A dendrimer compound, composition, preparation method and application thereof for nucleic acid delivery

Abstract

The present invention provides a dendrimer compound for nucleic acid delivery, a composition thereof, and a preparation method and application thereof. The preparation steps and operation steps of the dendrimer compound of the present invention are simple, the raw materials are simple and easy to obtain, the requirements for instrument and equipment are low, the reaction conditions are mild and safe, and the yield of the target product is high. The dendrimer compound obtained by the present invention has excellent biosafety; and it is used as a nucleic acid delivery vector with preventive or therapeutic properties, having a high transfection efficiency. The dendrimer compound of the present invention can be used in combination with other lipid components in a specific ratio to form a dendrimer-based lipid nanoparticle composition for delivering preventive or therapeutic agents (such as therapeutic nucleic acids), so as to achieve the purpose of delivering nucleic acids from outside the cell to inside the cell in vitro or in vivo.

Description

Technical Field

[0001] The present invention relates to a dendrimer compound, a composition thereof, a preparation method and an application thereof for nucleic acid delivery, and belongs to the technical field of dendrimer nucleic acid delivery carriers. Background Art

[0002] Nucleic acid drugs can act at the source of genetic information transmission, so they have advantages such as strong specificity, rich gene targets, and long-lasting efficacy, and avoid the complex synthesis and purification processes of traditional drugs, which can significantly reduce costs. However, naked nucleic acid drugs are negatively charged hydrophilic macromolecules and are difficult to enter cells due to electrostatic repulsion from the cell membrane and are easily degraded rapidly by ubiquitous nucleases. Therefore, nucleic acid drugs require a protective shell to enter cells. So, for nucleic acid drugs to successfully play their role in the body, they must rely on a suitable delivery carrier.

[0003] Currently, emerging dendrimers are leading in biomedical applications due to their excellent water solubility, low toxicity, easy functionalization, and topological tunability, such as gene editing and drug delivery. Lipid nanoparticles based on dendrimers consist of 1 to 3 lipid components in addition to dendrimers, including 0 to 2 kinds of auxiliary lipids and 0 to 1 kind of PEGylated lipid. Among them, the dendrimer compound plays a key role in nucleic acid encapsulation and release, so it is crucial to develop new and efficient dendrimer compounds.

[0004] There have been reports on the application of dendrimers in biomedicine in the prior art. For example, Chinese Patent Document CN1631936A discloses a polyamide-amine type dendrimer nanomaterial. Using methyl acrylate and ethylenediamine as starting monomers, a 5th-generation polyamide-amine type (PAMAM) dendrimer is synthesized. The obtained G5 PAMAM dendrimer is purified by dialysis to obtain a class of monodisperse nanoparticles with a highly branched spherical structure, with a molecular size of 50 - 90 nanometers, a very high density of positive functional groups on the surface, and a cavity inside. After mixing this nanoparticle with plasmid DNA in a certain proportion, the target gene can be transfected into hematopoietic stem cells purified by CD34 immunomagnetic beads, and the protein synthesized by the target gene can be expressed in hematopoietic stem cells for a long time. However, the synthesis steps are complicated and the target product needs to be obtained through multiple steps of reaction; a strong base also needs to be added during the reaction process, which has a certain degree of danger. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a dendrimer compound for nucleic acid delivery, a composition thereof, a preparation method thereof and an application thereof. The preparation steps and operation steps of the dendrimer compound of the present invention are simple, the raw materials are simple and easy to obtain, the requirements for instruments and equipment are low, the reaction conditions are mild and safe, and the yield of the target product is high. The dendrimer compound obtained by the present invention has excellent biosafety; and it is used as a nucleic acid delivery vector with preventive or therapeutic effects, and has high transfection efficiency. The dendrimer compound of the present invention can be used in combination with other lipid components in a specific ratio to form a dendrimer-based lipid nanoparticle composition for delivering preventive or therapeutic agents (such as therapeutic nucleic acids), so as to achieve the purpose of delivering nucleic acids from outside the cell to inside the cell in vitro or in vivo.

[0006] The technical solution of the present invention is as follows:

[0007] A dendrimer compound for nucleic acid delivery has a structure shown in Formula I or Formula II as follows:

[0008]

[0009] Wherein, in Formula I, R is selected from alkyl groups having 4-14 carbon atoms; in Formula II, R is selected from alkyl groups having 6-16 carbon atoms.

[0010] Preferably according to the present invention, in Formula I, R is selected from alkyl groups having 10 or 12 carbon atoms; in Formula II, R is selected from alkyl groups having 12 or 14 carbon atoms.

[0011] The preparation method of the above-mentioned dendrimer compound for nucleic acid delivery includes the steps of: uniformly mixing an ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) with 1,2-epoxyalkane or acrylate; obtaining the dendrimer compound for nucleic acid delivery after reaction, dialysis and drying;

[0012] The ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) has the following structure:

[0013]

[0014] According to the present invention, the ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) can be obtained commercially or prepared by existing methods.

[0015] Preferably according to the present invention, the molar ratio of the ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) to 1,2-epoxyalkane or acrylate is 1:2x + 2, wherein x is the number of primary amines of the ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0).

[0016] Preferably according to the present invention, the mixing of ethylenediamine core-polyamide-amine generation 0 dendrimer (PAMAM-G0) and 1,2-epoxyalkane or acrylate is carried out at room temperature.

[0017] Preferably according to the present invention, the 1,2-epoxyalkane is 1,2-epoxyhexane, 1,2-epoxyoctane, 1,2-epoxydodecane, 1,2-epoxytetradecane or 1,2-epoxyhexadecane; preferably, the 1,2-epoxyalkane is 1,2-epoxydodecane or 1,2-epoxytetradecane.

[0018] Preferably according to the present invention, the acrylate is hexyl acrylate, octyl acrylate, dodecyl acrylate, tetradecyl acrylate or hexadecyl acrylate; preferably, the acrylate is dodecyl acrylate or tetradecyl acrylate.

[0019] Preferably according to the present invention, the reaction temperature is 80 - 100 °C and the reaction time is 2 - 3 days.

[0020] Use of the above dendrimer compound for nucleic acid delivery in a prophylactic or therapeutic nucleic acid delivery carrier.

[0021] Preferably according to the present invention, the dendrimer compound can targetedly deliver prophylactic or therapeutic nucleic acid to the liver as a carrier.

