CN116041697A - Dendrimer compound and composition for nucleic acid delivery, and preparation methods and applications thereof - Google Patents

Dendrimer compound and composition for nucleic acid delivery, and preparation methods and applications thereof Download PDF

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CN116041697A
CN116041697A CN202310068617.6A CN202310068617A CN116041697A CN 116041697 A CN116041697 A CN 116041697A CN 202310068617 A CN202310068617 A CN 202310068617A CN 116041697 A CN116041697 A CN 116041697A
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dendrimer
nucleic acid
lipid
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杨传旭
孙倩
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Shandong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/02—Polyamines
    • C08G73/028—Polyamidoamines
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a dendrimer compound for nucleic acid delivery, a composition, a preparation method and application thereof. The preparation steps and the operation steps of the dendritic macromolecular compound are simple, the raw materials are simple and easy to obtain, the requirements on instruments and equipment are low, the reaction conditions are mild and safe, and the yield of target products is high. The dendrimer compound obtained by the invention has excellent biological safety; and for use in nucleic acid delivery vectors, either prophylactic or therapeutic, with high transfection efficiency. The dendrimer compounds of the present invention may be used in combination with other lipid components in specific proportions to form dendrimer-based lipid nanoparticle compositions for delivery of prophylactic or therapeutic agents (e.g., therapeutic nucleic acids), for the purpose of extracellular delivery of nucleic acids into cells in vitro or in vivo.

Description

Dendrimer compound and composition for nucleic acid delivery, and preparation methods and applications thereof
Technical Field
The invention relates to a dendritic macromolecular compound and composition for nucleic acid delivery, and a preparation method and application thereof, and belongs to the technical field of dendritic macromolecular nucleic acid delivery vectors.
Background
The nucleic acid medicine can play a role in the transmission source of genetic information, so that the nucleic acid medicine has the advantages of strong specificity, abundant gene targets, lasting curative effect and the like, avoids the complex synthesis and purification processes of the traditional medicine, and can obviously reduce the cost. However, naked nucleic acid medicine is a hydrophilic macromolecule with negative charges, is difficult to enter cells due to electrostatic repulsion of cell membranes, and is easily and rapidly degraded by ubiquitous nucleases. Thus, nucleic acid drugs require a protective housing to enter the cell. Nucleic acid drugs must therefore be delivered with a suitable delivery vehicle in order to function successfully in vivo.
Currently, emerging dendrimers are in the lead in biomedical applications, such as gene editing, drug delivery, due to their excellent water solubility, low toxicity, ease of functionalization, and topology adjustability. The lipid nanoparticle based on dendrimers consists of 1 to 3 lipid components, including 0 to 2 auxiliary lipids and 0 to 1 PEGylated lipid, in addition to the dendrimer. Wherein dendrimer compounds play a key role in nucleic acid entrapment and release, it is therefore important to develop new, efficient dendrimer compounds.
The application of dendrimers in biomedical applications has been reported in the prior art. For example, chinese patent document CN1631936a discloses a polyamide-amine dendritic polymer nanomaterial, which uses methyl acrylate and ethylenediamine as starting monomers to synthesize a 5-generation polyamide-amine (PAMAM) dendritic polymer (dendrimer), and uses a dialysis method to purify the obtained G5 PAMAM dendrimer to obtain a type of highly branched spherical monodisperse nanoparticle, wherein the molecular size is 50-90 nm, the surface has extremely high positive functional group density, and the interior has a cavity. After the nano particles are mixed with plasmid DNA with a certain proportion, the target genes can be transfected into hematopoietic stem cells purified by CD34 immunomagnetic beads, and the protein synthesized by the target genes can be expressed in the hematopoietic stem cells for a long time. However, the synthesis steps are complicated, and the target product can be obtained through multi-step reaction; strong alkali is also needed to be added in the reaction process, so that the method has certain danger.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a dendritic macromolecular compound and composition for nucleic acid delivery, and a preparation method and application thereof. The preparation steps and the operation steps of the dendritic macromolecular compound are simple, the raw materials are simple and easy to obtain, the requirements on instruments and equipment are low, the reaction conditions are mild and safe, and the yield of target products is high. The dendrimer compound obtained by the invention has excellent biological safety; and for use in nucleic acid delivery vectors, either prophylactic or therapeutic, with high transfection efficiency. The dendrimer compounds of the present invention may be used in combination with other lipid components in specific proportions to form dendrimer-based lipid nanoparticle compositions for delivery of prophylactic or therapeutic agents (e.g., therapeutic nucleic acids), for the purpose of extracellular delivery of nucleic acids into cells in vitro or in vivo.
The technical scheme of the invention is as follows:
a dendrimer compound for nucleic acid delivery having a structure according to formula I or formula II:
Figure BDA0004063015110000021
wherein, in the formula I, R is selected from alkyl with a carbon chain number of 4-14; in the formula II, R is selected from alkyl with a carbon chain number of 6-16.
According to a preferred embodiment of the invention, in formula I, R is selected from alkyl groups having a carbon chain number of 10 or 12; in formula II, R is selected from alkyl with carbon chain number of 12 or 14.
The preparation method of the dendrimer compound for nucleic acid delivery comprises the following steps: uniformly mixing ethylenediamine core-polyamide-amine type 0-generation dendritic macromolecule (PAMAM-G0) with 1, 2-alkylene oxide or acrylic ester; obtaining the dendritic macromolecular compound for nucleic acid delivery after reaction, dialysis and drying;
the ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0) has the following structure:
Figure BDA0004063015110000022
according to the present invention, ethylenediamine core-polyamide-amine type 0-generation dendrimer (PAMAM-G0) is commercially available or can be prepared according to the existing method.
According to a preferred embodiment of the invention, the molar ratio of ethylenediamine core-polyamide-amine type 0-substituted dendrimer (PAMAM-G0) to 1, 2-alkylene oxide or acrylate is 1:2x+2, where x is the primary amine number of ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0).
According to the invention, the mixing of ethylenediamine core-polyamide-amine type 0-generation dendrimers (PAMAM-G0) with 1, 2-alkylene oxide or acrylic esters is preferably carried out at room temperature.
According to a preferred embodiment of the present invention, the 1, 2-alkylene oxide is 1, 2-hexane oxide, 1, 2-octane oxide, 1, 2-dodecane oxide, 1, 2-tetradecane oxide or 1, 2-hexadecane oxide; preferably, the 1, 2-alkylene oxide is 1, 2-dodecane oxide or 1, 2-tetradecane oxide.
Preferably according to the invention, the acrylate is hexyl acrylate, octyl acrylate, dodecyl acrylate, tetradecyl acrylate or hexadecyl acrylate; preferably, the acrylate is dodecyl acrylate or tetradecyl acrylate.
