CN115957329A - Use of ionizable lipid compounds in the preparation of nucleic acid drug delivery systems - Google Patents
Use of ionizable lipid compounds in the preparation of nucleic acid drug delivery systems Download PDFInfo
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- CN115957329A CN115957329A CN202211314083.2A CN202211314083A CN115957329A CN 115957329 A CN115957329 A CN 115957329A CN 202211314083 A CN202211314083 A CN 202211314083A CN 115957329 A CN115957329 A CN 115957329A
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- nucleic acid
- acid drug
- drug delivery
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Classifications
-
- 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
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- Medicinal Preparation (AREA)
Abstract
The invention discloses application of an ionizable lipid compound in preparation of a nucleic acid drug delivery system, wherein the ionizable lipid compound is N ε -lauroyl lysine carboxamide. Said N is ε Lauroyl lysine carboxamide lysosome escape can be achieved by the N ε The nucleic acid drug delivery system prepared from lauroyl lysine formamide can deliver nucleic acid drug delivery molecules to tumor sites in vivo with high efficiency and can realize high-efficiency expression of nucleic acid.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of an ionizable lipid compound in preparation of a nucleic acid drug delivery system.
Background
Active molecules for nucleic acid drug delivery, including messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASO) or plasmid DNA (pDNA), have great potential for use in vaccines and gene therapy in vitro or in vivo. Most of the nano drug-loaded preparation enters cells in an endocytosis mode, and exists in an endosome after entering the cells, and the endosome can be gradually fused with lysosomes. The drug needs to exert the drug effect, and firstly can be released from the nano-carrier, and then some drugs can be released from an endosome according to the difference of the drug properties (the process is called endosome escape), otherwise, the drug can be degraded and inactivated by the lysosome after the endosome is fused with the lysosome; some drugs are not affected by degradation of lysosomes, and can be released from the lysosomes (the process is called lysosome escape), so that the drugs can be fully released to exert the drug effect. The lysosome escape of the existing ionizable lipid compound is poor, so that the ionizable lipid compound capable of escaping from the lysosome is urgently needed.
Disclosure of Invention
In a first aspect of the invention there is provided the use in the preparation of an ionizable lipid compound that is capable of lysosomal escapeUse of an ionizable lipid compound of N in a nucleic acid drug delivery system ε -lauroyl lysine carboxamide having the structural formula (I).
Further, the nucleic acid drug delivery system comprises a nucleic acid drug and a delivery liposome; the delivery liposome comprises the N ε -lauroyl lysine carboxamide, neutral lipids, structural lipids and polymer conjugated lipids; said N is ε -lauroyl lysine carboxamide may electrostatically adsorb the nucleic acid drug.
Further, the neutral lipid is hydrogenated soybean phosphatidylcholine; the structural lipid is cholesterol; the polymer conjugated lipid is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000.
Further, the delivery liposomes are composed of N ε -lauroyl lysine carboxamide: cholesterol: hydrogenated soy phosphatidylcholine: distearoylphosphatidylethanolamine-polyethylene glycol 2000 as 1mg:1mg:1mg:1mg of the raw materials.
Further, the nucleic acid drug is selected from one or more of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA and artificial nucleic acid.
Further, the nucleic acid drug is siRNA of Seq _1 sequence that inhibits Stat3 expression.
Still further, the nucleic acid drug delivery system is comprised of N epsilon-lauroyl lysine carboxamide: cholesterol: hydrogenated soy phosphatidylcholine: distearoyl phosphatidylethanolamine-polyethylene glycol 2000: siRNA was mixed at 1mg:1mg:1mg:1mg:8.9nmoL.
Furthermore, the nucleic acid drug delivery system is a nano-liposome particle, the average particle size of the nano-liposome particle is 50 nm-200 nm, and the polydispersity index of the nano-liposome particle is less than or equal to 0.4.
Furthermore, the average size of the nano-lipid particles is 50 nm-150 nm, and the polydispersity index of the nano-lipid particles is less than or equal to 0.3.
Further, the ionizable lipid compound can be selectively modified by a targeting substance, wherein the targeting substance is one or more of folic acid, a single-chain antibody or a targeting polypeptide.
Further, the nucleic acid drug delivery system is used for treating cancer.
Furthermore, the nucleic acid drug delivery system is used for inhibiting lung cancer cells and ovarian cancer cells.