[0022] Preferably according to the present invention, the prophylactic or therapeutic nucleic acid is selected from one or a combination of two or more of messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), RNA interference (RNAi), antisense oligonucleotide (ASO) or plasmid DNA. Preferably, it is one or a combination of two or more of messenger RNA, small interfering RNA, antisense oligonucleotide or plasmid DNA.

[0023] A dendrimer-based lipid nanoparticle composition (DLNP) comprising the above dendrimer compound and a prophylactic or therapeutic nucleic acid.

[0024] Preferably according to the present invention, the dendrimer compound is selected from one or a combination of two or more of Formula I or Formula II.

[0025] Preferably according to the present invention, the mass ratio of the dendrimer compound to the prophylactic or therapeutic nucleic acid is 1 - 22.5:1, preferably 7.5 - 16:1.

[0026] Preferably according to the present invention, the particle size of the dendrimer-based lipid nanoparticle composition is 60 nm - 300 nm.

[0027] Preferably according to the present invention, the dendrimer-based lipid nanoparticle composition further comprises a lipid component; the lipid component is one or a combination of two or more of a structural lipid, a steroid or a polymer-conjugated lipid.

[0028] Preferably, the structural lipid is selected from one or a combination of two or more of DOPC, DOPE, DSPC, DPPG, DSPE, DPPC, DMPC or POPC; the steroid is selected from one or a combination of two or more of cholesterol, β-sitosterol, saxisterol, stigmasterol, ergosterol or stigmastanol; the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid; further preferably, the polyethylene glycol-conjugated lipid is one or a combination of two or more of DMG-PEG, DSG-PEG, DSPE-PEG, DPPE-PEG, DMPE-PEG or TPGS-PEG.

[0029] Preferably, the mass ratio of the lipid component to the dendrimer compound is 1:1-4.

[0030] Preferably, the lipid component is a combination of a structural lipid, a steroid and a polymer-conjugated lipid; further preferably, the mass ratio of the dendrimer compound, the steroid, the structural lipid and the polymer-conjugated lipid is 4:1:1:2.

[0031] The preparation method of the above dendrimer-based lipid nanoparticle composition (DLNP) comprises the steps:

[0032] (1) Dissolve the dendrimer compound in ethanol to obtain a mixed solution; then dissolve the mixed solution in a sodium acetate buffer solution with a pH of 5-6 to obtain a dendrimer compound solution;

[0033] (2) Dissolve the prophylactic or therapeutic nucleic acid in enzyme-free water to obtain a nucleic acid solution;

[0034] (3) Mix the dendrimer compound solution and the nucleic acid solution evenly and incubate to obtain the dendrimer-based lipid nanoparticle composition.

[0035] Preferably according to the present invention, the method of step (1) is as follows: dissolve the dendrimer compound and the lipid component in ethanol to obtain a mixed solution; then drop the mixed solution into a sodium acetate buffer solution with a pH of 5-6 under vortex conditions to obtain a dendrimer compound solution.

[0036] Preferably, in the mixed solution, the concentration of the dendrimer compound is 80-120 mg / mL.

[0037] Preferably, the concentration of the sodium acetate buffer solution is 200 mmol / L.

[0038] Preferably, in the dendrimer compound solution, the concentration of the dendrimer compound is 0.05 - 5 mg / mL.

[0039] According to a preference of the present invention, in step (2), the concentration of the nucleic acid solution is 0.01 - 2 mg / mL.

[0040] According to a preference of the present invention, in step (3), the incubation temperature is room temperature and the incubation time is 5 - 10 min.

[0041] According to a preference of the present invention, after incubation in step (3), there is further a step of dilution to dilute to a required volume.

[0042] Use of the above dendrimer compound for nucleic acid delivery or a lipid nanoparticle composition based on a dendrimer in the prevention or treatment of diseases.

[0043] According to the present invention, the diseases are acute liver injury, acute liver failure, long - term liver fibrosis, etc.

[0044] Use of the above dendrimer compound for nucleic acid delivery or a lipid nanoparticle composition based on a dendrimer in a medicament for inducing protein expression in a subject.

[0045] According to a preference of the present invention, the subject is a mammal, preferably a non - primate animal, and more preferably a human.

[0046] The preparation route of the dendrimer compound for nucleic acid delivery according to the present invention is as follows:

[0047]

[0048] The technical features and beneficial effects of the present invention are as follows:

[0049] 1. The preparation steps and operation steps of the dendrimer compound of the present invention are simple, the raw materials are simple and easily available, the requirements for instrument and equipment are low, the reaction conditions are mild and safe, and the yield of the target product is high.

[0050] 2. The dendrimer compound of the present invention has low toxicity, so that the dendrimer has excellent biosafety; and it is used as a nucleic acid delivery vector with preventive or therapeutic properties and has high transfection efficiency. While the ethylenediamine core - polyamide - amine generation 0 dendrimer (PAMAM - G0) has high toxicity and poor transfection effect.

[0051] 3. 1,2-epoxyalkanes or acrylates with different structures in the present invention have an important impact on transfection efficiency. It can be seen from the examples of the present invention that from 1,2-epoxyhexane or hexyl acrylate to 1,2-epoxytetradecane or tetradecyl acrylate, the transfection efficiency increases with the increase of the chain length. However, when it comes to 1,2-epoxytetradecane or tetradecyl acrylate to 1,2-epoxyhexadecane or hexadecyl acrylate, the transfection efficiency decreases with the increase of the chain length. Therefore, 1,2-epoxytetradecane or tetradecyl acrylate is a node. Before this node, the transfection efficiency increases with the increase of the chain length, and after this node, the transfection efficiency decreases with the increase of the chain length. In summary, only 1,2-epoxyalkanes or acrylates with specific structures in the present invention can achieve the excellent effects of the present invention.

[0052] 4. The molar ratio of ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) to 1,2-epoxyalkane or acrylate in the present invention needs to be appropriate. For example, when the molar ratio of ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) to 1,2-epoxyalkane or acrylate is 1:7, other preparation methods and conditions are as described in Example 1 or 2; the obtained dendrimer compound is used to prepare a lipid nanoparticle composition according to the method of Example 6, and EGFP siRNA is delivered into Hela-GFP cells, and it is photographed with a fluorescence microscope. It is found that there is no gene silencing effect, indicating that its transfection effect is poor. Similarly, when the molar ratio of ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) to 1,2-epoxyalkane or acrylate is 1:8, the transfection effect is also not as good as that of the present invention. In conclusion, the molar ratio of ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) to 1,2-epoxyalkane or acrylate in the present invention needs to be appropriate. If it is not appropriate, the excellent effects of the present invention cannot be achieved.

