CN117982460A - Preparation and application of spherical rod-shaped lipid nano particles - Google Patents
Preparation and application of spherical rod-shaped lipid nano particles Download PDFInfo
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- CN117982460A CN117982460A CN202410262430.4A CN202410262430A CN117982460A CN 117982460 A CN117982460 A CN 117982460A CN 202410262430 A CN202410262430 A CN 202410262430A CN 117982460 A CN117982460 A CN 117982460A
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Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides spleen-targeted ionizable cationic lipid nanoparticles with a spherical rod-shaped structure, and a preparation method and application thereof. The spherical rod-shaped lipid nanoparticle can promote membrane fusion and enhance immune cell uptake; can form specific protein crown in the internal circulation, enhance the targeted delivery of spleen, can be rapidly degraded under the action of lysosomes after being phagocytized by cells, realizes the remarkable enhancement of transfection efficiency, has lower cytotoxicity and better biocompatibility, and is used as a delivery carrier of nucleic acid medicaments, thereby being beneficial to the clinical transformation of the nucleic acid medicaments.
Description
Technical Field
The present invention relates to the field of materials. In particular, the invention relates to organ targeting lipid nano-carrier materials, in particular to spleen targeting cationic lipid nano-particles with a ball-rod structure and transfection application of high-efficiency targeting spleen.
Background
Nucleic acid drugs have shown great potential in the field of disease prevention and treatment. Is affected by the global new coronavirus, and accelerates the research and development of nucleic acid medicaments, especially mRNA vaccines. However, free mRNA is easily digested by nucleases in plasma and is easily cleared by the body when entering the cell interior. To overcome the above drawbacks, mRNA is typically encapsulated inside LNP by lipid nanoparticle (Lipid Nanoparticle, LNP) technology, thereby preventing degradation of mRNA in plasma and improving mRNA uptake in cells.
The current non-liver-targeted LNP system is of great biological interest, especially in the treatment of regional diseases. However, limited by composition, formulation, preparation method, structure, targeting efficiency, transfection efficiency, etc. are all greatly affected, including but not limited to nucleic acid drug/LNP particle size, dispersity, surface charge, encapsulation efficiency, morphology, delivery efficiency, endosomal escape rate, organ targeting effect, etc. Based on the existing spleen targeting LNP technology, the LNP morphology is modified, so that the targeting efficiency and the delivery efficiency of an LNP system are improved.
Disclosure of Invention
The invention aims to provide a spherical-rod-shaped lipid nanoparticle, which can more efficiently target and deliver nucleic acid drugs to a treatment area after systemic administration through a system, reduce liver uptake and enhance spleen expression efficiency, thereby improving treatment effect.
The invention also aims to provide a pharmaceutical composition comprising the spherical-rod-shaped lipid nanoparticle.
The invention also aims to provide the application of the spherical-stick-shaped lipid nanoparticle or the pharmaceutical composition in disease treatment.
In a first aspect, the present invention provides a balloon-like lipid nanoparticle with spleen targeting, characterized in that the nanoparticle comprises:
1) An ionizable cationic lipid;
2) Neutral lipids;
3) A steroid compound;
4) Polyethylene glycol lipids; and
5) A bioactive agent;
The club-shaped lipid nanoparticle is composed of a spherical head part and a long club-shaped tail part.
In a specific embodiment, the club-like lipid nanoparticle is composed of one spherical head and one or more long club-like tails distributed at its edge.
In a specific embodiment, the ionizable cationic lipid is a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof,
Wherein:
G 1 is selected from the group consisting of: absent, substituted or unsubstituted C 1-C20 alkylene, substituted or unsubstituted C 2-C20 alkenylene, said substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 1 is selected from the group consisting of: H. substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, substituted or unsubstituted 4-8 membered heterocyclyl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted C 6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted 8-10 membered fused heterocyclyl 、-NRaRb、-NRaRbOH、-N(Ra(OH))(Rb(OH))、-C(O)OR、-OC(O)R、-C(O)NRaRb、-N(Ra)C(O)Rb、CN、-C(O)R、-OR、-O(CH2)mOR、-O(CH2)mNRaRb、-S(O)xR、-S-SR、-C(O)SR、-SC(O)R、-N(Ra)C(O)N(Rb)2、-OC(O)NRaRb、-N(Ra)C(O)ORb, containing 1,2 or 3 heteroatoms selected from N, O or S, said substitution being by 1,2 or 3C 1-C5 alkyl;
R, R a、Rb are each independently selected from the group consisting of: substituted or unsubstituted C 1-C20 alkyl, substituted or unsubstituted C 2-C20 alkenyl, substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
x is an integer from 0 to 2 (e.g., 0,1 or 2);
R 2 is selected from the group consisting of: H. a substituted or unsubstituted C 6-C10 aryl (preferably phenyl), a substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring, anthracycline), a substituted or unsubstituted C 1-C12 alkyl, a substituted or unsubstituted C 2-C12 alkenyl, a substituted or unsubstituted C 2-C12 alkynyl, and optionally substituted with 1, 2 or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is selected from the group consisting of: c 1-C5 alkyl, C 2-C5 alkenyl, C 2-C5 alkynyl;
Q is selected from the group consisting of: n, substituted or unsubstituted C 6-C10 aryl (preferably phenyl), substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring, anthracycline), substituted or unsubstituted C 4-C8 cycloalkyl (preferably cyclopentyl, cyclohexyl), substituted or unsubstituted 4-8 membered heterocyclyl (preferably pyrrole ring, pyrimidine ring) containing 1, 2 or 3 heteroatoms selected from N, O or S; the substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl, br;
Each G 2、G3、G4 is independently selected from the group consisting of: absent, substituted or unsubstituted C 1-C10 alkylene, substituted or unsubstituted C 2-C10 alkenylene, said substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl or Br;
L 1 and L 2 are each independently selected from the group consisting of: is not present in 、-O-、-C(O)-、-OC(O)-、-C(O)O-、-O(CH2)qC(O)O-、-O(CH2)qOC(O)-、-OC(O)O-、-S-、-S-S-、-C(O)NRd-、-NRdC(O)-、-S(＝O)x-、-SC(O)-、-C(O)S-;
R 3 and R 4 are each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-C20 alkyl, substituted or unsubstituted straight or branched C 2-C20 alkenyl, substituted or unsubstituted straight or branched C 2-C20 alkynyl, said substitution means substitution with 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
Each R d is independently selected from the group consisting of: H. c 1-C20 alkylene, C 2-C20 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl;
m and q are each independently integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
In specific embodiments, G 1 is C 1-C20 alkylene;
R 1 is selected from the group consisting of: substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, substituted or unsubstituted 4-8 membered heterocyclyl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted C 6-C10 aryl, 5-10 membered heteroaryl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted 8-10 membered fused heterocyclyl 、-NRaRb、-NRaRbOH、-N(Ra(OH))(Rb(OH))、-O(CH2)mNRaRb, containing 1,2 or 3 heteroatoms selected from N, O or S, said substitution referring to substitution with 1,2 or 3 groups selected from the group consisting of: c 1-C5 alkyl substitution;
R 2 is selected from the group consisting of: benzene rings, naphthalene rings, and optionally substituted with 1, 2, or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
Q is selected from the group consisting of: n, benzene ring;
Each G 2、G3、G4 is independently selected from the group consisting of: absent, C 1-C10 alkylene;
l 1 and L 2 are each independently selected from the group consisting of: absence, -O-, -C (O) -, -OC (O) -, -C (O) O-, -O (CH 2)qC(O)O-、-O(CH2)q OC (O) -, -OC (O) O-;
R 3 and R 4 are each independently a linear or branched C 1-C20 alkyl group;
R a、Rb are each independently selected from the group consisting of: c 1-C20 alkyl, C 1-C20 alkylene;
r c is C 1-C5 alkyl;
Each m and q is an integer independently selected from the group consisting of: 1.2, 3, 4, 5, 6, 7, 8.
