CN116370437A - Nucleic acid lipid nanoparticle compositions comprising cholesterol succinic acid monoesters and uses thereof - Google Patents

Nucleic acid lipid nanoparticle compositions comprising cholesterol succinic acid monoesters and uses thereof Download PDF

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CN116370437A
CN116370437A CN202310602246.5A CN202310602246A CN116370437A CN 116370437 A CN116370437 A CN 116370437A CN 202310602246 A CN202310602246 A CN 202310602246A CN 116370437 A CN116370437 A CN 116370437A
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lipid
nucleic acid
peg
nanoparticle composition
cholesterol
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潘兴华
王成
贾季雨
谢若天
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Beijing Innovic Pharmaceutical Technology Co ltd
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Abstract

The present invention relates to nucleic acid lipid nanoparticle compositions comprising cholesterol succinic acid monoesters and uses thereof. A nucleic acid lipid nanoparticle composition comprising an ionizable lipid, a helper lipid, a structural lipid, a polymer-bound lipid, and a nucleic acid, the structural lipid comprising a cholesterol succinic monoester. Compared with cholesterol, cholesterol succinic acid monoester can significantly improve the nucleic acid delivery efficiency of lipid nanoparticles, thereby improving the therapeutic effect of nucleic acid molecules.

Description

Nucleic acid lipid nanoparticle compositions comprising cholesterol succinic acid monoesters and uses thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a nucleic acid lipid nanoparticle composition containing cholesterol succinic acid monoester, a pharmaceutical preparation and application thereof.
Background
Cholesterol succinic acid monoester is a common acidic cholesterol ester, which has sensitivity to pH, and can be widely used in various pharmaceutical formulations, such as liposome (liposome), etc., but it has not been reported to be used in Lipid Nanoparticles (LNP) at present.
Liposomes, although similar in composition to LNP, are mostly small molecules, and LNP mainly encapsulates nucleic acid drugs, which have negative charge, so in order to bind the nucleic acid drugs to LNP, cationic lipids (ionizable lipids) are needed as core excipients. In addition, liposomes and LNPs are structurally distinct due to differences in encapsulation materials. Classical components of LNP include ionizable lipids, helper lipids, structural lipids, and polymer-bound lipids, among others, with cholesterol being a common structural lipid.
The present invention has been found to have an effect of enhancing LNP transfection compared to cholesterol by including cholesterol succinate monoester in structural lipid.
Disclosure of Invention
The present invention provides a nucleic acid lipid nanoparticle composition and a pharmaceutical formulation comprising a cholesterol succinate monoester that is capable of significantly improving the nucleic acid delivery efficiency of LNP compared to cholesterol, thereby improving the therapeutic effect of nucleic acid molecules.
In a first aspect, the invention provides a nucleic acid lipid nanoparticle composition comprising a lipid carrier and a nucleic acid, the lipid carrier comprising an ionizable lipid, a helper lipid, a structural lipid, and a polymer-bound lipid, the structural lipid comprising a cholesterol succinate monoester.
In some embodiments, the ionizable lipid is selected from the group consisting of 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester DLin-MC3-DMA (abbreviated as MC 3), ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) ALC-0315, 1, 2-diiodooxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleoxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolan DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1' - (2- ((2-hydroxydodecyl) amino) ethyl) piperazine-1-yl) dicarboxamide dol-2-dodecane-2-N, N-dicarboxamide, and at least one of 1,2- [ 2, 3] -dioxan-2-dimethyl-N-2- [ 2-dicarboxamide ] dimethyl-N-3-chloromethane, N-1- [ 2, 3] -dioxan-2-dimethyl-N-2- [ 2-chloromethane.
In some embodiments, the ionizable lipid is at least one selected from the group consisting of 4- (N, N-dimethylamino) butanoic acid (diimine) methyl ester DLin-MC3-DMA and ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) ALC-0315.