[0053] 5. The dendrimer compound of the present invention, or used in combination with other lipid components in a specific ratio, loads nucleic acid drugs with prophylactic or therapeutic effects; thereby achieving the purpose of delivering nucleic acid drugs from the outside of cells to the inside of cells in vitro or in vivo.

[0054] 6. The lipid nanoparticle composition (DLNP) based on dendrimers of the present invention can target and deliver nucleic acids into cells and escape from lysosomes, enabling the nucleic acids to function inside the cells, thereby enabling the cells to produce relevant proteins for therapeutic or prophylactic purposes. The structural lipids used in the present invention support the formation of the lamellar lipid bilayer structure and stabilize its structural arrangement; steroids have strong membrane fusion properties and promote intracellular uptake of nucleic acids; polymer-conjugated lipids are located on the surface of the lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation, and increasing stability. The ratio relationship between the lipid components and the dendrimer compounds in the present invention has a certain influence on the transfection effect. By controlling the only variable, three polymer-conjugated lipids with different structures were selected for experiments, namely: DMG-PEG2000, TPGS-PEG2000, and DSPE-PEG2000. The research results show that the transfection effects of DMG-PEG2000 and TPGS-PEG2000 are similar, and the transfection effect of DSPE-PEG2000 is significantly lower than the other two. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 1H-NMR spectrum of ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0). 1 1H-NMR spectrum.

[0056] Figure 2 1H-NMR spectrum of G0-O6. 1 1H-NMR spectrum.

[0057] Figure 3 1H-NMR spectrum of G0-O8. 1 1H-NMR spectrum.

[0058] Figure 4 1H-NMR spectrum of G0-O12. 1 1H-NMR spectrum.

[0059] Figure 5 1H-NMR spectrum of G0-O14. 1 1H-NMR spectrum.

[0060] Figure 6 1H-NMR spectrum of G0-O16. 1 1H-NMR spectrum.

[0061] Figure 7 Size diagrams of G0-O6, G0-O8, G0-O12, G0-O14, G0-O16, G0-A6, G0-A8, G0-A12, G0-A14, and G0-A16 in Example 3. The vertical axis represents the particle size.

[0062] Figure 8ζ-potential diagrams of G0-O6, G0-O8, G0-O12, G0-O14, G0-O16, G0-A6, G0-A8, G0-A12, G0-A14, and G0-A16 in Example 3. The ordinate is the ζ-potential.

[0063] Figure 9 Size diagrams 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, and G0-A16 / siRNA in Example 4. The ordinate is the particle size.

[0064] Figure 10 ζ-potential diagrams 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, and G0-A16 / siRNA in Example 4. The ordinate is the ζ-potential.

[0065] Figure 11 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, G0-A14 / siRNA, and G0-A16 / siRNA in Example 4. The ordinate is the siRNA encapsulation efficiency.

[0066] Figure 12 Size diagrams 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, and G0-A16 / mRNA in Example 5. The ordinate is the particle size.

[0067] Figure 13 ζ-potential diagrams 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, and G0-A16 / mRNA in Example 5. The ordinate is the ζ-potential.

[0068] Figure 14 For 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, G0-A14 / mRNA in Example 5. The vertical axis represents the mRNA encapsulation efficiency.

[0069] Figure 15 For the gene silencing efficiency of EGFP in cells after co-culturing the lipid nanoparticle composition with HeLa-GFP cells in Example 6. The vertical axis represents the gene silencing efficiency.

[0070] Figure 16 For the fluorescence quantification of EGFP in cells after co-culturing the lipid nanoparticle composition with HeLa-GFP cells in Example 6. The vertical axis represents the average fluorescence intensity.

[0071] Figure 17 For the gene expression efficiency of EGFP in cells after co-culturing the lipid nanoparticle composition with HeLa cells in Example 7. The vertical axis represents the expression efficiency of the EGFP gene in cells.

[0072] Figure 18 For the fluorescence quantification of EGFP in cells after co-culturing the lipid nanoparticle composition with HeLa cells in Example 7. The vertical axis represents the average fluorescence intensity.

[0073] Figure 19 For the expression of the PPIB gene in the liver after tail vein injection of PAMAM-G0-O14 / siRNA and PAMAM-G0-O14 / siNC for 48 h in Example 8. The vertical axis represents the expression level of PPIB mRNA in liver tissue.

[0074] Figure 20 For the in vivo bioluminescence images of mice after intravenous injection of G0-O12 / mLuc and G0-O14 / mLuc for 6 h in Example 9.

[0075] Figure 21 For the in vivo bioluminescence images of mice after intravenous injection of G0-A12 / mLuc and G0-A14 / mLuc for 6 h in Example 9.

[0076] Figure 22 For the quantitative results of in vivo bioluminescence of mice after intravenous injection of G0-O12 / mLuc, G0-O14 / mLuc, G0-A12 / mLuc and G0-A14 / mLuc for 6 h in Example 9. The vertical axis represents the total fluorescence intensity of the tissue.

[0077] Figure 23Enrichment of nanoparticles in major organs (heart, liver, spleen, lung, and kidney) 4 h after intravenous injection of G0-O14 / siRNA labeled with the fluorescent molecule DIR in Example 10.

[0078] Figure 24 Quantitative results of fluorescence signals of nanoparticles in major organs (heart, liver, spleen, lung, and kidney) 4 h after intravenous injection of G0-O14 / siRNA labeled with the fluorescent molecule DIR in Example 10. The ordinate is the efficiency of chemiluminescence.

[0079] Figure 25 Enrichment of nanoparticles in major organs (heart, liver, spleen, lung, and kidney) 4 h after intravenous injection of G0-O12 / mRNA and G0-O14 / mRNA labeled with the fluorescent molecule DIR in Example 11.

[0080] Figure 26 Enrichment of nanoparticles in major organs (heart, liver, spleen, lung, and kidney) 4 h after intravenous injection of G0-A12 / mRNA and G0-A14 / mRNA labeled with the fluorescent molecule DIR in Example 11.

[0081] Figure 27 Quantitative results of fluorescence signals of nanoparticles in major organs (heart, liver, spleen, lung, and kidney) 4 h after intravenous injection of G0-O12 / mRNA, G0-O14 / mRNA, G0-A12 / mRNA, and G0-A14 / mRNA labeled with the fluorescent molecule DIR in Example 11. The ordinate is the efficiency of chemiluminescence.

