HCl buffer system.

The mixing of organic phase and aqueous phase can be achieved by microfluidic and impinging flow reactors. The embedding efficiency of gene editing tool RNA can be optimized by adjusting the N/P ratio (molar ratio) of the system, and the N/P ratio is 1:1 to 9:1. The N/P ratio here specifically refers to the molar ratio of element N to element P in the LNP/mRNA system.

The optimization of embodiment 1.2 embedding rate

LNP was prepared by the method of Example 1.1, and LNP was prepared by regulating the molar ratio of ionizable lipid and cationic lipid, and its embedding rate for more than 2000bp mRNA was characterized. (RNA is derived from RNA extracted from yeast, purchased from McLean, Cat. No. R822593)

Characterization method of embedding rate: The embedding rate of prepared LNP is determined by Quant-iTTM RNA Reagent and Kit assay.

Specific detection method:

Solution configuration: Dilute an appropriate amount of 20×TE buffer solution to 1× with ultrapure water, which is enough for the day’s experiment. Dilute the concentrated dye solution with 1× TE (large range 25-1000ng/ml RNA) at a ratio of 1:200, (small range 1-50ng/ml RNA) at a ratio of 1:2000. Wrap it in gold foil or store it in a dark place away from light. Dilute TritonX-100 to 5% concentration with 1×TE buffer for use.

Sample processing: set ultrapure water with RNA concentration of 0 as the blank background, set TE buffer instead of LNP plus 5% TritonX-100 as the free RNA test sample, and set LNP plus 5% TritonX-100 as the total RNA test sample . Take the LNP sample to be determined and add an equal volume of 5% TritonX-100, 1×TE solution to the blank, and incubate at 50-60°C for 5-10 minutes.

The incubated LNP samples were diluted to 10-400 times with 1×TE buffer solution (appropriately adjusted according to the RNA concentration of the sample group). After dilution, 100 μl was added to a 96-well plate, and then 100 μl of the diluted concentrated dye solution was added in the dark, and the fluorescence absorbance was measured within 5 minutes. The measurement conditions are an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Calculation: Embedding rate=(absorbance of total RNA-absorbance of free RNA)/absorbance of total RNA×100%

The results are shown in Table 2. The SORT formula (#15&#16) is based on pH-responsive lipids, adding a certain proportion of cationic lipids so that the overall ionizable lipids content reaches more than 60% (higher charge). It affects the embedding rate of large fragments of mRNA. However, the formulas of the present invention (#1-#14) control the molar ratio of total ionizable lipids within 60%, ensuring a higher embedding rate of large fragment mRNA (above 90%).

Table 2 Preparation of LNP and determination of embedding rate

Example 2

The LNP constructed in Example 1.2 was placed in serum and vitreous simulated fluid respectively, and its particle size and zeta potential were detected. The results showed that when C/P=8/42, the LNP in the vitreous humor showed negative charges, and the particle size did not change much, indicating that it was easier to migrate to the positively charged corneal endothelial cells. And when C/P increases to 2/1, due to the increase in the adsorption of hyaluronic acid, its particle size increases (about 20nm larger), indicating that it is easier to be cleared by the retina through the choroid using the self-clearing effect in the eyeball, while enriched in the choroid.

Table 3 The delivery system of the present invention presents different potentials and particle sizes in serum and vitreous simulated fluid

Example 3

Utilize LNP formula number #1 (pure MC3-LNP) of table 2, #2, #14, intravitreal injection step:

Eight-week-old mice were injected intraperitoneally with a mixture of tetamine, zolazepam (1:1, 2.25 mg/kg body weight) and xylazine hydrochloride (0.7 mg/kg body weight) through anesthesia. Under an operating microscope, inject 2ul LNP (200ng/ul Cre mRNA-encapsulated lipid nanoparticles) (Leica Microsystems Ltd.) (Cre mRNA sequence is GenBank: AAL31698.1) into the vitreous using a Nanofil syringe with a 33G blunt needle. .

As shown in Figure 2, the results showed that the LNP#2 of the present application had a significant transfection effect in the eyeball; while common MC3LNP (#1) and #14 did not appear to be significantly enriched in corneal endothelial cells.