In a specific embodiment, the compound is a compound of formula (IA) or formula (IB):
wherein ring a is selected from the group consisting of: benzene ring, naphthalene ring, anthracene ring, cyclopentyl, cyclohexyl, pyrrole ring, pyrimidine ring;
R 2 is selected from the group consisting of: benzene ring, naphthalene ring, anthracene ring, C 1-C6 alkyl, and optionally substituted with 1,2, or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is C 1-C5 alkyl.
In a specific embodiment, the compound is a compound of formula (IA'),
Wherein,
P is an integer selected from the group consisting of: 2.3, 4 and 5;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
L 1 and L 2 are each independently selected from the group consisting of: absence of, -O-, -O (CH 2)qC(O)O-、-O(CH2)q OC (O) -;
q is an integer selected from the group consisting of: 4. 5, 6, 7, 8;
R 3 and R 4 are each independently a linear or branched C 8-C16 alkyl group;
R a、Rb is independently selected from the group consisting of: c 1-C3 alkyl, C 1-C3 alkylene.
In a specific embodiment, the compound is a compound of formula (IB'),
Wherein,
P is an integer selected from the group consisting of: 2.3, 4 and 5;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
Each G 2、G3、G4 is independently C 1-C10 alkylene;
l 1 and L 2 are each independently selected from the group consisting of: absence, -O-, -C (O) -, -OC (O) -, -C (O) O-, -OC (O) O-;
R 3 and R 4 are each independently a linear or branched C 8-C18 alkyl group;
R a、Rb is independently selected from the group consisting of: c 1-C3 alkyl, C 1-C3 alkylene.
In particular embodiments, the ionizable cationic lipid is selected from the group consisting of:
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/>
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In a specific embodiment, G 1 is a substituted or unsubstituted C 1-C10 alkylene, preferably a substituted or unsubstituted C 1-C4 alkylene, more preferably a substituted or unsubstituted ethylene, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
R a、Rb are each independently selected from the group consisting of: substituted or unsubstituted C 1-C10 alkyl, preferably substituted or unsubstituted C 1-C4 alkyl, more preferably substituted or unsubstituted methylene or ethylene, said substitution being by 1, 2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 2 is selected from the group consisting of: a substituted or unsubstituted C 6-C10 aryl (preferably phenyl), a substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring), and optionally substituted with 1, 2 or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is selected from the group consisting of: c 1-C5 alkyl;
Q is selected from the group consisting of: n, substituted or unsubstituted C 6-C10 aryl (preferably phenyl); the substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl, br;
Each G 2、G3、G4 is independently selected from the group consisting of: an absent, substituted or unsubstituted C 1-C3 alkylene group;
L 1 and L 2 are each independently selected from the group consisting of: absence of, -O-, -C (O) O-, -O (CH 2)q C (O) O-;
R 3 and R 4 are each independently a substituted or unsubstituted, linear or branched C 1-C16 alkyl group, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
q is an integer from 1 to 8 (e.g., 1,2,3, 4, 5, 6, 7, 8).
In particular embodiments, the ionizable cationic lipid is selected from the group consisting of:
Preferably, the ionizable cationic lipid is selected from the following compounds:
In a specific embodiment, the mole percentage of the ionizable cationic lipid is 40% -70%, preferably 45% -55%; the mole percentage of the neutral lipid is 5% -15%, preferably 8% -12%; the molar percentage of the steroid is 30% -50%, preferably 35% -45%; the polyethylene glycol lipid is 1-5%, preferably 1-3%; or alternatively
The mole ratio of the ionizable cationic lipid to the neutral lipid to the steroid to the polyethylene glycol lipid is (40-70): 5-15): 30-50): 1-5; preferably (45-55): (8-12): (35-45): (1-3); more preferably 50:10:38.5:1.5.
In a preferred embodiment, the neutral lipid is a neutral helper phospholipid.
In preferred embodiments, the neutral helper phospholipid is one or more of di-oleoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylcholine, di-palmitoyl phosphatidylethanolamine, di-stearoyl phosphatidylcholine, di-stearoyl-phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, di-oleoyl lecithin, dimyristoyl phosphatidylcholine, egg yolk lecithin, sphingomyelin, di-palmitoyl phosphatidylcholine, di-erucic phosphatidylcholine, di-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, di-oleoyl phosphatidylserine, di-oleoyl phosphatidylethanolamine DOPE, and distearoyl phosphatidylcholine DSPC; preferably di-oleoyl phosphatidylcholine, di-stearoyl phosphatidylcholine, di-oleoyl phosphatidylethanolamine DOPE or di-stearoyl phosphatidylcholine DSPC.
In preferred embodiments, the steroid is one or more of cholesterol, campesterol, β -sitosterol, stigmasterol, dehydrocholesterol, brassicasterol, stigmasterol, erythrinsterol, fucosterol, ergosterol, dehydroergosterol, dihydrocholesterol, campestanol, sitostanol, lotenol, cycloartenol; preferably cholesterol.
In a preferred embodiment, the polyethylene glycol lipid is phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), distearoyl phosphatidylglycerol-polyethylene glycol 2000, dilauroyl phosphatidylglycerol-polyethylene glycol 2000, di-oleoyl phosphatidylglycerol-polyethylene glycol 2000, dimyristoyl phosphatidylglycerol-polyethylene glycol 2000, di-linolenoyl phosphatidylglycerol-polyethylene glycol 2000, di-erucic phosphatidylglycerol-polyethylene glycol 2000, 1-palmitoyl-2-oleoyl phosphatidylglycerol-polyethylene glycol 2000, or di-palmitoyl phosphatidylglycerol-polyethylene glycol 2000; preferably phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000).
In particular embodiments, the bioactive agent is a therapeutic agent; including, but not limited to, nucleic acids, antibodies, protein drugs (including, but not limited to, antibodies), antibody drug conjugates, small molecule compounds; nucleic acids are preferred.
In specific embodiments, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rRNA), micrornas (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and micronuclear RNA (snRNA); mRNA is preferred.