[0082] Figure 28 Expression of Col1a1 mRNA in mouse liver tissue after treatment in Example 12. The ordinate is the expression level of Col1a1 mRNA in the liver.

[0083] Figure 29 Quantification of the content of hEPO in serum by ELISA experiment 6 h after tail vein injection of G0-O14 / mhEPO in Example 13. The ordinate is the expression level of hEPO protein in serum. Detailed implementation manners

[0084] The following details the implementation process and beneficial effects of the present invention through specific examples to help readers better understand the essence and characteristics of the present invention, which shall not be construed as a limitation on the scope of the examples of this case.

[0085] Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are all commercially available unless otherwise specified.

[0086] Example 1

[0087] Synthesis of Dendrimer Compound PAMAM-G0-An for Nucleic Acid Delivery

[0088] Steps: Mix ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) (40 mg) with acrylate (An, n = 6, 8, 12, 14, or 16, i.e., one of 120.9 mg hexyl acrylate, 140.5 mg octyl acrylate, 187.0 mg dodecyl acrylate, 207.8 mg tetradecyl acrylate, or 229.5 mg hexadecyl acrylate) evenly at room temperature, and react the mixture at 90 °C for 2.5 days. Put the reaction solution into a dialysis bag and dialyze it in ethanol for 24 hours (cut-off molecular weight: 1000 D), replacing the fresh ethanol every 1.5 h during this period, and then dry to remove ethanol to obtain a pale yellow oily liquid compound PAMAM-G0-An (abbreviation G0-An, n = 6, 8, 12, 14, or 16).

[0089] Example 2

[0090] Synthesis of Dendrimer Compound PAMAM-G0-On for Nucleic Acid Delivery

[0091] Steps: Mix ethylenediamine core-polyamidoamine generation 0 dendrimer (PAMAM-G0) (40 mg) with 1,2-epoxyalkane (On, n = 6, 8, 12, 14, or 16, i.e., one of 77.5 mg 1,2-epoxyhexane, 99.3 mg 1,2-epoxyoctane, 142.7 mg 1,2-epoxydodecane, 164.4 mg 1,2-epoxytetradecane, or 186.1 mg 1,2-epoxyhexadecane) evenly at room temperature, and react the mixture at 90 °C for 2.5 days. Put the reaction solution into a dialysis bag and dialyze it in ethanol for 24 hours (cut-off molecular weight: 1000 D), replacing the fresh ethanol every 1.5 h during this period, and then dry to remove ethanol to obtain a colorless oily liquid compound PAMAM-G0-On (abbreviation G0-On, n = 6, 8, 12, 14, or 16).

[0092] For the product PAMAM-G0-On 1 The 1H-NMR spectrum is as Figures 2 - 6 shown. At the same time, the compound PAMAM-G0 was also characterized, and its 1 1H-NMR spectrum is as Figure 1 shown. It can be seen from the figure that the target product was successfully prepared in the present invention.

[0093] Example 3

[0094] Preparation of a composition of a dendrimer compound and a lipid component, including the steps:

[0095] Dissolve the dendrimer compound (one of the dendrimer compounds prepared in Example 1 or Example 2), cholesterol, DOPE, and DMG-PEG2000 in ethanol respectively, and then mix them according to the ratio (the mass ratio of the dendrimer compound, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the dendrimer compound is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition, wherein the concentration of the dendrimer compound is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0096] Perform particle size detection and Zeta potential analysis on the obtained composition using standard detection methods on a Zetasizer nano instrument of Malvern Corporation. The particle size detection results are as Figure 7 shown and the Zeta potential results are as Figure 8 shown. It can be seen from the figure that the hydrated particle sizes of the compositions of the dendrimer compound and the lipid components are all between 80 - 250 nm. Except that the ζ-potential of G0-O6 is negative, the other ζ-potentials are all positive. Since nucleic acid molecules are negatively charged, the compositions of the dendrimer compound and the lipid components with positive ζ-potentials are more likely to bind to them through electrostatic interactions.

[0097] Example 4

[0098] Preparation of a lipid nanoparticle composition based on a dendrimer, comprising the steps:

[0099] Dissolve the dendrimer compound (one of the dendrimer compounds prepared in Example 1 or Example 2), cholesterol, DOPE, and DMG-PEG2000 in ethanol respectively, and then mix them according to the ratio (the mass ratio of the dendrimer compound, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the dendrimer compound is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of the dendrimer compound and lipid components, where the concentration of the dendrimer compound is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0100] Dissolve the enhanced green fluorescent protein-specific siRNA (siEGFP) in enzyme-free water to obtain an siRNA solution with a concentration of 20 μM.

[0101] Mix the above composition of the dendrimer compound and lipid components and the siRNA solution evenly (the mass ratio of the dendrimer compound to siRNA is 7.5:1), and incubate at room temperature for 10 min to obtain a dendrimer-based lipid nanoparticle composition (abbreviated as G0-On / siRNA or G0-An / siRNA, n = 6, 8, 12, 14, or 16).

[0102] According to the instructions of the Ribogreen kit, test and calculate the encapsulation efficiency of siRNA; use the standard detection method on the Zetasizer nano instrument of Malvern Company to detect the particle size and analyze the Zeta potential of the above dendrimer-based lipid nanoparticle composition.

[0103] The particle size detection results of the dendrimer-based lipid nanoparticle composition prepared in this example are as Figure 9 shown, the Zeta potential results are as Figure 10 shown, and the detection results of the siRNA encapsulation efficiency are as Figure 11 shown.

[0104] As can be seen from the figure, the hydrated particle sizes of dendrimer-based lipid nanoparticle compositions (DLNP) are all between 120 - 300 nm. Since siRNA molecules are negatively charged, after the composition of the dendrimer compound and the lipid component binds to it, the ζ-potential of most DLNP is negative. Additionally, overall, the encapsulation efficiency of G0-On / siRNA is a bit better.

[0105] Example 5

[0106] Preparation of dendrimer-based lipid nanoparticle compositions includes the steps:

[0107] Dissolve the dendrimer compound (one of the dendrimer compounds prepared in Example 1 or Example 2), cholesterol, DOPE, and DMG-PEG2000 in ethanol respectively, and then mix them according to the ratio (the mass ratio of the dendrimer compound, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the dendrimer compound is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of the dendrimer compound and the lipid component, where the concentration of the dendrimer compound is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0108] Enhanced green fluorescent protein specific mRNA (mEGFP) is dissolved in enzyme-free water to obtain an mRNA solution with a concentration of 0.1 mg / mL.