Example 4

Specific ZO-1 staining of mouse eye endothelial cells: After the mice were dissected, fresh eyeball tissues were washed with PBS, fixed in 4% paraformaldehyde for 48 hours, dehydrated in 30% sucrose overnight, OCT (Sakura Finetek ) embedded tissue, cut into about 8 μm thickness for immunofluorescent staining. Immunofluorescence antibody: soak slices in PBS for 5 minutes, repeat three times, use 3% BSA to block non-specific antigens, incubate at room temperature for 30 minutes, and then use ZO-1 antibody (Proteintech, 21773-1-AP, 1:1000) for 4 overnight incubations , wash the primary antibody three times with TBST, 5 min each time, incubate with FITC-labeled secondary antibody for 2 hours at room temperature, stain the nuclei with DAPI for 10 min, mount the slide, observe and take pictures with a fluorescent microscope (Olympus VS120 Automated Slide Scanner).

Since ZO-1 is only specifically expressed in eye endothelial cells in the eye, the specific enrichment of LNP can be judged by observing the co-localization of cre-mRNA (red) and ZO-1 cells (green) .

By staining endothelial-specific cells ZO-1, it was found that #2 transfected were indeed corneal endothelial cells. As shown in Figure 3, green (ZO-1) co-localizes with red cre-mRNA, indicating that #2 can efficiently transfect corneal endothelial cells, proving its efficient corneal endothelial enrichment.

As shown in Fig. 4, LNP (#2, #3, #4 and #5) can be enriched in corneal endothelial cells at C/P values between 1/24 and 2/1, while beyond this range as C/P If the P value is 24/1 (#6), it cannot be effectively enriched in the corneal endothelium, and if it is lower than this range (#1), it cannot be effectively transfected.

Example 5

As shown in Figure 4, #6 cannot be enriched into the corneal endothelial cells, but due to its high concentration of positively charged molecules cationic lipids, it can wrap hyaluronic acid, resulting in a larger particle size, and then hitchhike on the automatic clearance mechanism in the eye, From the retina into the choroid, and then achieve the enrichment of the choroid. As shown in Figure 5, on the right side of the white dotted line, the part indicated by the arrow is the choroid. When the C/P value is greater than or equal to 12, the choroid is obviously transfected, but the retina is not.