Further, the administration mode of the nucleic acid drug delivery system is injection.
Further, the nucleic acid drug delivery system is an injection.
Furthermore, the injection also comprises additives, and the additives comprise a stabilizer and/or a diluent.
Furthermore, the addition amount of the additive is 1-20% of the total mass of the injection.
Still further, the stabilizer includes, but is not limited to, sucrose or trehalose.
Still further, the diluent comprises one or more of phosphate buffer, acetate buffer, tris hydrochloride buffer.
Further, the nucleic acid drug delivery system is administered by local intramuscular, subcutaneous, endothelial, intravenous, intratumoral injection, or infusion.
The nucleic acid drug delivery system of the present invention can deliver nucleic acid drug delivery molecules such as mRNA, siRNA or pDNA into the body by various administration modes such as local muscular, subcutaneous, endothelial, intratumoral and perfusion, and systemic administration modes such as intravenous injection, and can effectively express therapeutic protein drugs or antigens in cells in the body to play a role in preventing or treating diseases
Compared with the prior art, the invention has the following beneficial effects:
said N is ε Lauroyl lysine carboxamide lysosome escape can be achieved by the N ε The nucleic acid drug delivery system prepared from lauroyl lysine formamide can deliver nucleic acid drug delivery molecules to tumor sites in vivo with high efficiency and can realize high-efficiency expression of nucleic acid.
The nucleic acid drug delivery system of the present invention can deliver nucleic acid drug delivery molecules such as mRNA, siRNA or pDNA into the body by various administration modes such as local intramuscular, subcutaneous, endothelial, intratumoral, and perfusion, and also by systemic administration modes such as intravenous injection, and can effectively express therapeutic protein drugs or antigens in cells in the body to play a role in preventing or treating diseases.
Drawings
FIG. 1 is N ε Lauroyl lysine carboxamide 1H NMR spectrum.
FIG. 2 is a mass spectrum of N epsilon-lauroyl lysine carboxamide;
FIG. 3 is a Western Blot plot of the drug encapsulation efficiency of a nucleic acid drug delivery system (WLNP nano-lipid particles).
Fig. 4 is a bar graph of drug encapsulation efficiency of nucleic acid drug delivery systems (WLNP nano-lipid particles).
Fig. 5 is a drug loading stability profile for nucleic acid drug delivery systems (WLNP nanoliposomes).
Fig. 6 is a graph of gene silencing efficiency detection for nucleic acid drug delivery systems (WLNP nano-lipid particles).
Figure 7 is an in vitro toxicity assay of nucleic acid drug delivery systems (WLNP nanoliposomes).
Figure 8 is a targeting assay in animals of the nucleic acid drug delivery system (WLNP nanoliposomes particle).
Figure 9 is a fluorescence plot of the lysosomal escape capacity of the nucleic acid drug delivery system (WLNP nanolipoparticles).
Fig. 10 is a bar graph of the lysosomal escape capacity of a nucleic acid drug delivery system (WLNP nanoliposome).
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
The term "liposome" as used herein refers to a bilayer nanoparticle composed of phospholipids, cholesterols and functional phospholipid components. Wherein the phospholipid is selected from one or two of soybean lecithin and egg yolk lecithin. The cholesterol component is selected from one or more of cholesterol and its derivatives, cholestane, cholic acid and bile acid. The functional phospholipid is one or more selected from distearoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol and distearoylphosphatidylethanolamine-polyethylene glycol.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Experimental example 1: n is a radical of hydrogen ε Preparation of lauroyl lysine carboxamide (LKN)
(1) Esterification reaction
Mixing 5g of N α -benzyloxycarbonyl lysine (N) α -Cbz-lys, lysine with alpha-amino protected by benzyloxycarbonyl group) was dissolved in 80mL of methanol (CH) 3 OH) is stirred and cooled for 10-30min in an ethanol bath at the temperature of-10 ℃, 6.4mL of thionyl chloride is added dropwise, and stirring is continued for 20h at room temperature. Evaporating under reduced pressure to remove excessive thionyl chloride, methanol and hydrogen chloride generated in the reaction until a white solid is obtained, and drying under vacuum at 50 ℃ to obtain an esterification reaction product N α -benzyloxycarbonyl lysine ester (N) α -Cbz-lysine ester)。