In a specific embodiment, the nanoparticle has a particle size of 100nm to 240nm, preferably 105nm to 200nm, more preferably 110nm to 160nm.
In a preferred embodiment, the nanoparticle has a polydispersity index (PDI) of 0.1 to 0.3, preferably 0.1 to 0.24, more preferably 0.1 to 0.20.
In a preferred embodiment, the nanoparticle has a zeta potential of from 1mV to-5 mV, preferably from 0mV to-5 mV, more preferably from-1 to-4 mV.
In specific embodiments, the ratio of nitrogen to phosphorus of the ionizable cationic lipid to the therapeutic agent is 9:1 to 36:1, e.g., 9:1, 12:1, 18:1, 24:1, 30:1, 36:1; preferably, the nitrogen to phosphorus ratio is 12:1, 18:1, 24:1, 30:1; more preferably, the nitrogen to phosphorus ratio is 12:1, 18:1.
In a second aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticle of the first aspect and a pharmaceutically acceptable excipient.
In a preferred embodiment, the pharmaceutical composition is a vaccine composition.
In a third aspect, the present invention provides the use of a lipid nanoparticle according to the first aspect or a pharmaceutical composition according to the second aspect in the manufacture of a medicament for the prevention or treatment of infectious diseases, tumours and autoimmune diseases.
In a preferred embodiment, the medicament is for delivering a therapeutic agent to the spleen.
In a fourth aspect, the present invention provides a method for preparing the lipid nanoparticle according to the first aspect, comprising the steps of:
s1: providing a mixture of an ionizable cationic lipid, optionally a neutral lipid, a steroid, a polyethylene glycol lipid, and an organic solvent;
S2: providing an aqueous solution of a bioactive agent;
S3: mixing the mixed solution and the aqueous solution in the steps S1 and S2 at a certain flow rate ratio (preferably 1:3) and a volume ratio (preferably 1:3) by a microfluidic system, and diluting the mixed solution (preferably 5 times) by water to obtain an intermediate solution;
s4: carrying out ultrafiltration concentration on the intermediate product solution to obtain concentrated solution;
S5: and (3) passing the concentrated solution through a filter membrane to obtain the lipid nanoparticle.
In a preferred embodiment, the organic solvent in step S1 is a water-miscible solvent, such as ethanol, acetone, isopropanol, methanol, etc.
In a preferred embodiment, the aqueous solution of step S2 comprises a buffer, preferably a physiologically acceptable buffer, such as a phosphate buffer, optionally a PBS buffer.
In a preferred embodiment, in step S3, the preparation of the intermediate solution is performed in a normal temperature environment, and the solution mixed by the microfluidic chip is immediately diluted with water, so as to reduce the concentration of ethanol and prevent the ethanol from damaging the particles.
In a preferred embodiment, in step S4, the intermediate solution is ultrafiltered using an ultrafiltration tube to remove a substantial amount of the ethanol present in the solution, while concentrating to obtain a concentrate of higher concentration, facilitating subsequent formulation of the desired target concentration of the cationic lipid nanoparticle solution.
In a preferred embodiment, in step S5, the concentrate is filtered with a 0.22 μm aqueous filter to remove large particles that have agglomerated within the system and to obtain cationic lipid nanoparticles that have a better dispersity and uniform size.
In a fifth aspect, the present invention provides a method of preventing or treating infectious diseases, tumors and autoimmune diseases, the method comprising the step of administering a prophylactically or therapeutically effective amount of the lipid nanoparticle of the first aspect or the pharmaceutical composition of the second aspect, or a medicament prepared from the lipid nanoparticle of the first aspect or the pharmaceutical composition of the second aspect, to a subject in need of prevention or treatment of infectious diseases, tumors and autoimmune diseases.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a SIM 7-lipid nanoparticle cryoelectron micrograph;
FIG. 2 is a SIM 40-lipid nanoparticle cryoelectron micrograph;
FIG. 3 is a SIM 45-lipid nanoparticle cryoelectron micrograph;
FIG. 4 is a SIM 74-lipid nanoparticle cryoelectron micrograph;
FIG. 5 is a scanning electron micrograph of SM102 lipid nanoparticles;
FIG. 6 is an in vitro image of the Fluc-mRNA expression of the SIM 7-lipid nanoparticle of the present invention;
FIG. 7 is an in vitro image of the Fluc-mRNA expression of a comparative example SM102 lipid nanoparticle of the present invention;
FIG. 8 is a chart showing the statistics of the Fluc-mRNA expression of the SIM 7-lipid nanoparticle of the present invention;
FIG. 9 is a chart showing the statistics of the Fluc-mRNA expression of the lipid nanoparticles of comparative example SM102 of the present invention;
FIG. 10 shows that the structure of the lipid nanoparticle of the present invention changes from spherical to cohesive to short to long rods as the nitrogen to phosphorus ratio increases;
FIGS. 11 to 23 show steps of synthesis of ionizable cationic lipid molecules (spleen-targeted imidazolyl lipid compounds) used in the present invention.
Detailed Description
The inventors have conducted extensive and intensive studies and have unexpectedly found that by controlling the nitrogen-to-phosphorus ratio in nanoparticles composed of ionizable cationic lipid molecules and nucleic acids, nanoparticles having a specific structure, i.e., club-like lipid nanoparticles, can be obtained. After systemic administration, the nanoparticle with a specific club-like structure can more efficiently target nucleic acid to a treatment area, such as a non-liver organ (e.g., spleen), so that liver uptake can be reduced, spleen expression efficiency can be enhanced, and therapeutic effect can be further improved. The present invention has been completed on the basis of this finding.
Spherical rod-shaped lipid nanoparticle
The spherical rod-shaped lipid nanoparticle is a spherical rod-shaped LNP system with high-efficiency organ targeted delivery and high-efficiency spleen expression. The club-like LNP system is largely affected by the composition ratio of ionizable cationic lipids to nucleic acid drugs in its constituent components. Thus, modulation of the nitrogen to phosphorus ratio of ionizable cationic lipid molecules to bioactive agents, such as therapeutic agents (e.g., nucleic acid drugs), is of great interest for preparing LNP systems for efficient spleen targeted delivery and expression.
The present inventors have unexpectedly found that the structure of the lipid nanoparticle changes from spherical to cohesive to short to long as the nitrogen to phosphorus ratio increases, as shown in fig. 10. Thus, by adjusting the nitrogen to phosphorus ratio of the ionizable cationic lipid molecules to the therapeutic agent in the nanoparticle, the present invention provides a club-like lipid nanoparticle. The spherical rod-shaped lipid nanoparticle consists of a spherical head part and a long rod-shaped tail part. In a specific embodiment, the club-like lipid nanoparticle is composed of one spherical head and one or more long club-like tails distributed at its edge.