In some embodiments, the helper lipid is at least one member selected from the group consisting of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dioleoyl phosphatidylserine DOPS, distearoyl phosphatidylserine DSPS, 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine DSPE, dipalmitoyl phosphatidylserine DPPS, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dipalmitoyl phosphatidylglycerol DPPG, oleoyl phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE, 1, 2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine DPPE, 1, 2-dimyristoyl-sn-glycero-3-phosphate ethanolamine DMPE, distearoyl phosphatidylethanolamine DSPE, and 1-stearoyl-2-soyl phosphatidylethanolamine DPPE.
In some embodiments, the helper lipid is at least one selected from the group consisting of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine DSPC.
In some embodiments, the structural lipid further comprises an additional structural lipid selected from at least one of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, fecal sterols, and corticosteroids.
In some embodiments, the additional structural lipid is cholesterol.
In some embodiments, the molar ratio of cholesterol succinic monoester to other structural lipids is (10-100): 90-0; illustratively, the molar ratio may be 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 100:0.
In some embodiments, the molar ratio of cholesterol succinic monoester to cholesterol is (10-100): (90-0); illustratively, the molar ratio may be 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 100:0.
In some embodiments, the polymer-bound lipid is selected from the group consisting of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), DMG-PEG 2000-mannose, cholesterol-PEG 2000, 1, 2-dimyristoyl-sn-glyceromoxy-polyethylene glycol PEG-DMG, dimyristoyl-polyethylene glycol PEG-C-DMG, polyethylene glycol-dimyristoyl-glycerol PEG-C14, PEG-1, 2-dimyristoyloxy propyl-3-amine PEG-C-DMA, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE, pegylated phosphatidylethanolamine PEG-PE, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol, tween-20, tween-80, 1, 2-dipalmitoyl-sn-methoxypolyethylene glycol PEG-DPG, 4-O- (2 ',3' -dimyristoyloxy-propyl-1-ethoxy-PEG-2 ' -di (tetradecyloxy) propyl-2-methoxy-ethylene succinate, and poly (PEG-ethoxy) propyl-O- (omega-methoxy) polyethylene glycol-A-PEG-poly (PEG-ethoxy) ethyl succinate At least one of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride PEG-c-DOMG and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propylcarbamate)) GalNAc-PEG-DSG.
In some embodiments, the polymer-bound lipid is at least one selected from the group consisting of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), 1, 2-dimyristoyl-sn-glycerinomethoxy-polyethylene glycol PEG-DMG, and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE.
In some embodiments, the nucleic acid is at least one selected from the group consisting of DNA, RNA, a complex comprising DNA or RNA (e.g., a complex of DNA and RNA, a complex of DNA and polypeptide/protein, a complex of RNA and polypeptide/protein), modified DNA, modified RNA, and a modified complex comprising DNA or RNA.
In some embodiments, the nucleic acid is RNA.
In some embodiments, the RNA is selected from mRNA, siRNA, dsRNA, rRNA, circRNA, saRNA, tRNA, snRNA or shRNA, preferably mRNA.
In some embodiments, the molar ratio of the ionizable lipid, the helper lipid, the structural lipid, and the polymer-bound lipid is (20-75): 2-25): 15-55): 0-15; illustratively, the molar ratio of ionizable lipids, helper lipids, structural lipids, and polymer-bound lipids may be 45:10:42:3, 30:25:30:10, 46:15:40:3, 50:10:38.5:1.5, 50:10:37:3, 50:9:38:3, 60:5:34.5:0.5, 75:5:19.5:0.5, 65:5:29.5:0.5, 55:8:36.5:0.5, 50:8:41.5:0.5, 50:10:39.5:0.5, 50:9.5:40:0.5, etc.
In some embodiments, the mass ratio of the nucleic acid to the lipid carrier (ionizable lipid, helper lipid, structural lipid, and polymer-bound lipid) is 1 (3-40). Illustratively, the mass ratio may be 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.
In a second aspect, the invention provides a pharmaceutical formulation comprising the nucleic acid lipid nanoparticle composition described above, and a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutical formulation may have a particle size of 30-500 nm, illustratively, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 200nm, 250nm, 300nm, 350nm, 500nm, etc.