[0109] Mix the above composition of the dendrimer compound and the lipid component and the mRNA solution evenly (the mass ratio of the dendrimer compound and mRNA is 15:1), and incubate at room temperature for 10 min to obtain a dendrimer-based lipid nanoparticle composition (abbreviated as G0-On / mRNA or G0-An / mRNA, n = 6, 8, 12, 14, or 16).

[0110] According to the instructions of the Ribogreen kit, test and calculate the encapsulation efficiency of mRNA; use the standard detection method on the Zetasizer nano instrument of Malvern Company to detect the particle size and analyze the Zeta potential of the above dendrimer-based lipid nanoparticle composition.

[0111] The particle size detection results of the dendrimer-based lipid nanoparticle composition prepared in this example are as follows Figure 12 shown, and the Zeta potential results are as follows Figure 13 shown, and the detection results of the mRNA encapsulation rate are as follows Figure 14 shown.

[0112] As can be seen from the figure, the hydrated particle sizes of the dendrimer-based lipid nanoparticle composition (DLNP) are all between 90 - 280 nm. The mRNA molecule is negatively charged, but after the combination of the dendrimer compound and the lipid component composition with it, the ζ-potential of DLNP is still positive, which may be because the mass ratios of the dendrimer compound and the lipid component composition to the two RNAs are different. In addition, except for G0-A14 / mRNA, the mRNA encapsulation rates of all DLNP are higher than 60%, and the encapsulation rates of G0-O8 / mRNA, G0-O12 / mRNA, and G0-O14 / mRNA are the best.

[0113] Example 6

[0114] Preparation of the dendrimer-based lipid nanoparticle composition, including the steps:

[0115] Dissolve the 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 then mix them according to the ratio (the mass ratio of the dendrimer compound, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the carrier sample is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain the composition of the dendrimer compound and the lipid component, where the concentration of the dendrimer compound is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0116] Dissolve enhanced green fluorescent protein-specific siRNA (siEGFP) in enzyme-free water to obtain an siRNA solution with a mass concentration of 20 μM.

[0117] Mix the composition of the above dendrimer compound and lipid component with the siRNA solution evenly (the mass ratio of the dendrimer compound to siRNA is 7.5:1), and incubate at room temperature for 10 min; then dilute it 5 times with 200 mM sodium acetate buffer with a pH of about 5.4 to obtain a lipid nanoparticle composition based on the carrier sample (abbreviated as G0-On / siRNA or G0-An / siRNA, n = 6, 8, 12, 14 or 16).

[0118] At the same time, use the commercially available positive control reagent RNAiMAX as the carrier, and the specific usage method is carried out according to the instructions to obtain a lipid nanoparticle composition based on the carrier sample, abbreviated as RNAiMAX / siRNA.

[0119] In vitro siRNA delivery efficiency

[0120] In a 37 °C carbon dioxide incubator, after culturing Hela-GFP cells in DMEM medium with 10% (volume fraction) FBS until they adhere, replace the fresh above-mentioned medium, add the lipid nanoparticle composition based on the carrier sample prepared above (the addition amount is about 50 μL / mL cell solution), and perform transfection under these conditions (37 °C) for 48 h. Test the in vitro siRNA delivery efficiency.

[0121] Evaluate the in vitro delivery efficiency of siRNA by the degree of EGFP gene silencing in cells. The test results of the in vitro siRNA delivery efficiency of different delivery carriers are shown in Figure 15 and the fluorescence quantitative results are as shown in Figure 16 .

[0122] Figure 15 It shows that the silencing effects of G0-O14, G0-A14, and G0-A12 in delivering siRNA are significantly better than those of the commercially available positive control reagent RNAiMAX. It can also be seen from Figure 16 that the fluorescence intensity of Hela-GFP cells added with G0-O14 / siRNA, G0-A14 / siRNA, and G0-A12 / siRNA is significantly weaker than that of the commercially available positive control reagent RNAiMAX, which also verifies Figure 15 's conclusion. In summary, the dendrimers PAMAM-G0-On and PAMAM-G0-An synthesized in the present invention can be used as siRNA delivery carriers and can achieve excellent transfection efficiency.

[0123] Example 7

[0124] Preparation of a lipid nanoparticle composition based on a dendrimer, including the steps:

[0125] Dissolve the dendritic macromolecule compound of the carrier sample (one of the dendritic macromolecule compounds prepared in Example 1 or Example 2), cholesterol, DOPE, and DMG-PEG2000 in ethanol respectively, and then mix them according to the ratio (the mass ratio of the dendritic macromolecule compound, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the carrier sample is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of the dendritic macromolecule compound and the lipid component, wherein the concentration of the dendritic macromolecule compound is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0126] Dissolve enhanced green fluorescent protein mRNA (mEGFP) in enzyme-free water to obtain an mRNA solution with a mass concentration of 0.1 mg / mL.

[0127] Mix the above composition of the dendritic macromolecule compound and the lipid component and the mRNA solution evenly (the mass ratio of the dendritic macromolecule compound to mRNA is 15:1), and incubate at room temperature for 10 min; then dilute it 5 times with a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a lipid nanoparticle composition based on the carrier sample (abbreviated as G0-On / mRNA or G0-An / mRNA, n = 6, 8, 12, 14, or 16).

[0128] At the same time, use the commercially available positive control reagent RNAiMAX as the carrier, and the specific usage method is carried out according to the instruction manual to obtain a lipid nanoparticle composition based on the carrier sample, abbreviated as RNAiMAX / mRNA.

[0129] In vitro mRNA delivery efficiency

[0130] In a 37 °C carbon dioxide incubator, after culturing Hela cells in DMEM medium containing 10% (volume fraction) FBS until they reach the adherent state, replace the fresh medium with the above-prepared lipid nanoparticle composition based on the carrier sample (the addition amount is about 50 μL / mL cell solution), and perform transfection for 24 hours under this condition (37 °C). Test the in vitro mRNA delivery efficiency.

[0131] The in vitro delivery efficiency of mRNA was evaluated by the high or low expression of the EGFP gene in cells. The test results of the in vitro mRNA delivery efficiency of different delivery vectors are shown in Figure 17 and the fluorescence quantitative results are as Figure 18 shown.