According to the invention, the reaction temperature is preferably from 80 to 100℃and the reaction time is from 2 to 3 days.
The use of a dendrimer compound as described above for nucleic acid delivery in a nucleic acid delivery vehicle having prophylactic or therapeutic properties.
According to a preferred aspect of the invention, the dendrimer compound may be targeted as a carrier to deliver a prophylactic or therapeutic nucleic acid to the liver.
According to a preferred embodiment of the present invention, the nucleic acid having prophylactic or therapeutic properties is selected from one or a combination of two or more of messenger RNA (mRNA), micro RNA (miRNA), small interfering RNA (siRNA), RNA interference (RNAi), antisense oligonucleotide (ASO) or plasmid DNA. Preferably one or a combination of more than two of messenger RNA, small interfering RNA, antisense oligonucleotides or plasmid DNA.
A dendrimer-based lipid nanoparticle composition (DLNP) comprising the dendrimer compound described above and a nucleic acid having prophylactic or therapeutic properties.
According to the present invention, preferably, the dendrimer compound is selected from one or a combination of two or more of the formulae I or II.
According to the invention, the mass ratio of the dendrimer compound to the nucleic acid having prophylactic or therapeutic properties is preferably 1 to 22.5:1, preferably 7.5-16:1.
According to a preferred aspect of the present invention, the dendrimer-based lipid nanoparticle composition has a particle size of 60nm to 300nm.
Preferably according to the present invention, the dendrimer-based lipid nanoparticle composition further comprises a lipid component; the lipid component is one or more than two of structural lipid, steroid or polymer conjugated lipid.
Preferably, the structural lipid is selected from one or a combination of more than two of DOPC, DOPE, DSPC, DPPG, DSPE, DPPC, DMPC or POPC; the steroid is selected from one or more than two of cholesterol, beta-sitosterol, rock soap sterol, stigmasterol, ergosterol or stigmastanol; the polymer conjugated lipid is a polyethylene glycol conjugated lipid; further preferably, the polyethylene glycol conjugated lipid is one or more of DMG-PEG, DSG-PEG, DSPE-PEG, DPPE-PEG, DMPE-PEG or TPGS-PEG.
Preferably, the mass ratio of lipid component to dendrimer compound is 1:1-4.
Preferably, the lipid component is a combination of a structural lipid, a steroid, and a polymer conjugated lipid; further preferred, the mass ratio of dendrimer compound, steroid, structural lipid and polymer conjugated lipid is 4:1:1:2.
the preparation method of the lipid nanoparticle composition (DLNP) based on the dendritic macromolecules comprises the following steps:
(1) Dissolving a dendrimer compound in ethanol to obtain a mixed solution; then dissolving the mixed solution in a sodium acetate buffer solution with the pH value of 5-6 to obtain a dendritic macromolecule compound solution;
(2) Dissolving nucleic acid with preventive or therapeutic property in enzyme-free water to obtain nucleic acid solution;
(3) Uniformly mixing the dendrimer compound solution and the nucleic acid solution, and incubating to obtain the lipid nanoparticle composition based on the dendrimer.
According to a preferred embodiment of the invention, the method of step (1) is as follows: dissolving a dendrimer compound and a lipid component in ethanol to obtain a mixed solution; then, under the vortex condition, the mixed solution is dripped into a sodium acetate buffer solution with the pH of 5-6 to obtain a dendritic macromolecule compound solution.
Preferably, the concentration of dendrimer compound in the mixture is 80-120mg/mL.
Preferably, the concentration of the sodium acetate buffer solution is 200mmol/L.
Preferably, the concentration of the dendrimer compound in the dendrimer compound solution is 0.05 to 5mg/mL.
According to the present invention, preferably, in the step (2), the concentration of the nucleic acid solution is 0.01-2mg/mL.
According to a preferred embodiment of the present invention, in step (3), the incubation temperature is room temperature and the incubation time is 5-10min.
According to the invention, in step (3), after incubation, a dilution step is further included to dilute to the desired volume.
Use of a dendrimer compound or a dendrimer-based lipid nanoparticle composition as described above for nucleic acid delivery in the prevention or treatment of a disease.
According to the invention, the disease is acute liver injury, acute liver failure, long-term liver fibrosis, etc.
Use of a dendrimer compound or a dendrimer-based lipid nanoparticle composition as described above for nucleic acid delivery in a medicament for inducing protein expression in a subject.
Preferably according to the invention, the subject is a mammal, preferably a non-primate, further preferably a human.
The preparation route of the dendrimer compound for nucleic acid delivery of the present invention is as follows:
Figure BDA0004063015110000051
the invention has the technical characteristics and beneficial effects that:
1. the preparation steps and the operation steps of the dendritic macromolecular compound are simple, the raw materials are simple and easy to obtain, the requirements on instruments and equipment are low, the reaction conditions are mild and safe, and the yield of target products is high.
2. The dendrimer compound of the present invention has low toxicity, so that the dendrimer has excellent biosafety; and for use in nucleic acid delivery vectors, either prophylactic or therapeutic, with high transfection efficiency. And ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0) has high toxicity and poor transfection effect.
3. The different structures of the 1, 2-alkylene oxide or acrylic ester of the invention have important influence on the transfection efficiency. From the examples of the present invention, the transfection efficiency increased with increasing chain length from 1, 2-epoxyhexane or hexyl acrylate to 1, 2-epoxytetradecane or tetradecyl acrylate, but decreased with increasing chain length from 1, 2-epoxytetradecane or tetradecyl acrylate to 1, 2-epoxyhexadecane or hexadecyl acrylate. Thus, 1, 2-epoxytetradecane or tetradecyl acrylate is a node, where the transfection efficiency increases with increasing chain length, and where the transfection efficiency decreases with increasing chain length. In summary, the excellent effects of the present invention can be achieved only by the 1, 2-alkylene oxide or acrylic acid ester of the specific structure of the present invention.
4. The molar ratio of ethylenediamine core-polyamide-amine type 0-generation dendrimer (PAMAM-G0) to 1, 2-alkylene oxide or acrylic ester according to the present invention needs to be suitable. Other preparation methods and conditions are described in examples 1 or 2, such as when the molar ratio of ethylenediamine core-polyamide-amine type 0-substituted dendrimer (PAMAM-G0) to 1, 2-alkylene oxide or acrylate is 1:7; lipid nanoparticle compositions were prepared using the resulting dendrimer compounds as described in example 6, and EGFP siRNA was delivered to Hela-GFP cells, which were photographed using a fluorescence microscope, and found to have no gene silencing effect, indicating poor transfection. As above, the transfection effect is not as good as that of the present invention when the molar ratio of ethylenediamine core-polyamide-amine type 0-substituted dendrimer (PAMAM-G0) to 1, 2-alkylene oxide or acrylate is 1:8. In view of the above, the molar ratio of ethylenediamine core-polyamide-amine type 0-substituted dendrimer (PAMAM-G0) to 1, 2-alkylene oxide or acrylic acid ester of the present invention needs to be suitable, and if not, the excellent effects of the present invention are not achieved.