In some embodiments, the encapsulation efficiency of the nucleic acid in the pharmaceutical formulation is greater than 50%. Illustratively, the encapsulation efficiency may be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.
In a third aspect, the present invention provides the use of a nucleic acid lipid nanoparticle composition as described above or a pharmaceutical formulation as described above in the preparation of a nucleic acid drug or genetic vaccine.
The present invention also provides a method for delivering a nucleic acid drug or genetic vaccine in vivo, comprising administering to a subject in need thereof the nucleic acid lipid nanoparticle composition described above or the pharmaceutical formulation described above.
The invention also provides the use of cholesterol succinic monoester for increasing the nucleic acid delivery efficiency of lipid nanoparticles.
The invention also provides the use of the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above for the manufacture of a medicament for delivering nucleic acid through the eye.
The present invention also provides a method of treating or preventing an ocular disease selected from the group consisting of diabetic retinopathy, macular edema, age-related macular degeneration, and retinitis pigmentosa by delivering nucleic acids through the eye, comprising administering to a subject in need thereof the above nucleic acid lipid nanoparticle composition or the above pharmaceutical formulation.
The present invention also provides a method of treating or preventing a liver disease selected from hepatitis, fatty liver, liver fibrosis, cirrhosis, alcoholic/non-alcoholic liver injury or hepatocellular carcinoma, comprising administering the above nucleic acid lipid nanoparticle composition or the above pharmaceutical formulation to a subject in need thereof.
In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered by one of the following routes of administration: oral, ocular, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered, for example, via an enteral or parenteral route of administration. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered, for example, via an ocular route. In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject at a dose of about 0.001mg/kg to about 10 mg/kg.
The invention has the following beneficial effects:
1) Lipid nanoparticles comprising cholesterol succinic acid monoesters in various molar ratios can significantly improve the delivery efficiency of nucleic acids;
2) The effect of cholesterol succinic acid monoester on improving the delivery efficiency of nucleic acid is not limited to specific ionizable lipids, such as MC3 and ALC-0315 (the ionizable lipids most commonly used at present and approved by FDA certification) each can achieve the effect of the present invention;
3) Cholesterol succinate can increase the efficiency of nucleic acid delivery in a variety of cell lines;
4) Cholesterol succinic acid monoester can also improve the delivery efficiency of nucleic acid in vivo, and in vivo experiments in mice confirm the feasibility of in vivo application.
Drawings
Fig. 1: chemiluminescence imaging diagram of the left eye of the mouse.
Fig. 2: chemiluminescence imaging diagram of the right eye of the mouse.
Detailed Description
For easier understanding of the present invention, certain technical and scientific terms are defined below in detail. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present specification, cholesterol succinic monoester has the following structure:
Figure SMS_1
。
in the present specification, the numerical range indicated by "numerical values a to B" means a range including the end point value A, B. Where lower and upper limits of a range of values are disclosed, any numerical value or any subrange falling within the range is indicated as being specifically disclosed. In particular, each numerical range (e.g., in the form of "about a to b", or equivalently "about a-b") of the parameters disclosed herein is to be understood as encompassing each numerical value and subrange therein.
The terms "comprising," "including," "having," or "containing," or any other variation thereof, are intended to cover a non-exclusive or open-ended inclusion. For example, a composition, method, or apparatus that comprises a list of elements is not necessarily limited to only those elements explicitly listed, but may also include other elements not explicitly listed or inherent to such composition, method, or apparatus.
In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
The term "pharmaceutically acceptable carrier" refers to an adjuvant that is administered with the above nucleic acid lipid nanoparticle composition or the above pharmaceutical formulation and which is suitable for contacting the tissues of humans and/or other animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable carriers that can be used in the present invention include, but are not limited to: a) A diluent; b) A lubricant; c) An adhesive; d) A disintegrant; e) Absorbents, colorants, flavors and/or sweeteners; f) Emulsifying or dispersing agents; and/or g) substances that enhance the absorption of the compound, etc.
In order to make the objects and technical solutions of the present invention more apparent, embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention.