As used herein, the term "nitrogen to phosphorus ratio" refers to the ratio of the amount of ionizable amino groups on an ionizable cationic lipid molecule to the amount of a substance of a bioactive agent, such as a phosphate on a nucleic acid molecule, in a lipid nanoparticle. In specific embodiments, the nitrogen to phosphorus ratio is from 9:1 to 36:1, such as 9:1, 12:1, 18:1, 24:1, 30:1, 36:1; preferably, the nitrogen to phosphorus ratio is 12:1, 18:1, 24:1, 30:1; more preferably, the nitrogen to phosphorus ratio is 12:1, 18:1.
The particle size of the spherical rod-shaped lipid nanoparticle of the present invention is 100nm to 240nm, preferably 105nm to 200nm, more preferably 110nm to 160nm; a Polydispersity (PDI) of 0.1 to 0.3, preferably 0.1 to 0.24, more preferably 0.1 to 0.20; the zeta potential is from 1mV to-5 mV, preferably from 0mV to-5 mV, more preferably from-1 to-4 mV.
To obtain the spherical rod-shaped lipid nanoparticle of the invention, the ionizable cationic lipid can be a compound shown in a formula (I), or pharmaceutically acceptable salt, prodrug or stereoisomer thereof,
Wherein the substituents are selected as described above.
Preferably, the mixture of formula (I) of the present invention may be a compound of formula (IA) or formula (IB):
Wherein the substituents are selected as described above.
More preferably, the mixture of the present invention may be a compound represented by the formula (IA ') or (IB'),
Wherein the substituents are selected as described above.
In particular embodiments, the ionizable cationic lipid may be any of the compounds shown in SIM1-SIM 244; preferably any one of the compounds shown in SIM7, SIM40, SIM45, SIM69, SIM74, SIM99, SIM120, SIM 121; more preferably SIM7 or SIM40.
In addition to the ionizable cationic lipid and the bioactive agent, e.g., nucleic acid, the inventive club-like lipid nanoparticle further comprises auxiliary materials such as neutral lipids, steroids, and polyethylene glycol lipids. Those skilled in the art know how to select these auxiliary materials. For example, the neutral lipid may be a neutral helper phospholipid; including, but not limited to, one or more of di-oleoyl phosphatidylcholine, palmitoyl oleoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylcholine, di-palmitoyl phosphatidylethanolamine, distearoyl phosphatidylcholine, distearoyl-phosphatidylethanolamine, hydrogenated soy phosphatidylcholine, di-oleoyl lecithin, dimyristoyl phosphatidylcholine, egg yolk lecithin, sphingomyelin, dipalmitoyl phosphatidylcholine, dityristoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, di-oleoyl phosphatidylserine, di-oleoyl phosphatidylethanolamine DOPE, and distearoyl phosphatidylcholine DSPC; preferably di-oleoyl phosphatidylcholine, di-stearoyl phosphatidylcholine, di-oleoyl phosphatidylethanolamine DOPE or di-stearoyl phosphatidylcholine DSPC. The steroid includes, but is not limited to, one or more of cholesterol, campesterol, beta-sitosterol, desmosterol, dehydrocholesterol, brassicasterol, stigmasterol, erythrinsterol, fucosterol, ergosterol, dehydroergosterol, dihydrocholesterol, campestanol, sitostanol, lotenol, cycloartanol; preferably cholesterol. The polyethylene glycol lipids include, but are not limited to, phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), distearoyl phosphatidylglycerol-polyethylene glycol 2000, dilauroyl phosphatidylglycerol-polyethylene glycol 2000, di-oleoyl phosphatidylglycerol-polyethylene glycol 2000, dimyristoyl phosphatidylglycerol-polyethylene glycol 2000, di-linolenoyl phosphatidylglycerol-polyethylene glycol 2000, di-erucic phosphatidylglycerol-polyethylene glycol 2000, 1-palmitoyl-2-oleoyl phosphatidylglycerol-polyethylene glycol 2000, or di-palmitoyl phosphatidylglycerol-polyethylene glycol 2000; preferably phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000).
Based on the teachings of the present invention, the man skilled in the art can adjust the ratio of said ionizable cationic lipids, neutral lipids, steroid and polyethylene glycol lipids in the nanoparticle, provided that a club-like nanoparticle is obtained. In a specific embodiment, in the club-like lipid nanoparticle of the present invention, the mole percentage of the ionizable cationic lipid is 40% -70%, preferably 45% -55%; the mole percentage of the neutral lipid is 5% -15%, preferably 8% -12%; the molar percentage of the steroid is 30% -50%, preferably 35% -45%; the polyethylene glycol lipid is 1-5%, preferably 1-3%; or the mole ratio of the ionizable cationic lipid, neutral lipid, steroid and polyethylene glycol lipid is (40-70): 5-15): 30-50): 1-5; preferably (45-55): (8-12): (35-45): (1-3); more preferably 50:10:38.5:1.5.
In this context, bioactive agent refers to a substance having biological activity encapsulated in a nanoparticle that needs to be delivered to a subject, e.g., a human body, using the nanoparticle. In a specific embodiment, the bioactive agent refers to a substance having therapeutic activity or therapeutic effect, i.e., a therapeutic agent. For example, the therapeutic agent may be a nucleic acid, an antibody, a protein drug (including but not limited to an antibody), an antibody drug conjugate, a small molecule compound, and the like.
In a specific embodiment, the bioactive agent in the club-like lipid nanoparticle of the present invention is a nucleic acid. In preferred embodiments, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rRNA), micrornas (miRNA), transfer RNA (tRNA), small interfering RNA (siRNA), and micronuclear RNA (snRNA); mRNA is preferred.
On the basis of the spherical-rod-shaped lipid nanoparticle, the invention provides a pharmaceutical composition, which comprises the lipid nanoparticle and a pharmaceutically acceptable excipient. The pharmaceutical composition may have various functions based on the bioactive agent in the nanoparticle. For example, the pharmaceutical composition of the invention may be a vaccine composition.
The lipid nanoparticle or pharmaceutical composition of the present invention can be used to deliver bioactive agents to non-liver organs, such as the spleen, thereby enabling the prevention or treatment of infectious diseases, tumors, and autoimmune diseases.
Based on the teachings of the present invention, one skilled in the art can prepare lipid nanoparticles of the present invention, provided that the nanoparticles produced are spherical-rod shaped lipid nanoparticles. In a specific embodiment, the club-like lipid nanoparticle of the invention is prepared by the following method:
s1: providing a mixture of an ionizable cationic lipid, optionally a neutral lipid, a steroid, a polyethylene glycol lipid, and an organic solvent;
S2: providing an aqueous solution of a bioactive agent;
S3: mixing the mixed solution and the aqueous solution in the steps S1 and S2 at a certain flow rate ratio (preferably 1:3) and a volume ratio (preferably 1:3) by a microfluidic system, and diluting the mixed solution (preferably 5 times) by water to obtain an intermediate solution;
s4: carrying out ultrafiltration concentration on the intermediate product solution to obtain concentrated solution;
S5: and (3) passing the concentrated solution through a filter membrane to obtain the lipid nanoparticle.