[0132] Figure 17 It was shown that the expression efficiencies of G0-O8, G0-O12, G0-O14, G0-O16, G0-A8, G0-A12, and G0-A14 in delivering mRNA were significantly better than those of the commercially available positive control reagent RNAiMAX. It can be seen from Figure 18 that the fluorescence intensities of Hela cells added with G0-O12 / mRNA, G0-O14 / mRNA, G0-O16 / mRNA, G0-A12 / mRNA, and G0-A14 / mRNA were significantly stronger than those of the commercially available positive control reagent RNAiMAX, which also confirmed and further defined the Figure 17 conclusion. In summary, the dendrimers PAMAM-G0-On and PAMAM-G0-An synthesized in the present invention can be used as delivery vectors for mRNA and can achieve excellent transfection efficiency.

[0133] Example 8

[0134] Preparation of a lipid nanoparticle composition based on a dendrimer, including the steps of:

[0135] Dissolve PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 prepared in Example 2 in ethanol respectively, and then mix them according to the ratio (the mass ratio of PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of PAMAM-G0-O14 is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of PAMAM-G0-O14 and lipid components, wherein the concentration of PAMAM-G0-O14 is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0136] Dissolve peptidylprolyl isomerase B siRNA (siPPIB) or scrambled siRNA (siNC) in enzyme-free water to obtain a 100 μM siRNA solution.

[0137] Mix the above composition and the siRNA solution evenly (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubate at room temperature for 10 min; then dilute it to the volume required for injection with 200 mM sodium acetate buffer with a pH of about 5.4 to obtain a lipid nanoparticle composition based on PAMAM-G0-O14.

[0138] In vivo siRNA delivery effect test

[0139] Select 8-week-old female C57 mice, and inject 0.2 mL of the lipid nanoparticle composition based on PAMAM-G0-O14 (the amount of siPPIB injected is 0.3 mg / kg, 0.6 mg / kg or 1.2 mg / kg, and the amount of siNC is 1.2 mg / kg) into each mouse through the tail vein as a negative control; 48 hours after administration, sacrifice the mice, dissect the liver tissue, extract RNA from it, reverse transcribe the RNA into DNA, and then analyze the gene silencing efficiency of GAPDH in the cells by a real-time fluorescence quantitative PCR instrument (Real time quantitative PCR, RT-qPCR).

[0140] According to the above process, when the dendrimer is PAMAM-G0-O14, the silencing efficiency of the PPIB gene in the mouse liver is measured and quantified as Figure 19 shown.

[0141] From Figure 19 it can be seen that 48 hours after injecting PAMAM-G0-O14 / siRNA, compared with the mice in the PAMAM-G0-O14 / siNC injection group, the expression of the PPIB gene in the mice in the PAMAM-G0-O14 / siPPIB injection group with different doses is significantly decreased, indicating that the dendrimer described in the present invention has excellent in vivo transfection effect of siRNA, and there is a positive linear relationship between the PPIB gene silencing effect and the dose of delivered siPPIB.

[0142] Example 9

[0143] Preparation of a lipid nanoparticle composition based on a dendrimer, including the steps:

[0144] The carrier samples prepared in Example 1 or 2 (PAMAM-G0-O12, PAMAM-G0-O14, PAMAM-G0-A12 or PAMAM-G0-A14), cholesterol, DOPE, and DMG-PEG2000 were respectively dissolved in ethanol, and then mixed according to the ratio (the mass ratio of the carrier sample, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the carrier sample is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, the mixed solution was dropped into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of the carrier sample and lipid components, wherein the concentration of the carrier sample is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0145] Dissolve firefly luciferase mRNA (Encoding firefly luciferase mRNA, mLuc) in enzyme-free water to obtain an mRNA solution with a concentration of 1 mg / mL.

[0146] Mix the above composition and the mRNA solution evenly (the mass ratio of the carrier sample to mRNA is 15:1), and incubate at room temperature for 10 min; then dilute with a 200 mM sodium acetate buffer solution with a pH of about 5.4 to the volume required for injection to obtain a lipid nanoparticle composition based on the carrier sample (abbreviated as G0-On / mRNA or G0-An / mRNA, n = 12 or 14).

[0147] In vivo mRNA delivery effect test

[0148] Select 8-week-old female BALB / c mice, and inject 0.2 mL of the lipid nanoparticle composition based on the carrier sample (the amount of mLuc injected is 0.2 mg / kg) into each mouse through the tail vein; 6 hours after administration, anesthetize the mice, and inject a PBS solution (160 μL, 25 mg / mL) of D-luciferin substrate subcutaneously, and dissect to obtain organs (the dissected organs are heart, liver, spleen, lung, and kidneys), and use a small animal imager to image and analyze the luciferase signal in the tissue.

[0149] According to the above process, when the dendrimers are respectively selected as PAMAM-G0-O14, O12, A14, and A12, the expression results of luciferase in each organ of the mice are measured and quantified as Figures 20 - 22 shown.

[0150] Figures 20 - 21 It was shown that 6 hours after injecting the lipid nanoparticle composition based on the dendritic macromolecule sample, strong fluorescence expression was observed in the mice, indicating that the dendritic macromolecule described in the present invention has excellent in vivo mRNA transfection effect. Figure 22 The fluorescence intensity expression in the liver showed an increasing trend from PAMAM-G0-O12 to PAMAM-G0-O14 and from PAMAM-G0-A12 to PAMAM-G0-A14. Therefore, it can be said that the chain length modified by the dendritic macromolecule has a certain influence on the in vivo mRNA transfection effect.

[0151] Example 10

[0152] Preparation of the lipid nanoparticle composition based on the dendritic macromolecule, including the steps:

[0153] Dissolve PAMAM-G0-O14, cholesterol, DOPE, DMG-PEG2000, and the fluorescent molecule DIR (labeled with the fluorescent molecule DIR) prepared in Example 2 in ethanol respectively, and then mix them according to the ratio (the mass ratio of PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of PAMAM-G0-O14 is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, the concentration of DMG-PEG2000 is 50 mg / mL, and the concentration of DIR is 1 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of PAMAM-G0-O14 and lipid components, wherein the concentration of PAMAM-G0-O14 is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, the concentration of DMG-PEG2000 is 0.5 mg / mL, and the concentration of DIR is 0.004 mg / mL.

[0154] Enhanced green fluorescent protein specific siRNA (siEGFP) was dissolved in enzyme-free water to obtain a siRNA solution with a concentration of 100 μM.