The reagents or apparatus used in the examples are all conventional products commercially available. Those not specifying the specific conditions were carried out according to the conventional conditions or the conditions recommended by the manufacturer. The term "room temperature" as used herein refers to 20 ℃ ± 5 ℃. As used herein, the term "about" when used in reference to a particular value or range of values is intended to encompass the value or range of values as well as ranges of errors that are acceptable to those skilled in the art of the value or range of values, such as, for example, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Example 1: preparation and characterization of nucleic acid lipid nanoparticles
1. Preparation of Lipid Nanoparticles (LNP)
The preparation of lipid nanoparticles first involved mixing and dissolving ionizable lipid MC3, helper lipid DSPC, polymer-bound lipid DMG-PEG2000 and structural lipids (cholesterol, cholesterol succinate monoester) in absolute ethanol at the molar ratios shown in table 1 to give a total lipid (i.e. lipid carrier) phase concentration of 5 mg/mL. The nucleic acid Luc-eGFP mRNA was dissolved in 50mM citrate buffer pH4.0 and prepared by rapid mixing with an ethanol solution of lipid. The mass ratio of mRNA to total lipid was 1:20.
LNP was prepared using a Micanna prescription screening chip with a total flow rate of 12ml/min and a lipid phase to aqueous phase flow rate ratio of 1:3. the collected lipid nanoparticles were dialyzed overnight with PBS at 4 ℃ to remove ethanol and acidic buffer salts. The volume of the dialyzate is more than 400 times of the volume of the sample. The dialyzed sample was filtered using a 0.2 μm filter. In addition to using microfluidic chips, lipid nanoparticles can be prepared by other means, such as injection mixing. After diafiltration, the sample was concentrated to 0.7 ug (mRNA)/ul using a 10kDa cellulose ultrafiltration membrane. The centrifugation parameters were 2000g to 3000g. The centrifugation temperature was 4 ℃. The ratio of nitrogen to phosphorus in the lipid nanoparticle was 6.87.
2. Particle size measurement: the particle size, polydispersity index (PDI) of the lipid nanoparticles prepared were measured using a malvern particle sizer.
3. mRNA encapsulation efficiency assay:
for mRNA-encapsulated lipid nanoparticles, quant-iT can be used TM RiboGreen ® A method for detecting RNA kit. The specific operation is as follows: the samples were diluted 10-20 fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). An equal volume of demulsifier (2% Triton X-100) was added to the sample to be tested and then diluted 10-20 times. RiboGreen is added ® Reagents were diluted 100-fold in TE buffer and 100 μl was added to 96-well plates. 100 microliters of the sample to be tested was added to the corresponding 96-well plate. And preparing a standard curve by using the mRNA standard substance. Fluorescence values at 520nm excited with 480nm excitation light were read in a microplate reader. The concentration of the encapsulated and free mRNA was calculated by standard curve. Encapsulation efficiency was calculated by the ratio of the concentration of the encapsulated mRNA to the concentration of the total mRNA.
The composition and characterization data of the lipid nanoparticles are shown in table 1.
TABLE 1
Figure SMS_2
Example 2: transfection effect of nucleic acid lipid nanoparticles at cellular level
1. MC 3-based nucleic acid lipid nanoparticles and cell transfection efficiency
The ionizable lipid MC3, helper lipid DSPC, polymer-conjugated lipid DMG-PEG2000, and structural lipids (cholesterol, cholesterol succinate monoester) were mixed in the proportions shown in table 2 to prepare an ethanol solution of 8 mg/mL. Luc-eGFP mRNA was dissolved in 50mM citric acid solution ph 4.0. The ethanol phase and the aqueous phase were rapidly mixed in a 1:3 ratio while ensuring a mass ratio of total lipid to mRNA of 20:1. After mixing, the mixture was allowed to stand for 1 hour, and then diluted with 30-fold volume of PBS, thereby preparing a nucleic acid lipid nanoparticle solution.