(2) Amidation reaction
Taking 2g of the product N alpha-benzyloxycarbonyl lysine ester obtained in the last step of esterification reaction, dissolving the product in 50ml of concentrated ammonia water, stirring and cooling the solution in an ethanol bath at the temperature of minus 5 ℃ for 24 hours, carrying out suction filtration to obtain a white solid, and carrying out vacuum drying at the temperature of 50 ℃ to obtain an amide reaction product N α -benzyloxycarbonyl lysinamide (N) α -benzyl oxycarbonyl lysine)。
(3) Esterification reaction
3g of N from the acylation product of the previous step α -benzyloxy groupDissolving carbonyl lysinamide and 3mL of triethylamine in 80mL of ethyl acetate, stirring and cooling for 10-30min in an ethanol bath at the temperature of-5 ℃, dropwise adding 3mL of lauroyl chloride, continuously stirring and reacting for 10min, stirring and reacting for 4h at room temperature, and removing the solvent by reduced pressure evaporation to obtain a crude esterification product. The crude product of the esterification reaction is reacted with saturated NaHCO 3 Stirring the solution for reaction for 15min, dissolving by-product fatty acid, extracting with equal volume of ethyl acetate, retaining the upper ethyl acetate phase, washing the ethyl acetate phase with equal volume of saturated NaCl solution, removing ethyl acetate by reduced pressure evaporation, and vacuum drying at 50 ℃ to obtain the acylation reaction product.
(4) Deprotection reaction
Dissolving 3g of the product of the acylation reaction in 40mL of methanol, adding HCl/methanol solution to adjust the pH of the reaction solution to be =3-4, and carrying out hydrogenation reaction for 4h at normal pressure by using palladium on carbon (10% Pd) as a catalyst. The catalyst was filtered off with celite, and the solvent was evaporated under reduced pressure to give a crude deprotected (benzyloxycarbonyl) product. The structure is correct through 1H NMR identification, and a representative 1H NMR spectrum is shown in the following figure 1, and specific data are as follows: 1H NMR 2 [400MHz ] DMSO-d6]δ8.17[s,3H(-NH 3 Cl)],8.01–7.69[m,2H(-CONH2)],7.52[s,1H(-NH-)],3.84–3.61[m,1H(–CH 2 -CH–CONH 2 )],3.00[d,2H(–CH 2 –CH 2 –CH 2 –CH 2 –NH–)],2.03[t,,2H(–CH 2 –CO–NH–)],1.71[d,2H(–CH 2 –CH 2 –CH 2 –CH 2 –NH–)],1.56–1.13[s,20H m,2H,(–CH 2 –CH 2 –CH 2 –CH 2 –NH–)],0.85[t,3H(CH 3 –CH 2 –CH 2 –)]。
Example 1 preparation of nucleic acid drug delivery System (WLNP Nanolipid particles)
The preparation method of the nucleic acid delivery system of this example used a conventional dropping method to prepare liposomes. Collecting Hydrogenated Soybean Phosphatidylcholine (HSPC) 1mg, cholesterol 1mg, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 1mg, and N1 mg ε -lauroyl lysine carboxamide (LKN) is dissolved in 1mL of methanol, 10 μ L of siRNA with a molar concentration of 200 μ M is mixed with the above solution in a mass ratio of 1Methanol was added to prepare 500. Mu.L of a solution, and 100. Mu.L of an acetic acid buffer solution with pH =5.0 was added to prepare a liposome mother liquor. At this time, N ε -lauroyl lysine carboxamide: cholesterol: hydrogenated soy phosphatidylcholine: distearoylphosphatidylethanolamine-polyethylene glycol 2000: the feeding ratio of siRNA is 1mg:1mg:1mg:1mg:8.9nmoL. The siRNA is siRNA of which the sequence for inhibiting Stat3 expression is Seq _ 1.
Seq_1：5’-TCGACTGCATAGGACGGAATGAACAAGCGTTCATTCC GTCCTATGCATTTTTT-3’。
Example 2 drug encapsulation efficiency assay of nucleic acid drug delivery systems (WLNP nano-lipid particles)
The control group was 4. Mu.L of siRNA dissolved in 200. Mu.L of methanol, the experimental group was WLNP liposome stock solution purified and equivalent to the control group siRNA, 2mg of heparin and 200. Mu.L of methanol were added, respectively, and ultrasonication was performed in an ice water bath for 5min with an ultrasonic cell disrupter. According to the volume ratio of 1:5 adding 6 times of loading buffer solution (loading buffer) into the control group and the experimental group respectively, mixing uniformly, performing electrophoresis for 20-40 min at a voltage of 70-120V, and shooting by using a gel imager. As shown in FIG. 2, the intensity of the free siRNA bands in the gel imager was high, indicating that the more free siRNA. Image J is used for counting the gray values of two parts to calculate the entrapment rate, and the result is shown in the right sides of figures 3 and 4, and the drug entrapment rate of the WLNP liposome can reach 65%.