5. The dendrimer compounds of the invention, or in combination with other lipid components in specific proportions, carry prophylactic or therapeutic nucleic acid agents; thereby achieving the purpose of delivering nucleic acid drugs from outside cells into cells in vitro or in vivo.
6. The dendrimer-based lipid nanoparticle composition (DLNP) of the present invention delivers nucleic acids into cells by targeting and can escape from lysosomes, enabling the nucleic acids to function within the cells, thereby enabling the cells to produce related proteins for therapeutic or prophylactic purposes. The structural lipid used in the invention has the functions of supporting the formation of a lamellar lipid bilayer structure and stabilizing the structural arrangement thereof; the steroid has stronger membrane fusion property and promotes the intracellular uptake of nucleic acid; the polymer conjugated lipid is positioned on the surface of the lipid nanoparticle, so that the hydrophilicity of the lipid nanoparticle is improved, the lipid nanoparticle is prevented from being rapidly cleared by an immune system, particle aggregation is prevented, and the stability is improved. The ratio relationship of the lipid component and the dendrimer compound has a certain influence on the transfection effect. By controlling the unique variables, three structurally different polymer conjugated lipids were selected for the experiment, respectively: the results of the researches on DMG-PEG2000, TPGS-PEG2000 and DSPE-PEG2000 show that the transfection effect of DMG-PEG2000 and TPGS-PEG2000 is almost similar, and the transfection effect of DSPE-PEG2000 is obviously lower than that of the other two.
Drawings
FIG. 1 is a schematic illustration of a dendrimer ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0) 1 H-NMR spectrum.
FIG. 2 is a diagram of G0-O6 1 H-NMR spectrum.
FIG. 3 is a diagram of G0-O8 1 H-NMR spectrum.
FIG. 4 is a diagram of G0-O12 1 H-NMR spectrum.
FIG. 5 is a diagram of G0-O14 1 H-NMR spectrum.
FIG. 6 is a diagram of G0-O16 1 H-NMR spectrum.
FIG. 7 is a dimensional chart of G0-O6, G0-O8, G0-O12, G0-O14, G0-O16, G0-A6, G0-A8, G0-A12, G0-A14, G0-A16 in example 3. The ordinate is particle size.
FIG. 8 is a zeta potential chart of G0-O6, G0-O8, G0-O12, G0-O14, G0-O16, G0-A6, G0-A8, G0-A12, G0-A14, G0-A16 in example 3. The ordinate is zeta potential.
FIG. 9 is a dimensional map of G0-O6/siRNA, G0-O8/siRNA, G0-O12/siRNA, G0-O14/siRNA, G0-O16/siRNA, G0-A6/siRNA, G0-A8/siRNA, G0-A12/siRNA, G0-A14/siRNA, G0-A16/siRNA in example 4. The ordinate is particle size.
FIG. 10 is a zeta potential map of G0-O6/siRNA, G0-O8/siRNA, G0-O12/siRNA, G0-O14/siRNA, G0-O16/siRNA, G0-A6/siRNA, G0-A8/siRNA, G0-A12/siRNA, G0-A14/siRNA, G0-A16/siRNA in example 4. The ordinate is zeta potential.
FIG. 11 shows encapsulation efficiency of G0-O6/siRNA, G0-O8/siRNA, G0-O12/siRNA, G0-O14/siRNA, G0-O16/siRNA, G0-A6/siRNA, G0-A8/siRNA, G0-A12/siRNA, and G0-A14/siRNA in example 4. The ordinate is siRNA encapsulation efficiency.
FIG. 12 is a plot of the size of G0-O6/mRNA, G0-O8/mRNA, G0-O12/mRNA, G0-O14/mRNA, G0-O16/mRNA, G0-A6/mRNA, G0-A8/mRNA, G0-A12/mRNA, G0-A14/mRNA, G0-A16/mRNA in example 5. The ordinate is particle size.
FIG. 13 is a zeta potential plot of G0-O6/mRNA, G0-O8/mRNA, G0-O12/mRNA, G0-O14/mRNA, G0-O16/mRNA, G0-A6/mRNA, G0-A8/mRNA, G0-A12/mRNA, G0-A14/mRNA, G0-A16/mRNA of example 5. The ordinate is zeta potential.
FIG. 14 shows the encapsulation efficiency of G0-O6/mRNA, G0-O8/mRNA, G0-O12/mRNA, G0-O14/mRNA, G0-O16/mRNA, G0-A6/mRNA, G0-A8/mRNA, G0-A12/mRNA and G0-A14/mRNA in example 5. The ordinate indicates the mRNA encapsulation efficiency.
FIG. 15 shows the gene silencing efficiency of EGFP in cells after co-culturing lipid nanoparticle composition of example 6 with HeLa-GFP cells. The ordinate is the gene silencing efficiency.
FIG. 16 is a fluorescent quantification of EGFP in cells after co-culturing lipid nanoparticle composition of example 6 with HeLa-GFP cells. The ordinate is the average fluorescence intensity.
FIG. 17 shows the gene expression efficiency of EGFP in HeLa cells after co-culturing the lipid nanoparticle composition of example 7. The ordinate indicates the expression efficiency of EGFP gene in cells.
FIG. 18 is a fluorescent quantification of EGFP in HeLa cells after co-culturing the lipid nanoparticle composition of example 7 with the cells. The ordinate is the average fluorescence intensity.
FIG. 19 shows the expression of PPIB gene in liver after tail-vein injection of PAMAM-G0-O14/siRNA, PAMAM-G0-O14/siNC for 48 h in example 8. The ordinate indicates the expression level of PPIB mRNA in liver tissue.
FIG. 20 is a graph showing bioluminescence in mice after intravenous injection of G0-O12/mLuc, G0-O14/mLuc for 6h in example 9.
FIG. 21 is a graph showing bioluminescence in mice after intravenous injection of G0-A12/mLuc, G0-A14/mLuc for 6h in example 9.
FIG. 22 shows the results of quantitative in vivo bioluminescence in mice after intravenous injection of G0-O12/mLuc, G0-O14/mLuc, G0-A12/mLuc and G0-A14/mLuc for 6h in example 9. The ordinate is the total fluorescence intensity of the tissue.
FIG. 23 shows the enrichment of nanoparticles in the major organs (heart, liver, spleen, lung and kidney) after 4h of intravenous injection of G0-O14/siRNA labeled with fluorescent molecule DIR in example 10.