ARPE-19 (human retinal pigment epithelial cells), huh7 (human hepatoma cells) and HepG2 (human hepatoma cells) were cultured in DMEM+10% FBS medium. Cells were plated in 24 well plates, 1E+5 viable cells per well, 1 ml in volume, prior to transfection. The prepared nucleic acid lipid nanoparticle was added in an amount of 0.33 ug mRNA/1 E+5 cells at 37℃with 5% CO 2 Luciferase expression was detected by luciferase substrate after 18-24 hours incubation under conditions.
TABLE 2
Figure SMS_3
As can be seen from table 2, lipid nanoparticles comprising cholesterol succinic acid monoester in structural lipid can significantly improve the delivery efficiency of nucleic acid in ARPE-19, hepG2 and Huh-7 cells, and have good potential for retinal epithelial cell transfection, liver targeting and high transfection in the liver, compared to lipid nanoparticles using cholesterol alone as structural lipid.
2. Nucleic acid lipid nanoparticle based on ALC-0315 and cell transfection efficiency
The ionizable lipid ALC-0315, helper lipid DSPC, polymer-conjugated lipid DMG-PEG2000, and structural lipid (cholesterol, cholesterol succinic monoester) were mixed in the proportions shown in Table 3 to prepare an ethanol solution of 6 mg/mL. Luc-eGFP mRNA was dissolved in 50mM citric acid solution ph 4.0. The ethanol phase and the aqueous phase were rapidly mixed in a 1:3 ratio while ensuring a mass ratio of total lipid to mRNA of 20:1. After mixing, the mixture was allowed to stand for 1 hour, and then diluted with 30-fold volume of PBS, thereby preparing a nucleic acid lipid nanoparticle solution.
Jurkat cells (human acute T-lymphoblastic leukemia cells) were cultured in RPMI-1640+10% FBS medium. Cells were plated in 24 well plates prior to transfection, with 1E+5 viable cells per well, 1 ml in volume. The prepared nucleic acid lipid nanoparticle was added in an amount of 0.25ug mRNA/1 E+5 cells at 37℃with 5% CO 2 Luciferase expression was detected by luciferase substrate after 18-24 hours incubation under conditions.
TABLE 3 Table 3
Figure SMS_4
As can be seen from table 3, the lipid nanoparticle comprising cholesterol succinic acid monoester in the structural lipid can significantly improve the delivery efficiency of nucleic acid in Jurkat cells, compared to the lipid nanoparticle using cholesterol alone as the structural lipid.
Example 3: animal in vivo delivery experiments
Mouse animal experiment:
the nucleic acid delivery efficiency of lipid nanoparticles was characterized by observing the manner of fluorescent expression in mice. Female C57/BL6 mice weighing 18-22g were taken and randomly grouped, 2 per group. After the adaptive feeding, lipid nanoparticles encapsulating Luc-eGFP mRNA were injected into mice via the vitreous cavity at a dose of 1. Mu.l per eye (0.7. Mu.g mRNA/. Mu.l). To observe luciferase expression, luciferase substrate was dissolved in sterile PBS solution to prepare a solution at a concentration of 30 mg/ml.
Mouse imaging: after 22-24 hours of lipid nanoparticle injection, each mouse was intraperitoneally injected with 150ul of luciferase substrate solution. Placing for 5 minutes, then anaesthetizing for 3 minutes by a carbon dioxide box, and placing the anaesthetized animal into a living animal imaging instrument for imaging.
TABLE 4 Table 4
Figure SMS_5
In fig. 1 and 2, the left mice are experimental condition 1 mice, and the right mice are experimental condition 2 mice.
As can be seen from table 4 and fig. 1-2, the lipid nanoparticles comprising cholesterol succinic acid monoester in structural lipid can significantly improve the delivery efficiency of nucleic acid in mice, confirming the feasibility of in vivo application, as compared to the lipid nanoparticles using cholesterol alone as structural lipid.

Claims (12)

1. A nucleic acid lipid nanoparticle composition comprising a lipid carrier and a nucleic acid, the lipid carrier comprising an ionizable lipid, a helper lipid, a structural lipid comprising a cholesterol succinate monoester, and a polymer-bound lipid.