Example 3 drug loading stability test of nucleic acid drug delivery systems (WLNP nano-lipid particles)
After the drug-loaded liposome stock solution is prepared, the drug-loaded liposome stock solution is respectively diluted by PBS with 0.01M and pH 7.4 and cell complete culture medium containing serum until the concentration of the drug-containing siRNA is 100 mu M, samples are taken every 24h for measuring the particle size and the potential of the liposome by a Malvern laser particle sizer, and the result is shown in figure 5, and N is used ε The particle size and the potential of the liposome of the lauroyl lysine formamide keep dynamic balance, which indicates that the siRNA is stably loaded in the liposome.
Found that N ε The liposome encapsulation efficiency of the lauroyl lysine formamide is high and the load is very stable, mainly N ε Lauroyl lysine carboxamide is a typical amphiphilic molecule (with long acyl chains that are apolar and polar end a-NH 2 ) And the water solubility is poor, so the affinity with phospholipid molecules is strong, the binding is tight, and the early leakage is not easy to occur.
Example 4 detection of Gene silencing efficiency of nucleic acid drug delivery System (WLNP Nanolipid particles)
The expression level of STAT3 mRNA after 293T cells are incubated by siRNA for 48h is specifically as follows:
293T cells at 3X 10 per well 5 The cell amount of (2) was inoculated into 6-well plates for incubation, after 24h, the culture medium was aspirated after the cells adhered to the wall and stably grown, the control group was free siRNA and the siRNA-free WLNP liposome prepared in reference example 1, the siRNA concentrations of the experimental group were 50nM and 100nM, respectively, and incubation was continued for 48h. Extracting cell RNA by using a Trizol method, and detecting the expression level of STAT3 siRNA of 293T cells by using qPCR.
As shown in fig. 6, the expression level of STAT3 siRNA in the drug group was significantly decreased compared to the control group and the siRNA-free group, and the expression level of siRNA was continuously decreased as the drug concentration was increased (difference in statistical analysis, indicated as P <0.01, indicated as P <0.001vs CON), indicating that WLNP liposomes could successfully deliver siRNA to cells and perform gene silencing.
Example 5 in vitro toxicity assay of WLNP Liposomal nanoparticles
The CCK-8 cell activity test of the medicine specifically comprises the following steps:
mixing ovarian cancer cells SKOV3 and eugonicOvarian epithelial cells IOSE in 10 per well 4 Respectively inoculating the amount of each cell into a 96-well plate for incubation, sucking out the culture medium after 24h and allowing the cells to adhere to the wall and stably grow, adding 100 mu L of liposome stock solution diluted by the culture medium, and allowing N to be generated ε Lauroyl lysine carboxamide final concentrations of 20. Mu.M, 40. Mu.M, 60. Mu.M, 80. Mu.M, 100. Mu.M, respectively, and incubation was continued for 48h. According to the method of the reagent instruction, the culture medium is aspirated, 100 μ L of 0.5mg/mL CCK-8 reagent is added to each well, after incubation for 1h, the absorbance of each well is recorded at 450nm using a microplate reader, and the cell viability is calculated according to the following formula:
wherein A0, A1 and A2 are absorbance of blank group (pure culture medium without liposome), drug-loaded liposome group (cells are treated by drug-loaded liposome) and control group (cells are treated by blank liposome).
The results are shown in fig. 7, when the drug concentration is increased, the survival rates of ovarian cancer cell SKOV3 and normal ovarian epithelial cell IOSE are hardly reduced except for the group with the drug concentration of 100 μ M, which indicates that WLNP nano-lipid particles have substantially no toxicity when used as a delivery system at a concentration of 80 μ M or less.