FIG. 24 shows the result of quantifying fluorescent signals of nanoparticles in each major organ (heart, liver, spleen, lung and kidney) after intravenous injection of fluorescent molecule DIR-labeled G0-O14/siRNA for 4h in example 10. The ordinate is the efficiency of chemiluminescence.
FIG. 25 shows the enrichment of nanoparticles in the major organs (heart, liver, spleen, lung and kidney) after 4h of intravenous injection of fluorescent molecule DIR-labeled G0-O12/mRNA, G0-O14/mRNA in example 11.
FIG. 26 shows the enrichment of nanoparticles in the major organs (heart, liver, spleen, lung and kidney) 4h after intravenous injection of fluorescent molecule DIR labeled G0-A12/mRNA, G0-A14/mRNA in example 11.
FIG. 27 shows the result of fluorescent signal quantification of nanoparticles in each major organ (heart, liver, spleen, lung and kidney) after 4 hours of intravenous injection of G0-O12/mRNA, G0-O14/mRNA, G0-A12/mRNA and G0-A14/mRNA labeled with fluorescent molecule DIR in example 11. The ordinate is the efficiency of chemiluminescence.
FIG. 28 shows the expression of Col1a1 mRNA in liver tissue of mice after treatment in example 12. The ordinate indicates the expression level of Col1a1 mRNA in the liver.
FIG. 29 is a graph showing the quantitative hEPO content of serum by ELISA after tail vein injection of G0-O14/mhEPO6 h in example 13. The ordinate indicates the amount of hEPO protein expressed in serum.
Detailed Description
The following detailed description of the invention and the advantages of the invention will be presented to assist the reader in better understanding the nature and characteristics of the invention and is not intended to limit the scope of the invention.
Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials, unless otherwise specified, are all commercially available.
Example 1
Synthesis of dendrimer PAMAM-G0-An for nucleic acid delivery
The steps are as follows: ethylenediamine core-polyamide-amine type 0-substituted dendrimer (PAMAM-G0) (40 mg) was uniformly mixed with acrylic acid ester (An, n=6, 8, 12, 14 or 16, i.e., acrylic acid ester was one of 120.9mg of hexyl acrylate, 140.5mg of octyl acrylate, 187.0mg of dodecyl acrylate, 207.8mg of tetradecyl acrylate or 229.5mg of hexadecyl acrylate) at room temperature, and the mixture was reacted at 90 ℃ for 2.5 days. The reaction solution was put into a dialysis bag and dialyzed in ethanol for 24 hours (molecular weight cut-off: 1000D), during which fresh ethanol was changed every 1.5 hours, followed by drying to remove the ethanol to give PAMAM-G0-An (abbreviated as G0-An, n=6, 8, 12, 14 or 16) as a pale yellow oily liquid compound.
Example 2
Synthesis of dendrimer PAMAM-G0-On for nucleic acid delivery
The steps are as follows: ethylenediamine core-polyamide-amine type 0-generation dendrimer (PAMAM-G0) (40 mg) was mixed with 1, 2-alkylene oxide (On, n=6, 8, 12, 14 or 16, i.e., 1, 2-alkylene oxide was 77.5mg of 1, 2-hexane oxide, 99.3mg of 1, 2-octane oxide, 142.7mg of 1, 2-dodecane oxide, 164.4mg of 1, 2-tetradecane oxide, or 186.1mg of one of 1, 2-hexadecane oxide) at room temperature uniformly, and the mixture was reacted at 90 ℃ for 2.5 days. The reaction solution was put into a dialysis bag and dialyzed in ethanol for 24 hours (molecular weight cut-off: 1000D), during which fresh ethanol was changed every 1.5 hours, followed by drying to remove ethanol to obtain a colorless oily liquid compound PAMAM-G0-On (abbreviated as G0-On, n=6, 8, 12, 14 or 16).
The product PAMAM-G0-On 1 The H-NMR spectra are shown in FIGS. 2-6, and the compound PAMAM-G0 is also characterized 1 The H-NMR spectrum is shown in FIG. 1. The graph shows that the invention successfully prepares the target product.
Example 3
Preparation of a composition of a dendrimer compound and a lipid component comprising the steps of:
dissolving a dendrimer compound (one of the dendrimer compounds prepared in example 1 or example 2), cholesterol, DOPE and DMG-PEG2000 in ethanol respectively, and mixing according to a ratio (the mass ratio of the dendrimer compound to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the dendrimer compound is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixture was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition, wherein the concentration of the dendrimer compound was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
The composition obtained above was subjected to particle size detection and Zeta potential analysis using standard detection methods on a zetasizer ano instrument from malvern. The particle size measurement results are shown in FIG. 7 and the Zeta potential results are shown in FIG. 8. From the figure, it is clear that the hydrated particle size of the composition of the dendrimer compound and the lipid component is between 80 and 250 nm. Except that the zeta potential of G0-O6 is negative, the zeta potential is positive. Because the nucleic acid molecule is negatively charged, the composition of dendrimer compounds and lipid components, having a positive zeta potential, is more easily bound thereto by electrostatic action.
Example 4
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
dissolving a dendrimer compound (one of the dendrimer compounds prepared in example 1 or example 2), cholesterol, DOPE and DMG-PEG2000 in ethanol respectively, and mixing according to a ratio (the mass ratio of the dendrimer compound to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the dendrimer compound is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortex conditions to obtain a composition of a dendrimer compound and a lipid component, wherein the concentration of the dendrimer compound was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Enhanced green fluorescent protein specific siRNA (Enhanced green fluorescent protein specific siRNA, siEGFP) was dissolved in enzyme-free water to give an siRNA solution at a concentration of 20 μm.
Uniformly mixing the composition of the dendrimer compound and the lipid component with the siRNA solution (the mass ratio of the dendrimer compound to the siRNA is 7.5:1), and incubating for 10min at room temperature to obtain the lipid nanoparticle composition based On the dendrimer (G0-On/siRNA or G0-An/siRNA for short, wherein n=6, 8, 12, 14 or 16).
According to the description of the Ribogreen kit, the encapsulation efficiency of the siRNA is tested and calculated; the above dendrimer-based lipid nanoparticle compositions were subjected to particle size detection and Zeta potential analysis using standard detection methods on a zetasizer rno instrument from malvern.
The particle size detection result of the dendrimer-based lipid nanoparticle composition prepared in this example is shown in fig. 9, the Zeta potential result is shown in fig. 10, and the siRNA encapsulation efficiency detection result is shown in fig. 11.
From the figure, it can be seen that the hydrated particle size of the dendrimer-based lipid nanoparticle composition (DLNP) was between 120 and 300 nm. Because siRNA molecules are negatively charged, the zeta potential of most DLNPs is negative after the combination of dendrimer compounds and lipid components. In addition, overall, G0-On/siRNA was more encapsulated.