2. The nucleic acid lipid nanoparticle composition of claim 1, wherein the structural lipid further comprises an additional structural lipid selected from at least one of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, fecal sterols, and corticosteroids.
3. The nucleic acid lipid nanoparticle composition of claim 2, wherein the additional structural lipid is cholesterol.
4. The nucleic acid lipid nanoparticle composition of claim 2, wherein the molar ratio of cholesterol succinic monoester to other structural lipids is (10-100): 90-0.
5. The nucleic acid lipid nanoparticle composition of claim 1 or 2, wherein the ionizable lipid is at least one member selected from the group consisting of 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester DLin-MC3-DMA, ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) ALC-0315, 1, 2-dioleyloxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-dioleylene-4- (2-dimethylaminoethyl) - [1,3] -dioxolane DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1' - (2- ((2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) piperazine-1-dioleyl-piperazine-dioleyl-dode, and 1-dioleyl-2-dol-N, N-2- [ 2-dimethylcarbamoyl ] -2-N-d-N, 2- [ d-chlorohydrin-N, N-2-d-N-dimethylcarbamoyl ] -2-d.
6. The nucleic acid lipid nanoparticle composition according to claim 1 or 2, wherein the helper lipid is at least one selected from the group consisting of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DSPC, dioleoyl phosphatidylserine DOPS, distearoyl phosphatidylserine DSPS, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine DSPE, dipalmitoyl phosphatidylserine DPPS, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dipalmitoyl-phosphatidylglycerol DPPC, oleoyl phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE, 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE, 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine DPPE, stearoyl phosphatidylethanolamine DPPE, and stearoyl phosphatidylethanolamine DSPE 1-soyl pe.
7. The nucleic acid lipid nanoparticle composition of claim 1 or 2, wherein the polymer-conjugated lipid is selected from 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 DMG-PEG2000, DMG-PEG 2000-mannose, cholesterol-PEG 2000, 1, 2-dimyristoyl-sn-glyceromo-polyethylene glycol PEG-DMG, dimyristoyl glycerol-polyethylene glycol PEG-C-DMG, polyethylene glycol-dimyristoyl glycerol PEG-C14, PEG-1, 2-dimyristoyloxy propyl-3-amine PEG-C-DMA, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE, pegylated phosphatidylethanolamine PEG-PE, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, tween-20, tween-80, 1, 2-diyl-sn-methoxypolyethylene glycol-g, 4- (2, 4 ' - (2-O-dpo-2 '; 3' -Di (tetradecanoyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, at least one of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride PEG-c-DOMG and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propylcarbamate)) GalNAc-PEG-DSG.
8. The nucleic acid lipid nanoparticle composition of claim 1 or 2, wherein the molar ratio of the ionizable lipid, helper lipid, structural lipid, and polymer-bound lipid is (20-75): (2-25): (15-55): (0-15).
9. The nucleic acid lipid nanoparticle composition according to claim 1 or 2, wherein the nucleic acid is at least one selected from the group consisting of DNA, RNA, a complex containing DNA or RNA, modified DNA, modified RNA, and a modified complex containing DNA or RNA.
10. The nucleic acid lipid nanoparticle composition according to claim 1 or 2, wherein the mass ratio of the nucleic acid to the lipid carrier is 1 (3-40).
11. A pharmaceutical formulation comprising the nucleic acid lipid nanoparticle composition of any one of claims 1-10, and a pharmaceutically acceptable carrier.