The reagents and process conditions used in each step can be determined by one skilled in the art. For example, the organic solvent used in step S1 is a solvent that is miscible with water, such as ethanol, acetone, isopropanol, methanol, and the like. The aqueous solution in step S2 may comprise a buffer, preferably a physiologically acceptable buffer, such as a phosphate buffer, optionally a PBS buffer. In step S3, the preparation of the intermediate solution is performed in a normal temperature environment, and the solution mixed by the microfluidic chip is immediately diluted with water, so that the concentration of ethanol is reduced, and the ethanol is prevented from damaging particles. In step S4, the intermediate solution is ultrafiltered by using an ultrafiltration tube, so as to remove a large amount of ethanol existing in the solution, and concentrate the solution to obtain a concentrated solution with higher concentration, thereby facilitating the subsequent preparation of the cationic lipid nanoparticle solution with the required target concentration. In step S5, the concentrated solution is filtered by using a water-based filter membrane with the size of 0.22 mu m so as to remove large particles which are agglomerated in the system, and the cationic lipid nano particles with better dispersity and uniform size are obtained.
According to the invention, the lipid nanoparticle is regulated and controlled from a spherical shape to a spherical shape by regulating the nitrogen-phosphorus ratio of the ionizable cationic lipid molecules and the nucleic acid molecules in the spleen-targeted lipid nanoparticle. Compared with the prior art, the beneficial effects are as follows:
1. The formation of the club-like structure enhances the uptake of lipid nanoparticles by cells;
2. the formation of the ball rod-shaped structure enhances the expression of living bodies;
3. the formation of the ball rod-shaped structure improves the capacity of LNP targeting the spleen, so that an LNP system can more stably and efficiently deliver nucleic acid medicines to the spleen;
4. the spherical rod-shaped lipid nanoparticle can activate immune cells more efficiently, cause immune response and improve the immune effect of organisms.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1
This example 1 provides a method for preparing an imidazolyl lipid compound SIM7 with spleen targeting, the chemical formula of SIM7 is shown below:
Referring to fig. 11 and 12, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM7 is as follows:
Step 1: after dissolving compound A1-1 (15.1 g,78.0 mmol) in 100mL of anhydrous Dichloromethane (DCM), the mixture was placed in a nitrogen-protected flask, and compound A1-2 (29.0 g,156.0 mmol) and 4-Dimethylaminopyridine (DMAP) (11.4 g,93.8 mmol) were added to the mixture, and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) (78.1 g,407.0 mmol) was added in portions, and the reaction was continued after the reaction solution was cooled to room temperature. After 18 hours of reaction, the reaction mixture was washed twice with 500mL of a 0.4N hydrochloric acid/10% sodium chloride mixed solution, and once with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography on silica gel, and the target eluate was collected and concentrated to give Compound A1-3 (22.5 g).
Step 2: compound A1-4 (2.8 g,10 mmol) was dissolved in Dimethylformamide (DMF), A1-3 (21.8 g,30 mmol) and potassium carbonate (4.1 g,30 mmol) were added, stirred 24h at 80℃and TLC was followed until the material was consumed, after completion of the reaction extracted twice with 100mL of water and twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography on silica gel, and the target eluate was collected and concentrated to give compound A1 (16.6 g).
Step 3: compound A1 (14.8 g,21.0 mmol) and compound B1 (3.1 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM7 with spleen targeting (18.8 g, 89.6% yield).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM7 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.27(s,1H),8.06(s,1H),7.95(s,1H),7.87(s,1H),7.77(d,J＝15.5Hz,2H),7.51(s,2H),7.36(dd,J＝7.5,2.0Hz,1H),7.25(d,J＝2.0Hz,1H),6.94(d,J＝7.4Hz,1H),4.18(s,2H),4.13-4.08(m,8H),3.61(d,J＝5.1Hz,4H),2.84(d,J＝12.4Hz,1H),2.76(s,1H),2.67(s,4H),2.23(s,4H),1.79(s,4H),1.66(s,4H),1.61(s,4H),1.45(s,4H),1.38(s,4H),1.32(d,J＝7.6Hz,8H),1.28(dd,J＝4.6,1.6Hz,24H),0.90(s,6H).
Example 2
This example 2 provides a method for preparing an imidazolyl lipid compound SIM40 with spleen targeting, the SIM40 chemical formula is shown below:
Referring to fig. 13 and 14, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM40 is as follows:
Step 1: after dissolving compound A2-1 (15.1 g,78.0 mmol) in 100mL of anhydrous DCM, the mixture was placed in a nitrogen-protected flask, compound A2-2 (20.3 g,156.0 mmol) and DMAP (11.4 g,93.8 mmol) were added to the mixture, and EDC (78.1 g,407.0 mmol) was added in portions, and the reaction was continued until the reaction solution cooled to room temperature. After 18h of reaction, the reaction mixture was washed twice with 500mL of a 0.4N HCl/10% NaCl mixture, once with saturated brine, and the organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product. The crude product was purified by column chromatography on silica gel, and the target eluate was collected and concentrated to give compound A2-3 (21.6 g).
Step 2: compound A2-4 (2.8 g,10 mmol) was dissolved in DMF, A2-3 (9.18 g,30 mmol) and potassium carbonate (4.1 g,30 mmol) were added, stirred for 24h at 80℃followed by TLC until the material was consumed, after completion of the reaction extracted twice with 100mL of water and twice with 100mL of DCM, the combined organic phases were dried over anhydrous magnesium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography on silica gel, and the target eluate was collected and concentrated to give compound A2 (15.2 g).
Step 3: compound A2 (12.4 g,21.0 mmol) and compound B2 (3.4 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM40 with spleen targeting (13.1 g, yield 69.3%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM40 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.27(s,1H),8.06(s,1H),7.95(s,1H),7.87(s,1H),7.78(s,1H),7.64(s,1H),7.51(s,2H),7.36(dd,J＝7.5,1.8Hz,1H),7.27(d,J＝2.0Hz,1H),6.94(d,J＝7.4Hz,1H),4.13-4.08(m,10H),3.61(d,J＝5.0Hz,4H),2.63(d,J＝12.3Hz,2H),2.59-2.53(m,4H),2.23(s,4H),2.01(s,2H),1.79(s,4H),1.66(s,4H),1.61(s,4H),1.45(s,4H),1.38(s,4H),1.33(s,4H),1.32-1.27(m,12H),0.90(s,6H).
Example 3
This example 3 provides a method for preparing an imidazolyl lipid compound SIM45 with spleen targeting, the chemical formula of SIM45 is shown below:
Referring to fig. 15, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM45 is as follows:
compound A1 (14.8 g,21.0 mmol) and compound B2 (3.4 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM45 with spleen targeting (15.5 g, yield 73.1%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM45 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.27(s,1H),8.06(s,1H),7.95(s,1H),7.87(s,1H),7.78(s,1H),7.64(s,1H),7.51(s,2H),7.36(dd,J＝7.5,1.8Hz,1H),7.27(d,J＝2.0Hz,1H),6.94(d,J＝7.4Hz,1H),4.13-4.08(m,10H),3.61(d,J＝5.1Hz,4H),2.63(d,J＝12.3Hz,2H),2.56(d,J＝12.3Hz,2H),2.53(s,2H),2.23(s,4H),2.01(s,2H),1.79(s,4H),1.66(s,4H),1.61(s,4H),1.45(s,4H),1.38(s,4H),1.32(d,J＝7.6Hz,8H),1.28(dd,J＝4.6,1.6Hz,24H),0.90(s,6H).