[0155] Mix the above composition and siRNA solution evenly (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubate at room temperature for 10 min; then dilute it to the volume required for injection with 200 mM sodium acetate buffer at about pH 5.4 to obtain a lipid nanoparticle composition based on PAMAM-G0-O14.

[0156] Organ distribution of siRNA dendrimer nanoparticles

[0157] Select 8-week-old female C57 mice, and inject 0.2 mL of the lipid nanoparticle composition based on PAMAM-G0-O14 (the dose of siRNA injected is 0.6 mg / kg) into each mouse through the tail vein; 4 hours after administration, dissect and take organs (the dissected organs are heart, liver, spleen, lung and kidneys), and use a small animal imager to perform imaging analysis on the tissues.

[0158] The chemiluminescence intensity and its quantification value in each organ of the mice are as Figures 23 - 24 . According to Figures 23 - 24 the results, the dendrimer of the present invention can targetedly deliver siRNA to the liver and has a relatively high chemiluminescence intensity.

[0159] Example 11

[0160] Preparation of a lipid nanoparticle composition based on a dendrimer, comprising the steps of:

[0161] Dissolve the carrier samples (PAMAM-G0-O12, PAMAM-G0-O14, PAMAM-G0-A12 or PAMAM-G0-A14) prepared in Example 1 or 2, cholesterol, DOPE, DMG-PEG2000, and the fluorescent molecule DIR (labeled with the fluorescent molecule DIR) in ethanol respectively, and then mix them according to the ratio (the mass ratio of the carrier sample, cholesterol, DOPE, DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of the carrier sample is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, the concentration of DMG-PEG2000 is 50 mg / mL, and the concentration of DIR is 1 mg / mL; then, under vortex conditions, drop the mixed solution into 200 mM sodium acetate buffer at about pH 5.4 to obtain a composition of the carrier sample and lipid components, wherein the concentration of the carrier sample is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, the concentration of DMG-PEG2000 is 0.5 mg / mL, and the concentration of DIR is 0.004 mg / mL.

[0162] Dissolve firefly luciferase mRNA (Encoding firefly luciferase mRNA, mLuc) in enzyme-free water to obtain an mRNA solution with a concentration of 1 mg / mL.

[0163] Mix the above composition and the mRNA solution evenly (the mass ratio of the carrier sample to mRNA is 15:1), and incubate at room temperature for 10 min; then dilute with 200 mM sodium acetate buffer with a pH of about 5.4 to the volume required for injection to obtain a lipid nanoparticle composition based on the carrier sample (abbreviated as G0-On / mRNA or G0-An / mRNA, n = 12 or 14).

[0164] Organ distribution of mRNA dendrimer nanoparticles

[0165] Select 8-week-old female BALB / c mice, and inject 0.2 mL of the lipid nanoparticle composition based on the carrier sample (the dose of injected mRNA is 0.2 mg / kg) into each mouse through the tail vein. After 6 hours of administration, dissect and take the organs (the dissected organs are heart, liver, spleen, lung, and kidneys), and use a small animal imager to perform imaging analysis on the tissues.

[0166] The chemiluminescence intensity and its quantification value in each organ of the mice are as Figures 25 - 27 . According to Figures 25 - 27 the results, it shows that the dendrimer of the present invention can targetedly deliver mRNA to the liver and has a relatively high chemiluminescence intensity.

[0167] Example 12

[0168] Preparation of a lipid nanoparticle composition based on a dendrimer, including the steps:

[0169] Dissolve the PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 prepared in Example 2 in ethanol respectively, and then mix them according to the ratio (the mass ratio of PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 is 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of PAMAM-G0-O14 is 100 mg / mL, the concentration of cholesterol is 25 mg / mL, the concentration of DOPE is 25 mg / mL, and the concentration of DMG-PEG2000 is 50 mg / mL; then, under vortex conditions, drop the mixed solution into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of PAMAM-G0-O14 and lipid components, wherein the concentration of PAMAM-G0-O14 is 1 mg / mL, the concentration of cholesterol is 0.25 mg / mL, the concentration of DOPE is 0.25 mg / mL, and the concentration of DMG-PEG2000 is 0.5 mg / mL.

[0170] Dissolve procollagen α1(I) siRNA (siCol1a1) or scrambled siRNA (siNC) in enzyme-free water to obtain a siRNA solution with a concentration of 100 μM.

[0171] Mix the above composition and the siRNA solution evenly (the mass ratio of PAMAM-G0-O14 to siRNA is 7.5:1), and incubate at room temperature for 10 min; then dilute it to the volume required for injection with a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a lipid nanoparticle composition based on PAMAM-G0-O14 (abbreviated as G0-O14 / siNC or G0-O14 / siCol1a1).

[0172] Treatment of animal models of liver fibrosis

[0173] To establish a long-term animal model of liver fibrosis, inject a mixture of corn oil and CCl4 (the injection dose of CCl4 is 1 μL / g, and the volume ratio of corn oil to CCl4 is 2:1) into mice by intraperitoneal injection, inject once on each of the 1st, 4th, 8th, 11th, and 15th days, 0.06 mL each time, and use mice injected with only 0.04 mL of corn oil as a blank control. At the same time, use wild-type mice without any treatment as the WT group.

[0174] Eight-week-old female C57 mice were selected. Each mouse was intravenously injected through the tail vein with 0.2 mL of a lipid nanoparticle composition based on PAMAM-G0-O14 (the doses of siNC and siCol1a1 injected were 0.8 mg / kg) into the mouse body. The mice injected with G0-O14 / siNC were used as negative controls and were injected once each on days 2, 9, and 16. Finally, the mice were sacrificed on day 18, the liver tissues were dissected, RNA was extracted from them, the RNA was reverse transcribed into DNA, and then the gene silencing efficiency of GAPDH in the cells was analyzed by RT-qPCR experiments.

[0175] According to the above process, the silencing efficiency of the PPIB gene in the mouse liver was measured and quantified as Figure 28 shown. It can be seen from Figure 28 that compared with the mice in the G0-O14 / siNC injection group, the expression of the Col1a1 gene in the mice in the G0-O14 / siCol1a1 injection group was significantly reduced, indicating that the dendrimer described in the present invention has excellent in vivo transfection effect of siRNA and can achieve an obvious liver fibrosis treatment effect by inhibiting the expression of the Col1a1 gene.