12. Use of the nucleic acid lipid nanoparticle composition of any one of claims 1-10 or the pharmaceutical formulation of claim 11 in the preparation of a nucleic acid drug or genetic vaccine.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117427174A (en) * 2023-12-20 2024-01-23 百达联康生物科技(深圳)有限公司 Composite material for nucleic acid medicine and preparation method and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011134675A1 (en) * 2010-04-27 2011-11-03 Salama Zoser B Carrier and targeting system comprising a siosomal composition for intracellular delivery and targeting of active substance
CN102481256A (en) * 2009-07-09 2012-05-30 玛瑞纳生物技术有限公司 Amphoteric liposomes comprising imino lipids
CN104382853A (en) * 2008-10-16 2015-03-04 玛瑞纳生物技术有限公司 Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
CN106794141A (en) * 2014-07-16 2017-05-31 诺华股份有限公司 Nucleic acid is encapsulated in the method in lipid nano particle main body
US20190062788A1 (en) * 2017-08-22 2019-02-28 Rubius Therapeutics, Inc. Lipid nanoparticle methods and compositions for producing engineered erythroid cells
CN115957187A (en) * 2021-10-09 2023-04-14 北京启辰生生物科技有限公司 Lipid nanoparticle composition and drug delivery system prepared from same
CN115960180A (en) * 2021-10-09 2023-04-14 北京启辰生生物科技有限公司 2019-nCoV S protein mutant and genetically engineered mRNA and vaccine composition thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104382853A (en) * 2008-10-16 2015-03-04 玛瑞纳生物技术有限公司 Processes and Compositions for Liposomal and Efficient Delivery of Gene Silencing Therapeutics
CN102481256A (en) * 2009-07-09 2012-05-30 玛瑞纳生物技术有限公司 Amphoteric liposomes comprising imino lipids
WO2011134675A1 (en) * 2010-04-27 2011-11-03 Salama Zoser B Carrier and targeting system comprising a siosomal composition for intracellular delivery and targeting of active substance
CN106794141A (en) * 2014-07-16 2017-05-31 诺华股份有限公司 Nucleic acid is encapsulated in the method in lipid nano particle main body
US20190062788A1 (en) * 2017-08-22 2019-02-28 Rubius Therapeutics, Inc. Lipid nanoparticle methods and compositions for producing engineered erythroid cells
CN115957187A (en) * 2021-10-09 2023-04-14 北京启辰生生物科技有限公司 Lipid nanoparticle composition and drug delivery system prepared from same
CN115960180A (en) * 2021-10-09 2023-04-14 北京启辰生生物科技有限公司 2019-nCoV S protein mutant and genetically engineered mRNA and vaccine composition thereof

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
N. MIGNET等: "Anionic pH-sensitive pegylated lipoplexes to deliver DNA to tumors", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》, vol. 361, no. 01, pages 194 - 201, XP023316077, DOI: 10.1016/j.ijpharm.2008.05.017 *
SHAGUNA SETH等: "RNAi-based Therapeutics Targeting Survivin and PLK1 for Treatment of Bladder Cancer", 《MOLECULAR THERAPY》, vol. 19, no. 05, pages 928 - 935, XP055188584, DOI: 10.1038/mt.2011.21 *
XINWEI CHENG等: "The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery", 《ADVANCED DRUG DELIVERY REVIEWS》, vol. 99, pages 22 - 33 *
YANG FAN等: "Study of the pH-sensitive mechanism of tumor-targeting liposomes", 《COLLOIDS AND SURFACES B: BIOINTERFACES》, vol. 151, pages 19 - 25, XP029905793, DOI: 10.1016/j.colsurfb.2016.11.042 *
孙霁等: "pH敏感脂质体作为基因载体的研究进展", 《解放军药学学报》, vol. 26, no. 06, pages 553 - 557 *
张雪敬: "寡聚荷正电氨基酸修饰的基因递送纳米脂质载体的构建和初步评价", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 09, pages 079 - 42 *
曾慧琳等: "pH 敏感脂质体在药物传递系统中的应用", 《医药导报》, vol. 33, no. 03, pages 348 - 351 *
游利江等: "低 pH 插入肽修饰的载 siＲNA 脂质体的制备与体外评价", 《中国药学杂志》, vol. 55, no. 04, pages 312 - 316 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117427174A (en) * 2023-12-20 2024-01-23 百达联康生物科技(深圳)有限公司 Composite material for nucleic acid medicine and preparation method and application thereof
CN117427174B (en) * 2023-12-20 2024-05-31 百达联康生物科技(深圳)有限公司 Composite material for nucleic acid medicine and preparation method and application thereof

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