Example 5
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
dissolving a dendrimer compound (one of the dendrimer compounds prepared in example 1 or example 2), cholesterol, DOPE and DMG-PEG2000 in ethanol respectively, and mixing according to a ratio (the mass ratio of the dendrimer compound to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the dendrimer compound is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortex conditions to obtain a composition of a dendrimer compound and a lipid component, wherein the concentration of the dendrimer compound was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Enhanced green fluorescent protein specific mRNA (Enhanced green fluorescent protein specific mRNA, mEGFP) was dissolved in enzyme-free water to give an mRNA solution at a concentration of 0.1 mg/mL.
Uniformly mixing the composition of the dendrimer compound and the lipid component with the mRNA solution (the mass ratio of the dendrimer compound to the mRNA is 15:1), and incubating for 10min at room temperature to obtain the lipid nanoparticle composition based On the dendrimer (G0-On/mRNA or G0-An/mRNA for short, wherein n=6, 8, 12, 14 or 16).
The encapsulation efficiency of mRNA is tested and calculated according to the specification of a Ribogreen kit; the above dendrimer-based lipid nanoparticle compositions were subjected to particle size detection and Zeta potential analysis using standard detection methods on a zetasizer rno instrument from malvern.
The particle size detection result of the dendrimer-based lipid nanoparticle composition prepared in this example is shown in fig. 12, the Zeta potential result is shown in fig. 13, and the mRNA encapsulation efficiency detection result is shown in fig. 14.
From the figure, it can be seen that the hydrated particle size of the dendrimer-based lipid nanoparticle composition (DLNP) was between 90 and 280 nm. The mRNA molecules are negatively charged, but the zeta potential of DLNPs is still positive after the combination of dendrimer compound and lipid component is combined with it, probably because the mass ratio of dendrimer compound and lipid component combination and the two RNAs are different. In addition, the mRNA encapsulation efficiency of all DLNPs except G0-A14/mRNA was higher than 60%, with the encapsulation efficiency of G0-O8/mRNA, G0-O12/mRNA and G0-O14/mRNA being the best.
Example 6
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
dissolving a carrier sample dendrimer compound (one of the dendrimer compounds prepared in example 1 or example 2), cholesterol, DOPE and DMG-PEG2000 in ethanol respectively, and mixing according to a ratio (the mass ratio of the dendrimer compound to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the carrier sample is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortex conditions to obtain a composition of a dendrimer compound and a lipid component, wherein the concentration of the dendrimer compound was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Enhanced green fluorescent protein specific siRNA (Enhanced green fluorescent protein specific siRNA, siEGFP) was dissolved in enzyme-free water to give an siRNA solution with a mass concentration of 20 μm.
Uniformly mixing the composition of the dendrimer compound and the lipid component with the siRNA solution (the mass ratio of the dendrimer compound to the siRNA is 7.5:1), and incubating for 10min at room temperature; then diluted 5-fold with 200mM sodium acetate buffer with pH of about 5.4 to obtain lipid nanoparticle composition based On carrier sample (G0-On/siRNA or G0-An/siRNA for short, n=6, 8, 12, 14 or 16).
Meanwhile, a commercially available positive control reagent RNAiMAX is used as a carrier, and the specific use method is carried out according to the specification, so that a lipid nanoparticle composition based on a carrier sample is obtained, which is called RNAiMAX/siRNA for short.
In vitro siRNA delivery efficiency
After Hela-GFP cells were cultured in DMEM medium containing 10% (volume fraction) FBS to an adherent state in a carbon dioxide incubator at 37℃and the fresh medium was replaced, the lipid nanoparticle composition based on the carrier sample prepared as described above (addition amount of about 50. Mu.L/mL cell solution) was added thereto, and transfection was performed under this condition (37 ℃) for 48 hours. In vitro siRNA delivery efficiency was tested.
The in vitro siRNA delivery efficiency was evaluated by how much EGFP gene was silenced in the cells, and the results of in vitro siRNA delivery efficiency tests for different delivery vehicles are shown in FIG. 15 and the results of fluorescence quantification are shown in FIG. 16.
FIG. 15 shows that the silencing effect of G0-O14, G0-A14 and G0-A12 delivered siRNA was significantly better than that of the commercially available positive control reagent RNAiMAX. From FIG. 16, it can also be seen that the fluorescence intensity of Hela-GFP cells to which G0-O14/siRNA, G0-A14/siRNA and G0-A12/siRNA were added was significantly weaker than that of the commercially available positive control reagent RNAiMAX, which also confirms the conclusion of FIG. 15. In conclusion, the dendrimers PAMAM-G0-On and PAMAM-G0-An synthesized by the invention can be used as delivery vectors of siRNA and can achieve excellent transfection efficiency.
Example 7
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
dissolving a carrier sample dendrimer compound (one of the dendrimer compounds prepared in example 1 or example 2), cholesterol, DOPE and DMG-PEG2000 in ethanol respectively, and mixing according to a ratio (the mass ratio of the dendrimer compound to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the carrier sample is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortex conditions to obtain a composition of a dendrimer compound and a lipid component, wherein the concentration of the dendrimer compound was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
The enhanced green fluorescent protein mRNA (Enhanced green fluorescent protein mRNA, mEGFP) was dissolved in enzyme-free water to give an mRNA solution with a mass concentration of 0.1 mg/mL.
Uniformly mixing the composition of the dendrimer compound and the lipid component with the mRNA solution (the mass ratio of the dendrimer compound to the mRNA is 15:1), and incubating for 10min at room temperature; then diluted 5-fold with 200mM sodium acetate buffer with pH of about 5.4 to obtain lipid nanoparticle composition based On carrier sample (G0-On/mRNA or G0-An/mRNA for short, n=6, 8, 12, 14 or 16).
Meanwhile, a commercially available positive control reagent RNAiMAX is used as a carrier, and the specific use method is carried out according to the specification, so that a lipid nanoparticle composition based on a carrier sample is obtained, which is called RNAiMAX/mRNA for short.
In vitro mRNA delivery efficiency
After Hela cells were cultured in DMEM medium containing 10% (volume fraction) FBS to an adherent state in a carbon dioxide incubator at 37℃and the fresh medium was replaced, the lipid nanoparticle composition based on the carrier sample prepared as described above (addition amount of about 50. Mu.L/mL cell solution) was added, and transfection was performed under this condition (37 ℃) for 24 hours. In vitro mRNA delivery efficiency was tested.
The in vitro delivery efficiency of mRNA was evaluated by the level of EGFP gene expression in cells, and the results of in vitro mRNA delivery efficiency tests for different delivery vehicles are shown in FIG. 17 and the results of fluorescence quantification are shown in FIG. 18.