Claims

A method for delivering foreign substances targeting specific cells in the eye, comprising administering lipid nanoparticles (LNP) to the vitreous, and the lipid nanoparticles comprise the following components in terms of molar percentage: ionizable lipid 20% -70%, cholesterol-based lipids 30%-60%, structured lipids 25%-50%, PEGylated lipids 1%-30%, wherein the ionizable lipids include pH sensitive lipids and permanent The cationic lipid, the molar ratio (C/P value) of the permanent cationic lipid and the pH responsive lipid is in the range of 1:24 to 24:1.
The method according to claim 1, wherein said ocular specific cells comprise corneal endothelial cells or choroidal cells.
The method according to any one of claims 1-2, wherein the LNP is targeted to different cells of the eye by adjusting the C/P value in the LNP.
The method according to any one of claims 1-3, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 1:24 to 2:1.
The method according to any one of claims 1-4, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 8:42 to 32:18.
The method according to any one of claims 1-5, wherein said LNP targets choroidal cells when said C/P value is not less than 12:1.
The method according to any one of claims 1-6, wherein said LNP targets choroidal cells when said C/P value is not less than 24:1.
The method according to any one of claims 1-7, wherein said LNP targets choroidal cells when said C/P value is in the range of 12:1 to 24:1.
The method according to any one of claims 1-8, wherein the content of the ionizable lipid is about 40%-60% by mole percentage.
The method of claim 9, wherein said ionizable lipid is present in an amount of no more than about 50% by mole percent.
The method according to any one of claims 1-10, wherein the content of the ionizable lipid is about 40%-50% by mole percentage.
The method according to any one of claims 1-10, wherein the content of the ionizable lipid is about 45%-55% by mole percentage.
The method according to any one of claims 1-12, wherein the content of the ionizable lipid is about 50% by mole percentage.
The method according to any one of claims 1-13, calculated by molar percentage, wherein the content of the ionizable lipid is about 40-60%, and the C/P value is between 8:42 and 32: In the range of 18, the LNP targets corneal endothelial cells cell.
The method according to any one of claims 1-13, calculated by molar percentage, wherein the content of the ionizable lipid is about 40-60%, and when the C/P value is not less than 12:1, The LNP targets choroidal cells.
The method according to any one of claims 1-15, wherein the permanent cationic lipid is selected from the group consisting of DOTAP, DDAB, DOTMA, DC-Chol and combinations thereof.
The method according to any one of claims 1-16, wherein the pH sensitive lipid is selected from MC3, LPO1 and combinations thereof.
The method according to any one of claims 1-17, wherein the structural lipid is selected from DPPC, DSPC, DOPE and combinations thereof.
The method according to any one of claims 1-18, wherein the pegylated lipid is selected from the group consisting of DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG-PEG, Ceramide PEG and combinations thereof.
The method of any one of claims 1-19, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.
The method according to any one of claims 1-20, wherein the exogenous substance comprises nucleic acid and/or protein.
The method according to claim 21, wherein the nucleic acid is selected from siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR) related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA) , one or more of single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
The method of claim 22, wherein the nucleic acid comprises mRNA and/or sgRNA.
The method according to claim 23, wherein said mRNA comprises a nucleic acid sequence encoding an enzyme.
The method according to claim 24, wherein the mRNA comprises a nucleic acid sequence encoding a Cas protein.
The method according to any one of claims 1-25, wherein the lipid nanoparticle is a nucleic acid lipoplex, wherein the molar ratio of element N to element P is 1:1 to 9:1.
An LNP delivery system targeting specific cells in the eye, wherein the LNP comprises the following components: ionizable lipid 20%-70%, cholesterol-based lipid 30%-60%, structural lipid 25%-50%, PEGylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio of permanent cationic lipids to pH-responsive lipids (C/P value ) at 1:24 to 24:1.
The LNP delivery system according to claim 27, wherein said ocular specific cells comprise corneal endothelial cells or vascular Choroid cells.
The LNP delivery system according to any one of claims 27-28, wherein the LNP is targeted to different cells of the eye by adjusting the C/P value in the LNP.
The LNP delivery system according to any one of claims 27-29, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 1:24 to 2:1.
The LNP delivery system according to any one of claims 27-30, wherein said LNP targets corneal endothelial cells when said C/P value is in the range of 8:42 to 32:18.
The LNP delivery system according to any one of claims 27-31, wherein said LNP targets choroidal cells when said C/P value is not less than 12:1.
The LNP delivery system according to any one of claims 27-32, wherein said LNP targets choroidal cells when said C/P value is not less than 24:1.
The LNP delivery system according to any one of claims 27-32, wherein said LNP targets choroidal cells when said C/P value is in the range of 12:1 to 24:1.
The LNP delivery system according to any one of claims 27-34, wherein said ionizable lipid is present in an amount of about 40%-60% by molar percentage.
The LNP delivery system of any one of claims 27-35, wherein the ionizable lipid is present in an amount of no more than about 50%, calculated as a molar percentage.
The LNP delivery system according to any one of claims 27-36, wherein said ionizable lipid is present in an amount of about 40%-50% by molar percentage.