Example 4
This example 4 provides a method for preparing an imidazolyl lipid compound SIM69 with spleen targeting, the SIM69 chemical formula is shown below:
referring to fig. 16 and 17, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM69 is as follows:
Step 1: k 2CO3 (13.3 g,96 mmol) and KI (2.0 g,12 mmol) were introduced into a solvent mixture containing A3-2 (19.8 g,80 mmol) and A3-1 (2.4 g,40 mmol) in methanol and acetonitrile (100 mL,30:70, v/v). The reaction mixture was refluxed in an oil bath for 48 hours. The solvent was washed three times with water. The organic layer was further washed with diethyl ether several times to remove any residual A3-2. The organic solvent was then dried over anhydrous Na 2SO4 and the solvent was removed under reduced pressure. The crude compound A3 obtained was directly used without additional purification.
Step 2: compound A3 (8.3 g,21.0 mmol) and compound B1 (3.1 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM69 with spleen targeting (11.1 g, yield 76.3%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM69 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.10(s,1H),8.06(s,1H),7.96(s,1H),7.87(s,1H),7.62(d,J＝5.6Hz,2H),7.51(s,2H),4.12(s,2H),3.79(s,2H),3.61(d,J＝5.1Hz,4H),2.77(s,2H),2.67(s,4H),2.52(s,4H),1.53(s,3H),1.33-1.27(m,37H),0.90(s,6H).
Example 5
This example 5 provides a method for preparing an imidazolyl lipid compound SIM99 with spleen targeting, the chemical formula of SIM99 is shown below:
referring to fig. 18, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM99 is as follows:
Compound A3 (8.3 g,21.0 mmol) and compound B2 (3.4 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM99 with spleen targeting (12.0 g, yield 81.2%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM99 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.10(s,1H),8.06(s,1H),7.96(s,1H),7.87(s,1H),7.62(d,J＝5.8Hz,2H),7.51(s,2H),4.05(s,2H),3.79(s,2H),3.61(d,J＝5.1Hz,4H),2.63(d,J＝12.3Hz,2H),2.59-2.50(m,8H),1.94(s,2H),1.53(s,3H),1.33-1.26(m,37H),0.90(s,6H).
Example 6
This example 6 provides a method for preparing an imidazolyl lipid compound SIM74 with spleen targeting, the SIM74 chemical formula is shown below:
referring to fig. 19 and 20, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM74 is as follows:
Step 1: a4-1 (21.3 g,30 mmol) was dissolved in 100mL anhydrous DCM and PCC (8.6 g,40 mmol) was slowly added over 15min and stirred at room temperature for 2h and the solvent was removed under reduced pressure to give crude compound A4 which was used without further purification.
Step 2: compound A4 (14.8 g,21.0 mmol) and compound B1 (3.1 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM74 with spleen targeting (12.5 g, yield 59.2%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM74 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.10(s,1H),8.06(s,1H),7.96(s,1H),7.87(s,1H),7.62(d,J＝5.6Hz,2H),7.51(s,2H),4.78(s,1H),4.12(d,J＝3.3Hz,4H),3.79(s,2H),3.61(d,J＝5.1Hz,4H),2.77(s,2H),2.67(s,4H),2.52(s,4H),2.28(s,2H),2.24(s,2H),1.71-1.64(m,4H),1.61-1.51(m,10H),1.41-1.36(m,8H),1.33-1.27(m,40H),0.90(s,9H).
Example 7
This example 7 provides a method for preparing an imidazolyl lipid compound SIM120 with spleen targeting, the SIM120 chemical formula is shown below:
Referring to fig. 21 and 22, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM120 is as follows:
Step 1: a5-2 (11.6 g,60 mmol), A5-1 (4.1 g,30 mmol), K 2CO3 (10.0 g,72 mmol) and KI (1.7 g,10 mmol) were dissolved in anhydrous DMF and stirred overnight in a glove box at 80 ℃. The reaction progress was monitored by thin layer chromatography throughout the reaction (developing reagent: dichloromethane, DCM). After the reaction was completed, 30ml of DCM was added, washed three times with 50ml of water and the organic phase was collected. Subsequently, purification was performed using a flash column chromatography system with DCM as eluent to give compound A5 as a white solid.
Step 2: compound A5 (7.6 g,21.0 mmol) and compound B2 (3.4 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM120 with spleen targeting (11.8 g, yield 83.6%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM120 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.27(s,1H),8.06(s,1H),7.95(s,1H),7.87(s,1H),7.78(s,1H),7.64(s,1H),7.51(s,2H),7.36(dd,J＝7.5,1.8Hz,1H),7.27(d,J＝2.0Hz,1H),6.94(d,J＝7.4Hz,1H),4.10(d,J＝2.0Hz,6H),3.61(d,J＝5.0Hz,4H),2.63(d,J＝12.3Hz,2H),2.56(d,J＝12.3Hz,2H),2.53(s,2H),2.01(s,2H),1.80(d,J＝0.7Hz,4H),1.45(s,4H),1.32(d,J＝10.5Hz,8H),1.29(d,J＝3.5Hz,8H),0.90(s,6H).
Example 8
This example 8 provides a method for preparing an imidazolyl lipid compound SIM121 with spleen targeting, the chemical formula of SIM121 is shown below:
referring to fig. 23, the specific procedure for preparing the spleen-targeted imidazolyl lipid compound SIM121 is as follows:
Compound A5 (7.6 g,21.0 mmol) and compound B1 (3.1 g,21.0 mmol) were dissolved in 100mL of DCM and stirred for 20min. C1 (6.7 g,21.0 mmol) and piperazine (0.2 g,2.1 mmol) were added sequentially and the resulting reaction mixture was stirred at room temperature for 24h. The compound was purified by flash column chromatography system to give imidazolyl lipid compound SIM121 with spleen targeting (10.1 g, yield 77.3%).
The main data of nuclear magnetic hydrogen spectrum of the imidazolyl lipid compound SIM121 with spleen targeting are as follows:
1H NMR(500MHz,CDCl3)δ8.27(s,1H),8.06(s,1H),7.95(s,1H),7.87(s,1H),7.77(d,J＝15.5Hz,2H),7.51(s,2H),7.36(dd,J＝7.5,2.0Hz,1H),7.25(d,J＝1.9Hz,1H),6.94(d,J＝7.4Hz,1H),4.18(s,2H),4.10(d,J＝2.2Hz,4H),3.61(d,J＝5.0Hz,4H),2.84(d,J＝12.5Hz,1H),2.76(s,1H),2.67(s,4H),1.80(d,J＝0.7Hz,4H),1.45(s,4H),1.35-1.27(m,16H),0.90(s,6H).