FIG. 17 shows that the expression efficiency of delivery of mRNA by G0-O8, G0-O12, G0-O14, G0-O16, G0-A8, G0-A12 and G0-A14 is significantly better than that of the commercially available positive control reagent RNAiMAX. As can be seen from FIG. 18, the fluorescence intensity of HeLa cells added with G0-O12/mRNA, G0-O14/mRNA, G0-O16/mRNA, G0-A12/mRNA and G0-A14/mRNA was significantly higher than that of the commercially available positive control reagent RNAiMAX, which also confirms and further defines the conclusion of FIG. 17. In conclusion, the dendrimers PAMAM-G0-On and PAMAM-G0-An synthesized by the invention can be used as delivery vectors of mRNA and can achieve excellent transfection efficiency.
Example 8
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
respectively dissolving PAMAM-G0-O14, cholesterol, DOPE and DMG-PEG2000 prepared in example 2 in ethanol, and mixing according to a proportion (the mass ratio of the PAMAM-G0-O14, the cholesterol, the DOPE and the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the PAMAM-G0-O14 is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixture was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of PAMAM-G0-O14 and a lipid component, wherein the concentration of PAMAM-G0-O14 was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
The peptidyl prolyl isomerase B siRNA (peptidylprolyl isomerase B siRNA, sippiib) or disordered siRNA (scrambled siRNA, siNC) was dissolved in enzyme-free water to give 100 μm siRNA solution.
Uniformly mixing the composition and siRNA solution (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer with pH around 5.4 to obtain lipid nanoparticle composition based on PAMAM-G0-O14.
In vivo siRNA delivery efficacy test
Female C57 mice of 8 weeks of age were selected, each of which was injected into the mice by tail vein with a volume of 0.2mL of PAMAM-G0-O14-based lipid nanoparticle composition (the amount of injected siPPIB was 0.3mg/kg, 0.6mg/kg or 1.2mg/kg, and the amount of siNC was 1.2 mg/kg) to inject siNC as a negative control; mice were sacrificed 48 hours after dosing, liver tissue was dissected, RNA was extracted therefrom, RNA was reverse transcribed into DNA, and the gene silencing efficiency of GAPDH in cells was analyzed by a real-time fluorescent quantitative PCR instrument (Real time quantitative PCR, RT-qPCR).
According to the above procedure, the silencing efficiency of PPIB gene in mouse liver was measured and quantified when PAMAM-G0-O14 was used as the dendrimer, as shown in FIG. 19.
From FIG. 19, it can be seen that after 48 hours of PAMAM-G0-O14/siRNA injection, PPIB gene expression was significantly reduced in mice injected with different doses of PAMAM-G0-O14/siPPIB compared to mice injected with PAMAM-G0-O14/siNC groups, indicating that dendrimers of the present invention have excellent in vivo siRNA transfection effects, and PPIB gene silencing effect has a positive linear relationship with the doses of delivered siPPIB.
Example 9
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
dissolving the carrier samples (PAMAM-G0-O12, PAMAM-G0-O14, PAMAM-G0-A12 or PAMAM-G0-A14), cholesterol, DOPE and DMG-PEG2000 prepared in the example 1 or 2 in ethanol respectively, and mixing according to the proportion (the mass ratio of the carrier samples to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the carrier samples is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of a carrier sample and a lipid component, wherein the concentration of the carrier sample was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Firefly luciferase mRNA (Encoding firefly luciferase mRNA, mLuc) was dissolved in enzyme-free water to obtain a 1mg/mL mRNA solution.
Uniformly mixing the composition and the mRNA solution (the mass ratio of the carrier sample to the mRNA is 15:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer with pH around 5.4 to obtain lipid nanoparticle composition based On carrier sample (abbreviated as G0-On/mRNA or G0-An/mRNA, n=12 or 14).
In vivo mRNA delivery efficacy test
Female BALB/c mice of 8 weeks of age were selected, each of which was injected into the mice via tail vein with a volume of 0.2mL of the carrier sample-based lipid nanoparticle composition (the amount of mLuc injected was 0.2 mg/kg); 6 hours after dosing, mice were anesthetized, organs were dissected (heart, liver, spleen spleens, lung and kidney kidneys, respectively) by subcutaneous injection of a PBS solution of D-luciferin substrate (160. Mu.L, 25 mg/mL), and luciferase signals in the tissues were analyzed by imaging with a small animal imager.
According to the above procedure, when PAMAM-G0-O14, O12, A14 and A12 were used as dendrimers, the results of luciferase expression in each organ of mice were measured and quantified as shown in FIGS. 20 to 22.
FIGS. 20-21 show that the lipid nanoparticle composition based on the vector sample has strong fluorescence expression in mice after 6 hours of injection, indicating that the dendrimer of the present invention has excellent in vivo mRNA transfection effect. In FIG. 22, the expression level of fluorescence intensity of liver is gradually increased from PAMAM-G0-O12 to PAMAM-G0-O14 and from PAMAM-G0-A12 to PAMAM-G0-A14, so that the modified chain length of dendrimer has a certain effect on the in vivo transfection effect of mRNA.
Example 10
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
PAMAM-G0-O14, cholesterol, DOPE, DMG-PEG2000 and fluorescent molecule DIR (marked by fluorescent molecule DIR) prepared in example 2 are respectively dissolved in ethanol, and then mixed according to the proportion (the mass ratio of the PAMAM-G0-O14 to the cholesterol to the DOPE to the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the PAMAM-G0-O14 is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL, the concentration of the DMG-PEG2000 is 50mg/mL and the concentration of the DIR is 1mg/mL; then, the mixture was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of PAMAM-G0-O14 and a lipid component, wherein the concentration of PAMAM-G0-O14 was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, the concentration of DMG-PEG2000 was 0.5mg/mL, and the concentration of DIR was 0.004mg/mL.
Enhanced green fluorescent protein specific siRNA (Enhanced green fluorescent protein specific siRNA, siEGFP) was dissolved in enzyme-free water to give an siRNA solution at a concentration of 100 μm.
Uniformly mixing the composition and siRNA solution (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer with pH around 5.4 to obtain lipid nanoparticle composition based on PAMAM-G0-O14.
Organ distribution of siRNA dendrimer nanoparticles
Female C57 mice of 8 weeks of age were selected, each of which was injected into the mice via tail vein with a volume of 0.2mL of PAMAM-G0-O14-based lipid nanoparticle composition (injected siRNA at a dose of 0.6 mg/kg); after 4 hours of administration, organs were dissected (heart, liver, spleen, lung and kidney kidneys, respectively) and tissues were analyzed by imaging with a small animal imager.
The intensity of chemiluminescence in each organ of the mice and their quantification are shown in FIGS. 23-24. According to the results of FIGS. 23-24, the dendrimers of the present invention can target siRNA delivery to the liver and have higher chemiluminescent intensities.