Example 6: WLNP-siRNA-cy5 nano lipid particle animal in vivo targeting detection
The WLNP-siRNA-cy5 nano-lipid particles in the experimental group are prepared according to the preparation method of example 1 by adopting a tail vein injection mode, 200 mu L of neutral PBS buffer solution is adopted to quickly switch the liposome environment, the control group is free siRNA-cy5, the other conditions are the same, and the WLNP-Cy nano-lipid particles are injected into the tail vein of a female Babl/c mouse with lung cancer cells A549 tumors successfully in 6-8 weeks, and the intravenous injection of 50 mu g of siRNA is controlled. The fluorescence expression of each organ of mice 4h after intravenous injection of WLNP-siRNA-cy is shown in FIG. 8. The results show that the fluorescence expression of the siRNA-cy5 in the control group in the mouse tumor is almost zero, while the WLNP-siRNA-cy5 nano lipid particle in the experimental group in the mouse tumor expresses, which indicates that the WLNP-siRNA-cy5 nano lipid particle has a certain targeting effect.
Example 7 detection of lysosomal escape Capacity of WLNP-siRNA-cy5 Nanolipid particles
A549 cells (lung cancer cells) were seeded at 8X 10 per well 3 Respectively inoculating the cell amount of the cells into a 96-well plate for incubation, after 24 hours, sucking out the culture medium after the cells adhere to the wall and stably grow, adding Hoechst 33342 for staining for 10min, adding Lyso-Tracker Green for staining for 15min, adding 100 mu L of liposome stock solution diluted by the culture medium into an experimental group, and replacing N with cationic liposome as a control group ε Lauroyl lysine carboxamide, the rest of the conditions being identical, was washed off by incubation for 10 minutes and photographed every 20 minutes under high connotation for 2h.
The results are shown in fig. 9, 10, with the green position lysosomes and the red WLNP-siRNA-cy5 nano-lipid particles. The red part which is not overlapped with the green part is the lipid particles, lysosome escape is carried out, the lysosome escape rate of the drug in the experimental group is about 42 percent, the lysosome escape rate of the control group is about 7 percent, and the experimental group is 6 times that of the control group, so that the WLNP-siRNA-cy5 nanometer lipid particles are proved to have good lysosome capacity.
In summary, the following steps: n is a radical of ε The escape of lysosomes by lauroyl lysine carboxamide, from N ε The nucleic acid drug delivery system prepared from lauroyl lysine formamide can deliver nucleic acid drug delivery molecules to tumor sites in vivo with high efficiency and can realize high expression of nucleic acid.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. Use of an ionizable lipid compound in the manufacture of a nucleic acid drug delivery system, wherein said ionizable lipid compound is N ε -lauroyl lysine carboxamide having the formula (I),
2. the use of claim 1, wherein the nucleic acid drug delivery system comprises a nucleic acid drug and a delivery liposome; the delivery liposome comprises the N ε -lauroyl lysine carboxamide, neutral lipids, structural lipids and polymeric conjugated lipids; said N is ε Lauroyl lysine carboxamide may electrostatically adsorb the nucleic acid drug.
3. Use according to claim 2, wherein the neutral lipid is hydrogenated soy phosphatidylcholine; the structural lipid is cholesterol; the polymer conjugated lipid is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000.
4. The use of claim 3, wherein said delivery liposome is composed of N ε -lauroyl lysine carboxamide: cholesterol: hydrogenated soy phosphatidylcholine: distearoylphosphatidylethanolamine-polyethylene glycol 2000 as 1mg:1mg:1mg:1mg charge ratio.
5. The use according to claim 2, wherein the nucleic acid drug is selected from the group consisting of one or more of mRNA, siRNA, sgRNA, ASO, circRNA, microRNA, DNA, ecDNA, and artificial nucleic acid.
6. The use according to claim 5 characterized in that the nucleic acid drug is siRNA with sequence Seq _1 that inhibits Stat3 expression.
7. The use of any one of claims 4 or 6, wherein the nucleic acid drug delivery system consists of N ε -lauroyl lysine carboxamide: cholesterol: hydrogenated soy phosphatidylcholine: distearoylphosphatidylethanolamine-polyethylene glycol 2000: siRNA was mixed at 1mg:1mg:1mg:1mg:8.9nmoL.
8. The use of any one of claims 1-7, wherein the nucleic acid drug delivery system is for the treatment of cancer.
9. The use of claim 8, wherein the nucleic acid drug delivery system is for inhibiting lung cancer cells, ovarian cancer cells.
10. The use of any one of claims 1-7, wherein the nucleic acid drug delivery system is administered by injection.
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