EXAMPLE 9 preparation of mRNA-lipid nanoparticles
MRNA-lipid nanoparticles were prepared with a nitrogen to phosphorus ratio of 1:1 of ionizable cationic lipid molecules to nucleic acid molecules.
Firstly, SIM7 is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SIM7, DSPC, cholesterol and DMG-PEG2000 are respectively 50%, 10%, 38.5% and 1.5%. The lipid molecules are simultaneously dissolved in absolute ethanol to be used as an organic phase solution. For in vivo transfection experiments, the aqueous phase solution was prepared as follows: luc-mRNA (firefly luciferase mRNA) was dispersed in citrate buffer (10 mm, ph=4.0) and the nitrogen-to-phosphorus ratio of SIM7 to Luc-mRNA was controlled to 1:1. And (3) rapidly mixing the organic phase solution and the aqueous phase solution at room temperature by utilizing a microfluidic technology, mixing the organic solution and the aqueous solution at a flow rate of 1:3 and a volume of 1:3, and diluting the mixed solution by using water for 5 times to obtain an intermediate solution. And (3) carrying out ultrafiltration concentration on the intermediate solution by using an ultrafiltration tube to obtain concentrated solution. Finally, filtering the sample by adopting a 0.22 mu m water-based filter membrane, and performing aseptic treatment to obtain a final product.
The method for preparing mRNA-lipid nanoparticles at a nitrogen to phosphorus ratio of SIM7 to nucleic acid molecule of 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1 is similar to that at a nitrogen to phosphorus ratio of 1:1, except that the nitrogen to phosphorus ratio of ionizable cationic lipid SIM7 to nucleic acid molecule is altered.
SIM40, SIM45, SIM69, SIM74, SIM99, SIM120, SIM121 produced mRNA-lipid nanoparticles at nitrogen to phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, and SIM7 produced mRNA-lipid nanoparticles at nitrogen to phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1 were similar except that the species of ionizable cationic lipid was changed in the step of producing the initial organic phase.
Example 10
For the above nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, and 36:1, respectively, the Luc-SIM 7-lipid nanoparticles, luc-SIM 40-lipid nanoparticles, luc-SIM 45-lipid nanoparticles, luc-SIM 69-lipid nanoparticles, luc-SIM 74-lipid nanoparticles, luc-SIM 99-lipid nanoparticles, luc-SIM 120-lipid nanoparticles, luc-SIM 121-lipid nanoparticles were detected using a dynamic light scattering particle sizer, the detection medium was a buffer solution at a detection temperature of 25 ℃, thereby determining the particle size, polydisperse coefficient (PDI), and surface potential (zeta) of the cationic lipid nanoparticles LNP contained in the final dispersion; the encapsulation efficiency of mRNA was determined using RiboGreen (Thermofisher) method, excitation wavelength 480nm, emission wavelength 520nm, and the results are shown in tables 1-8, with almost all formulations falling in the size range of about 100nm to about 300nm, while the polydispersity index varied from about 0.2 to about 0.4, indicating a relatively uniform size; the surface potential gradually increases from about-10 mV to about-1 mV with the increase of the nitrogen-phosphorus ratio; the encapsulation efficiency gradually increases with the increase of the nitrogen-to-phosphorus ratio, and has higher encapsulation efficiency after the nitrogen-to-phosphorus ratio is 12:1.
TABLE 1 physicochemical parameters of Luc-SIM 7-lipid nanoparticle LNP
TABLE 2 physicochemical parameters of Luc-SIM 40-lipid nanoparticle LNP
Table 3 physicochemical parameters of Luc-SIM 45-lipid nanoparticle LNP
Table 4 physicochemical parameters of Luc-SIM 69-lipid nanoparticle LNP
Table 5-physico-chemical parameters of Luc-SIM 74-lipid nanoparticle LNP
TABLE 6 physicochemical parameters of Luc-SIM 99-lipid nanoparticle LNP
TABLE 7 physicochemical parameters of Luc-SIM 120-lipid nanoparticle LNP
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TABLE 8 physicochemical parameters of Luc-SIM 121-lipid nanoparticle LNP
Example 11
Lipid nanoparticles with SIM7, SIM40, SIM45, and SIM74 as constituent components were used for cryo-electron microscopy. Images were taken at 45000 x magnification using Glacios freeze electron microscope, the results are shown in fig. 1-4. The results showed that at a nitrogen to phosphorus ratio of 1:1, SIM7/SIM40/SIM45/SIM 74-lipid nanoparticles were spherical, at nitrogen to phosphorus ratios of 3:1, 6:1, SIM7/SIM40/SIM45/SIM 74-lipid nanoparticles were in an adhering state, at a nitrogen to phosphorus ratio of 9:1, SIM7/SIM40/SIM45/SIM 74-lipid nanoparticles began to exhibit short club-like structures, at nitrogen to phosphorus ratios of 12:1, 18:1, 24:1, 30:1, 36:1, sim7-lipid nanoparticles were in long club-like structures, at nitrogen to phosphorus ratios of 12:1, 18:1, sim40-lipid nanoparticles were in long club-like structures, at nitrogen to phosphorus ratios of 12:1, 18:1, sim45-lipid nanoparticles were in long club-like structures, and at nitrogen to phosphorus ratios of 12:1, 18:1, sim74-lipid nanoparticles were in long club-like structures (as shown in fig. 10).
Example 12
Lipid nanoparticles composed of SIM7, SIM40, SIM45, SIM74 were used for in vivo Luc-mRNA delivery testing.
It is reported in the literature that lipid nanoparticles may differ in vivo and in vitro delivery characteristics, often due to differences in vivo barriers, organ distribution, cellular properties, etc. Therefore, transfection conditions of in vitro cell experiments may not be used for predicting in vivo activity or tissue tropism, and thus the present example directly performs delivery of Luc-mRNA in vivo by lipid nanoparticles to study in vivo mRNA transport efficiency and organ targeting selectivity.
The particles used were Luc-SIM7/SIM40/SIM45/SIM 74-lipid nanoparticles encapsulated as described in example 1.
In order to examine the mRNA transfer efficiency and the targeting selection of the lipid nanoparticle with the spherical rod-shaped structure, the experiment takes Luc-mRNA stably expressing luciferase as a model, and examines the mRNA expression efficiency and the targeting delivery condition of the lipid nanoparticle with different nitrogen-phosphorus ratios of Luc-SIM7/SIM40/SIM45/SIM 74.
Experimental animals: balb/c mice, females, 6 weeks old, weigh approximately 20g.
Blank control group: group 1, 3 mice, tail vein injected with PBS buffer.
Experimental group: 36 groups of 3 mice, and tail veins were each injected with an injection containing lipid nanoparticles of the present invention.