Example 11
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
the carrier samples (PAMAM-G0-O12, PAMAM-G0-O14, PAMAM-G0-A12 or PAMAM-G0-A14), cholesterol, DOPE, DMG-PEG2000 and fluorescent molecule DIR (marked by fluorescent molecule DIR) prepared in example 1 or 2 were dissolved in ethanol respectively, and mixed in proportion (the mass ratio of the carrier sample to the cholesterol, DOPE and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the carrier sample is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL, the concentration of the DMG-PEG2000 is 50mg/mL and the concentration of the DIR is 1mg/mL; then, the mixed solution was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of a carrier sample and a lipid component, wherein the concentration of the carrier sample was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, the concentration of DMG-PEG2000 was 0.5mg/mL, and the concentration of DIR was 0.004mg/mL.
Firefly luciferase mRNA (Encoding firefly luciferase mRNA, mLuc) was dissolved in enzyme-free water to obtain an mRNA solution having a concentration of 1 mg/mL.
Uniformly mixing the composition and the mRNA solution (the mass ratio of the carrier sample to the mRNA is 15:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer with pH around 5.4 to obtain lipid nanoparticle composition based On carrier sample (abbreviated as G0-On/mRNA or G0-An/mRNA, n=12 or 14).
Organ distribution of mRNA dendrimer nanoparticles
Female BALB/c mice of 8 weeks of age were selected, each of which was injected into the mice via tail vein with a volume of 0.2mL of the lipid nanoparticle composition based on the carrier sample (injected mRNA dose of 0.2 mg/kg). 6 hours after administration, organs were dissected (heart, liver, spleen, lung and kidney kidneys, respectively) and tissues were analyzed by imaging with a small animal imager.
The intensity of chemiluminescence in each organ of the mice and their quantification are shown in FIGS. 25-27. From the results of FIGS. 25-27, it is demonstrated that dendrimers of the present invention can target delivery of mRNA to the liver and have higher chemiluminescent intensities.
Example 12
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
respectively dissolving PAMAM-G0-O14, cholesterol, DOPE and DMG-PEG2000 prepared in example 2 in ethanol, and mixing according to a proportion (the mass ratio of the PAMAM-G0-O14, the cholesterol, the DOPE and the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the PAMAM-G0-O14 is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixture was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of PAMAM-G0-O14 and a lipid component, wherein the concentration of PAMAM-G0-O14 was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Procollagen α1 (I) siRNA (protocol a1 (I) siRNA, sicl 1a 1) or disordered siRNA (scrambled siRNA, siNC) was dissolved in enzyme-free water to give an siRNA solution at a concentration of 100 μm.
Uniformly mixing the composition and siRNA solution (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer solution with pH of about 5.4 to obtain lipid nanoparticle composition based on PAMAM-G0-O14 (G0-O14/siNC or G0-O14/siCol1a 1).
Treatment of animal models of liver fibrosis
To create a long-term liver fibrosis animal model, corn oil and CCl were injected intraperitoneally 4 Is a mixture (CCl) 4 Is injected at a dosage of 1 μl/g, corn oil and CCl 4 The volume ratio of (2): 1) Mice injected into the body of mice were injected once on days 1, 4, 8, 11, and 15, with 0.06mL of corn oil injected each time, and mice injected with only 0.04mL of corn oil served as blank controls. Wild-type mice without any treatment were also used as WT group.
Female C57 mice of 8 weeks of age were selected, each mouse was injected with PAMAM-G0-O14-based lipid nanoparticle composition (injected siNC, siCol1a1 at a dose of 0.8 mg/kg) at a volume of 0.2mL by tail vein into the body of the mouse, G0-O14/siNC-injected mice were used as negative controls, each injection was made once on days 2, 9, 16, and finally the mice were sacrificed on day 18, liver tissues were dissected, RNA was extracted therefrom, RNA was reverse transcribed into DNA, and gene silencing efficiency of GAPDH in cells was analyzed by RT-qPCR experiments.
The silencing efficiency of the PPIB gene in mouse livers was measured and quantified as described above and shown in FIG. 28. As can be seen from fig. 28, compared with the mice injected with the G0-O14/siNC group, the expression of the Col1a1 gene in the mice injected with the G0-O14/siNC 1 group was significantly reduced, indicating that the dendrimer of the present invention has excellent siRNA in-vivo transfection effect, and can achieve significant liver fibrosis treatment effect by inhibiting the expression of the Col1a1 gene.
Example 13
Preparation of a dendrimer-based lipid nanoparticle composition comprising the steps of:
respectively dissolving PAMAM-G0-O14, cholesterol, DOPE and DMG-PEG2000 prepared in example 2 in ethanol, and mixing according to a proportion (the mass ratio of the PAMAM-G0-O14, the cholesterol, the DOPE and the DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution, wherein the concentration of the PAMAM-G0-O14 is 100mg/mL, the concentration of the cholesterol is 25mg/mL, the concentration of the DOPE is 25mg/mL and the concentration of the DMG-PEG2000 is 50mg/mL; then, the mixture was dropped into 200mM sodium acetate buffer solution having a pH of about 5.4 under vortexing conditions to obtain a composition of PAMAM-G0-O14 and a lipid component, wherein the concentration of PAMAM-G0-O14 was 1mg/mL, the concentration of cholesterol was 0.25mg/mL, the concentration of DOPE was 0.25mg/mL, and the concentration of DMG-PEG2000 was 0.5mg/mL.
Human erythropoietin (hEPO) mRNA was dissolved in enzyme-free water to give an mRNA solution at a concentration of 1 mg/mL.
Uniformly mixing the composition and mRNA solution (the mass ratio of PAMAM-G0-O14 to mRNA is 15:1), and incubating for 10min at room temperature; then diluted to the volume required for injection with 200mM sodium acetate buffer solution with pH of about 5.4 to obtain lipid nanoparticle composition (abbreviated as G0-O14/mhEPO) based on PAMAM-G0-O14.
Determination of expression and Effect of human erythropoietin (hEPO) mRNA in mice
Female BALB/c mice of 8 weeks old were selected, each of which was injected with PAMAM-G0-O14-based lipid nanoparticle composition (injected mhEPO at doses of 0.2mg/kg, 0.4mg/kg and 0.6 mg/kg) in a volume of 0.2mL by tail vein, and mice injected with only sodium acetate buffer solution were used as blank controls (buffer group). After 6 hours of administration, mice were subjected to eyeball blood collection, and serum was isolated. The serum was analyzed for hEPO protein content by Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA).
A standard curve was prepared using hEPO protein standard samples, and the expression of hEPO in serum corresponding to the experimental group was calculated from the standard curve as shown in fig. 29. As is evident from fig. 29, the expression of the mouse hEPO protein of the G0-O14/mhEPO group was significantly better than that of the control group, and it can be seen that the expression of hEPO had a positive linear relationship with the dose of mRNA delivered according to the effect of delivering different doses of mhEPO.