The LNP delivery system according to any one of claims 27-37, wherein said ionizable lipid is present in an amount of about 45%-55%, calculated as a molar percentage.
The LNP delivery system according to any one of claims 27-38, wherein said ionizable lipid is present in an amount of about 50%, calculated as a molar percentage.
The LNP delivery system according to any one of claims 27-39, calculated as a molar percentage, wherein the content of the ionizable lipid is about 40-60%, and the C/P value is between 8:42 and In the range of 32:18, the LNP targets corneal endothelial cells.
The LNP delivery system according to any one of claims 27-40, calculated as a molar percentage, wherein the content of the ionizable lipid is about 50%, and the C/P value is between 8:42 and 32: 18, the LNP targets corneal endothelial cells.
The LNP delivery system according to any one of claims 27-41, calculated as a molar percentage, wherein said electrically When the lipid content is about 40-60%, and the C/P value is not less than 12:1, the LNP targets the choroidal cells.
The LNP delivery system according to any one of claims 27-42, calculated as a molar percentage, wherein said ionizable lipid content is about 50%, and said C/P value is not less than 24:1, so The LNPs target choroidal cells.
The LNP delivery system according to any one of claims 27-43, wherein said permanent cationic lipid is selected from the group consisting of: DOTAP, DDAB, DOTMA, DC-Chol, and combinations thereof.
The LNP delivery system according to any one of claims 27-44, wherein the pH sensitive lipid is selected from MC-3, LP-01 and combinations thereof.
The LNP delivery system according to any one of claims 27-45, wherein the structural lipid is selected from the group consisting of DPPC, DSPC, DOPE and combinations thereof.
The LNP delivery system according to any one of claims 27-46, wherein the pegylated lipid is selected from DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG- PEG, ceramide PEG, and combinations thereof.
The LNP delivery system of any one of claims 27-47, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.
The LNP delivery system according to any one of claims 27-48, when said LNP is selected from #2, #3, #4 and #5 in Table 1, said LNP targets corneal endothelial cells.
The LNP delivery system according to any one of claims 27-49, when said LNP is selected from #6 in Table 1, said LNP targets choroidal cells.
The LNP delivery system of any one of claims 27-50 configured as a liquid for intravitreal administration.
The LNP delivery system according to any one of claims 27-51 , said administering comprising intravitreal injection.
LNP delivery system for targeted delivery of specific cells in the eye, wherein the LNP comprises the following components: ionizable lipid 20%-70%, cholesterol-based lipid 30%-60%, structured lipid 25 %-50%, PEGylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio of permanent cationic lipids and pH-responsive lipids (C/P value) in the range of 1:24 to 24:1.
Use of the LNP delivery system according to any one of claims 27-52 in the preparation of a medicament for the prevention and/or treatment of eye diseases or disorders.
A pharmaceutical composition for the treatment of ocular diseases or conditions, comprising lipid nanoparticles comprising a therapeutic agent, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70%, based on cholesterol 30%-60% of lipids, 25%-50% of structured lipids, 1%-30% of pegylated lipids, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is between 1:24 and 24:1.
A pharmaceutical composition for treating corneal endothelial cell diseases or disorders, comprising lipid nanoparticles containing therapeutic agents, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70%, based on Cholesterol lipid cholesterol 30%-60%, structured lipid 25%-50%, pegylated lipid 1%-30%, wherein the ionizable lipids include pH sensitive lipids and permanent cationic lipids, The molar ratio (C/P value) of the permanent cationic lipid to the pH responsive lipid is 1:24 to 2:1.
A pharmaceutical composition for treating a choroidal cell disease or disorder, comprising lipid nanoparticles comprising a therapeutic agent, wherein the lipid nanoparticles comprise the following components: ionizable lipid 20%-70%, based on cholesterol Lipid cholesterol 30%-60%, structured lipid 25%-50%, pegylated lipid 1%-30%, wherein the ionizable lipid includes pH sensitive lipid and permanent cationic lipid, so The molar ratio (C/P value) of the permanent cationic lipid and the pH responsive lipid is not less than 12:1.
The composition of claim 57, wherein the therapeutic agent is selected from the group consisting of:

i) a. a Cas protein or a polynucleotide encoding a Cas protein; and b. a guide RNA that hybridizes to a target site of a gene, wherein the gene encodes a protein that contributes to an eye disease or disorder;

ii) antisense oligonucleotides; and

iii) mRNA encoding a protein whose deficiency causes said eye disease disease or condition.
A method of treating an ocular disease or condition in a subject, the method comprising administering to the subject's eye a lipid nanoparticle comprising a therapeutic agent, the lipid nanoparticle comprising the following component: ionizable lipid 20%-70%, cholesterol-based lipids 30%-60%, structured lipids 25%-50%, pegylated lipids 1%-30%, wherein the ionizable lipids include pH-sensitive lipids and permanent cationic lipids, the molar ratio (C/P value) of the permanent cationic lipids and pH-responsive lipids is between 1:24 and 24:1.
The method of claim 59, wherein the therapeutic agent is selected from the group consisting of:

i) a. a Cas protein or a polynucleotide encoding a Cas protein; and b. a guide RNA that hybridizes to a target site of a gene, wherein the gene encodes a protein that contributes to an eye disease or disorder;

ii) antisense oligonucleotides; and

iii) mRNA encoding a protein whose deficiency causes said eye disease disease or condition.