[0176] Example 13

[0177] Preparation of a lipid nanoparticle composition based on a dendrimer, including the steps:

[0178] PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 prepared in Example 2 were respectively dissolved in ethanol, and then mixed according to the ratio (the mass ratio of PAMAM-G0-O14, cholesterol, DOPE, and DMG-PEG2000 was 4:1:1:2) to obtain a mixed solution. In the mixed solution, the concentration of PAMAM-G0-O14 was 100 mg / mL, the concentration of cholesterol was 25 mg / mL, the concentration of DOPE was 25 mg / mL, and the concentration of DMG-PEG2000 was 50 mg / mL; then, under vortex conditions, the mixed solution was dropped into a 200 mM sodium acetate buffer solution with a pH of about 5.4 to obtain a composition of PAMAM-G0-O14 and lipid components, wherein the concentration of PAMAM-G0-O14 was 1 mg / mL, the concentration of cholesterol was 0.25 mg / mL, the concentration of DOPE was 0.25 mg / mL, and the concentration of DMG-PEG2000 was 0.5 mg / mL.

[0179] Human erythropoietin (hEPO) mRNA was dissolved in enzyme-free water to obtain an mRNA solution with a concentration of 1 mg / mL.

[0180] Mix the above composition and the mRNA solution evenly (the mass ratio of PAMAM-G0-O14 to mRNA is 15:1), and incubate at room temperature for 10 min; then dilute it with 200 mM sodium acetate buffer with a pH of about 5.4 to the volume required for injection to obtain a lipid nanoparticle composition based on PAMAM-G0-O14 (abbreviated as G0-O14 / mhEPO).

[0181] Expression and Effect Determination of Human Erythropoietin (hEPO) mRNA in Mice

[0182] Select 8-week-old female BALB / c mice, and inject 0.2 mL of the lipid nanoparticle composition based on PAMAM-G0-O14 (the doses of injected mhEPO are 0.2 mg / kg, 0.4 mg / kg, and 0.6 mg / kg) into each mouse through the tail vein. Mice injected only with sodium acetate buffer solution are used as blank controls (buffer group). Six hours after administration, blood is collected from the eyes of the mice, and serum is separated. The content of hEPO protein in the serum is analyzed by enzyme-linked immunosorbent assay (ELISA).

[0183] Use the hEPO protein standard sample to make a standard curve, and calculate the expression of hEPO in the serum corresponding to the experimental group through the standard curve as Figure 29 . It can be clearly seen from Figure 29 that the expression of hEPO protein in mice in the G0-O14 / mhEPO group is significantly better than that in the control group, and it can be seen from the effects of delivering different doses of mhEPO that the expression of hEPO has a positive linear relationship with the dose of delivered mRNA.

Claims

1. A dendrimer-based lipid nanoparticle composition, characterized in that, Comprising a dendrimer compound, a lipid component, and a prophylactic or therapeutic nucleic acid; The dendrimer compound has a structure represented by the following Formula I or Formula II: In Formula I, R is selected from alkyl groups having 10 or 12 carbon atoms; in Formula II, R is selected from alkyl groups having 12 or 14 carbon atoms; The method for preparing the dendrimer compound includes the steps of: uniformly mixing an ethylenediamine core-polyamide-amine generation 0 dendrimer with 1,2-epoxyalkane or acrylate; obtaining the dendrimer compound after reaction, dialysis, and drying; The ethylenediamine core-polyamide-amine generation 0 dendrimer has the following structure: The molar ratio of the ethylenediamine core-polyamide-amine generation 0 dendrimer to 1,2-epoxyalkane or acrylate is 1:2x + 2, where x is the number of primary amines of the ethylenediamine core-polyamide-amine generation 0 dendrimer; the 1,2-epoxyalkane is 1,2-epoxydodecane or 1,2-epoxytetradecane; the acrylate is dodecyl acrylate or tetradecyl acrylate; The lipid component is a combination of a structural lipid, a steroid, and a polymer-conjugated lipid; the structural lipid is selected from one or a combination of two or more of DOPC, DOPE, DSPC, DPPG, DSPE, DPPC, DMPC, or POPC; the steroid is selected from one or a combination of two or more of cholesterol, β-sitosterol, saxisterol, stigmasterol, ergosterol, or stigmastanol; the polymer-conjugated lipid is a polyethylene glycol-conjugated lipid DMG-PEG; The mass ratio of the lipid component to the dendrimer compound is 1:1 - 4.

2. The lipid nanoparticle composition based on dendrimer according to claim 1, wherein Including one or more of the following conditions: i. The mixing of the ethylenediamine core-polyamide-amine generation 0 dendrimer with 1,2-epoxyalkane or acrylate is carried out at room temperature; ii. The reaction temperature is 80 - 100 °C, and the reaction time is 2 - 3 days.

3. The lipid nanoparticle composition based on dendrimer according to claim 1, characterized in that, Including one or more of the following conditions: i. The mass ratio of the dendrimer compound to the prophylactic or therapeutic nucleic acid is 1 - 22.5:1; ii. The particle size of the dendrimer-based lipid nanoparticle composition is 60 nm - 300 nm; iii. The mass ratio of the dendrimer compound, steroid, structural lipid, and polymer-conjugated lipid is 4:1:1:

2.

4. The method for preparing the dendrimer-based lipid nanoparticle composition according to claim 1, including the steps of: (1) Dissolving the dendrimer compound in ethanol to obtain a mixed solution; then dissolving the mixed solution in a sodium acetate buffer solution with a pH of 5 - 6 to obtain a dendrimer compound solution; (2) Dissolving the prophylactic or therapeutic nucleic acid in enzyme-free water to obtain a nucleic acid solution; (3) Mixing the dendrimer compound solution and the nucleic acid solution uniformly and incubating to obtain the dendrimer-based lipid nanoparticle composition.

5. The preparation method of the dendrimer-based lipid nanoparticle composition according to claim 4, characterized in that, Including one or more of the following conditions: i. The method of step (1) is as follows: Dissolve the dendrimer compound and the lipid component in ethanol to obtain a mixed solution; then, under vortex conditions, drop the mixed solution into a sodium acetate buffer solution with a pH of 5-6 to obtain a dendrimer compound solution; in the mixed solution, the concentration of the dendrimer compound is 80-120 mg / mL; the concentration of the sodium acetate buffer solution is 200 mmol / L; in the dendrimer compound solution, the concentration of the dendrimer compound is 0.05-5 mg / mL; ii. In step (2), the concentration of the nucleic acid solution is 0.01-2 mg / mL; iii. In step (3), the incubation temperature is room temperature, and the incubation time is 5-10 min; iv. In step (3), after incubation, a dilution step is further included.

Images