Claims (10)

1. A dendrimer compound for nucleic acid delivery, characterized by having a structure represented by formula I or formula II:
Figure FDA0004063015100000011
wherein, in the formula I, R is selected from alkyl with a carbon chain number of 4-14; in the formula II, R is selected from alkyl with a carbon chain number of 6-16;
Preferably, in formula I, R is selected from alkyl groups having a carbon chain number of 10 or 12; in formula II, R is selected from alkyl with carbon chain number of 12 or 14.
2. The method for preparing a dendrimer compound for nucleic acid delivery according to claim 1, comprising the steps of: uniformly mixing ethylenediamine core-polyamide-amine type 0-generation dendritic macromolecule (PAMAM-G0) with 1, 2-alkylene oxide or acrylic ester; obtaining the dendritic macromolecular compound for nucleic acid delivery after reaction, dialysis and drying;
the ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0) has the following structure:
Figure FDA0004063015100000012
3. the method of preparing a dendrimer compound for nucleic acid delivery according to claim 2, comprising one or more of the following conditions:
i. the molar ratio of the ethylenediamine core-polyamide-amine type 0-generation dendritic macromolecule (PAMAM-G0) to the 1, 2-alkylene oxide or acrylic ester is 1:2x+2, wherein x is the primary amine number of ethylenediamine core-polyamide-amine type 0 generation dendrimer (PAMAM-G0);
ii. The mixing of ethylenediamine core-polyamide-amine type 0-generation dendrimer (PAMAM-G0) with 1, 2-alkylene oxide or acrylate is carried out at room temperature;
iii, the 1, 2-alkylene oxide is 1, 2-alkylene oxide, 1, 2-dodecylene oxide, 1, 2-tetradecane oxide or 1, 2-hexadecane oxide; preferably, the 1, 2-alkylene oxide is 1, 2-epoxydodecane or 1, 2-epoxytetradecane;
iv, the acrylic ester is hexyl acrylate, octyl acrylate, dodecyl acrylate, tetradecyl acrylate or hexadecyl acrylate; preferably, the acrylic ester is dodecyl acrylate or tetradecyl acrylate;
v, the reaction temperature is 80-100 ℃, and the reaction time is 2-3 days.
4. Use of a dendrimer compound for nucleic acid delivery according to claim 1 in a nucleic acid delivery vehicle having prophylactic or therapeutic properties;
preferably, the dendrimer compound may be targeted as a carrier to deliver a prophylactic or therapeutic nucleic acid to the liver;
preferably, the nucleic acid having prophylactic or therapeutic properties is selected from one or more than two of messenger RNA (mRNA), microrna (miRNA), small interfering RNA (siRNA), RNA interference (RNAi), antisense oligonucleotide (ASO) or plasmid DNA; preferably one or a combination of more than two of messenger RNA, small interfering RNA, antisense oligonucleotides or plasmid DNA.
5. A dendrimer-based lipid nanoparticle composition (DLNP) comprising the dendrimer compound according to claim 1 and a nucleic acid having prophylactic or therapeutic properties.
6. The dendrimer-based lipid nanoparticle composition (DLNP) according to claim 5, comprising one or more of the following conditions:
i. the dendrimer compound is selected from one or more than two of the formula I or the formula II;
ii. The mass ratio of the dendrimer compound to the nucleic acid having prophylactic or therapeutic properties is 1 to 22.5:1, preferably 7.5-16:1;
iii the particle size of the lipid nanoparticle composition based on dendrimers is 60nm to 300nm;
iv, the dendrimer-based lipid nanoparticle composition further comprises a lipid component; the lipid component is one or more than two of structural lipid, steroid or polymer conjugated lipid;
preferably, the structural lipid is selected from one or a combination of more than two of DOPC, DOPE, DSPC, DPPG, DSPE, DPPC, DMPC or POPC; the steroid is selected from one or more than two of cholesterol, beta-sitosterol, rock soap sterol, stigmasterol, ergosterol or stigmastanol; the polymer conjugated lipid is a polyethylene glycol conjugated lipid; further preferably, the polyethylene glycol conjugated lipid is one or more of DMG-PEG, DSG-PEG, DSPE-PEG, DPPE-PEG, DMPE-PEG or TPGS-PEG;
Preferably, the mass ratio of lipid component to dendrimer compound is 1:1-4;
preferably, the lipid component is a combination of a structural lipid, a steroid, and a polymer conjugated lipid; further preferred, the mass ratio of dendrimer compound, steroid, structural lipid and polymer conjugated lipid is 4:1:1:2.
7. a method of preparing a dendrimer-based lipid nanoparticle composition (DLNP) according to claim 5 or 6, comprising the steps of:
(1) Dissolving a dendrimer compound in ethanol to obtain a mixed solution; then dissolving the mixed solution in a sodium acetate buffer solution with the pH value of 5-6 to obtain a dendritic macromolecule compound solution;
(2) Dissolving nucleic acid with preventive or therapeutic property in enzyme-free water to obtain nucleic acid solution;
(3) Uniformly mixing the dendrimer compound solution and the nucleic acid solution, and incubating to obtain the lipid nanoparticle composition based on the dendrimer.
8. The method of preparing a dendrimer-based lipid nanoparticle composition (DLNP) according to claim 7, comprising one or more of the following conditions:
i. the method of the step (1) is as follows: dissolving a dendrimer compound and a lipid component in ethanol to obtain a mixed solution; then dripping the mixed solution into a sodium acetate buffer solution with the pH of 5-6 under the vortex condition to obtain a dendritic macromolecule compound solution;
Preferably, the concentration of the dendrimer compound in the mixed solution is 80-120mg/mL;
preferably, the concentration of the sodium acetate buffer solution is 200mmol/L;
preferably, the concentration of the dendrimer compound in the dendrimer compound solution is 0.05-5mg/mL;
ii. In the step (2), the concentration of the nucleic acid solution is 0.01-2mg/mL;
iii, in the step (3), the incubation temperature is room temperature, and the incubation time is 5-10min;
iv, in step (3), after incubation, a dilution step is further included.
9. Use of a dendrimer compound for nucleic acid delivery according to claim 1 or a dendrimer-based lipid nanoparticle composition according to claim 5 for the prophylaxis or treatment of a disease.
10. Use of a dendrimer compound for nucleic acid delivery according to claim 1 or a dendrimer-based lipid nanoparticle composition according to claim 5 in a medicament for inducing protein expression in a subject;
preferably, the subject is a mammal, preferably a non-primate, further preferably a human.
CN202310068617.6A 2023-02-06 2023-02-06 Dendrimer compound and composition for nucleic acid delivery, and preparation methods and applications thereof Pending CN116041697A (en)

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