The Luc-SIM7/SIM40/SIM45/SIM 74-lipid nanoparticle injection was prepared at the above nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1 by tail vein injection into Balb/c mice (mRNA dose of 0.5 mg/kg), 6 hours later by intraperitoneal injection of 15mg/mL of D-Luciferin fluorescein at a dose of 100. Mu.L, and then after 5 minutes, the mice were sacrificed using cervical dislocation to strip heart, liver, spleen, lung and kidneys.
Using a small animal living body imager (Xenogen)Spectrum; perkinElmer inc., waltham, MA, USA) by bioluminescence mode. Fig. 6 shows in vitro images of luciferases in major organs 6 hours after intravenous injection of Luc-SIM 7-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, fig. 8 is a statistical plot of average expression of mLuc mRNA in spleens 6 hours after intravenous injection of Luc-SIM 7-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, table 11 to table 14 are average expression in major organs mLuc mRNA after intravenous injection of Luc-SIM 7/40/45/74-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, respectively.
TABLE 9 in vivo transfection Performance test of Luc-SIM 7-lipid nanoparticle LNP
TABLE 10 in vivo transfection Performance test of Luc-SIM 40-lipid nanoparticle LNP
TABLE 11 in vivo transfection Performance test of Luc-SIM 45-lipid nanoparticle LNP
Table 12-Luc-SIM 74-lipid nanoparticle LNP in vivo transfection performance test
As shown in fig. 6, 8, 9, 10, 11, and 12, LNP with nitrogen to phosphorus ratio of 12:1, 18:1, 24:1, 30:1, 36:1, SIM7/SIM40/SIM45/SIM74 involved in preparation had higher expression in spleen than LNP with nitrogen to phosphorus ratio of 1:1, 3:1, 6:1, 9:1, SIM7/SIM40/SIM45/SIM74 involved in preparation; besides the nitrogen-phosphorus ratio of 1:1, under the condition of other nitrogen-phosphorus ratios, the enrichment rate of LNP prepared by the SIM7/SIM40/SIM45/SIM74 in spleen is up to 98%, and the data further show that the lipid nanoparticle with the spherical rod-shaped structure can greatly improve the spleen site-specific transfection efficiency of the lipid nanoparticle.
Comparative example 1 preparation of mRNA-lipid nanoparticles
MRNA-lipid nanoparticles were prepared at a nitrogen to phosphorus ratio of 1:1 for commercial lipid molecules to nucleic acid molecules.
Firstly, SM102 is selected as ionizable cationic lipid, 1, 2-distearoyl-sn-glycerophosphorylcholine (DSPC) is selected as neutral auxiliary phospholipid, cholesterol is selected as steroid, 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000) is selected as PEG phospholipid, and the mole percentages of SM102, DSPC, cholesterol and DMG-PEG2000 are respectively 50%, 10%, 38.5% and 1.5%. The lipid molecules are simultaneously dissolved in absolute ethanol to be used as an organic phase solution. For in vivo transfection experiments, the aqueous phase solution was prepared as follows: luc-mRNA (firefly luciferase mRNA) was dispersed in citrate buffer (10 mm, ph=4.0) and the nitrogen-to-phosphorus ratio of SM102 to Luc-mRNA was controlled to 1:1. And (3) rapidly mixing the organic phase solution and the aqueous phase solution at room temperature by utilizing a microfluidic technology, mixing the organic solution and the aqueous solution at a flow rate of 1:3 and a volume of 1:3, and diluting the mixed solution by using water for 5 times to obtain an intermediate solution. And (3) carrying out ultrafiltration concentration on the intermediate solution by using an ultrafiltration tube to obtain concentrated solution. Finally, filtering the sample by adopting a 0.22 mu m water-based filter membrane, and performing aseptic treatment to obtain a final product.
The method for preparing mRNA-lipid nanoparticles at 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1 to nucleic acid molecules was similar to that at 1:1 to nitrogen-phosphorus ratio, except that the nitrogen-phosphorus ratio of commercial lipid molecules SM102 to nucleic acid molecules was altered.
Comparative example 2.
For the Luc-SM 102-lipid nanoparticles prepared at the nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, respectively, detection was performed by using a dynamic light scattering particle sizer, the detection medium was PBS buffer, the detection temperature was 25deg.C, and the particle size, polydispersity index (PDI), and surface potential (zeta) of the cationic lipid nanoparticles LNP contained in the final dispersion were determined; the encapsulation efficiency of mRNA was determined using RiboGreen (Thermofisher) method, excitation wavelength 480nm, emission wavelength 520nm, and the results are shown in table 13, with almost all formulations falling in the size range of about 50nm to about 150nm, and polydispersity index varying from about 0.1 to about 0.4; the surface potential varies between-3 mV and-7 mV; the encapsulation efficiency gradually increased to be stable with the increase of the nitrogen-phosphorus ratio, and the encapsulation efficiency tended to be stable after the nitrogen-phosphorus ratio was 6:1.
TABLE 13 physicochemical parameters of Luc-SM 102-lipid nanoparticle LNP
Comparative example 3.
Lipid nanoparticles composed of SM102 were used for cryo-electron microscopy. Images were taken at 45000 x magnification using Glacios freeze electron microscope, the results are shown in fig. 5. The results showed that SM 102-lipid nanoparticles were spherical regardless of the nitrogen to phosphorus ratio.
Comparative example 4.
Lipid nanoparticles composed of SM102 were used for in vivo Luc-mRNA delivery assays.
It is reported in the literature that lipid nanoparticles may differ in vivo and in vitro delivery characteristics, often due to differences in vivo barriers, organ distribution, cellular properties, etc. Therefore, transfection conditions of in vitro cell experiments may not be used for predicting in vivo activity, nor for predicting tissue tropism, and thus the present comparative example directly performs delivery of Luc-mRNA in vivo of lipid nanoparticles to study in vivo mRNA transport efficiency and organ targeting selectivity thereof.
The particles used were Luc-SM 102-lipid nanoparticles entrapped in the particles prepared in comparative example (1).
In order to examine the mRNA transfer efficiency and the targeting selection of the lipid nanoparticle with the spherical rod-shaped structure, the experiment takes Luc-mRNA stably expressing luciferase as a model, and examines the mRNA expression efficiency and the targeting delivery condition of the lipid nanoparticle of Luc-SM102 under different nitrogen-phosphorus ratios.
Experimental animals: balb/c mice, females, 6 weeks old, weigh approximately 20g.
Blank control group: group 1, 3 mice, tail vein injected with PBS buffer.
Experimental group: 9 groups of 3 mice were injected with injections containing the lipid nanoparticles of the present invention, respectively, by tail vein.
The Luc-SM 102-lipid nanoparticle injection was prepared at the nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1 by tail vein injection of Balb/c mice (mRNA dose of 0.5 mg/kg), 6 hours later by intraperitoneal injection of 15mg/mL of D-Luciferin fluorescein at a dose of 100. Mu.L, and then after 5 minutes, the mice were sacrificed by cervical dislocation, and heart, liver, spleen, lung and kidney were exfoliated.
Using a small animal living body imager (Xenogen)Spectrum; perkinElmer inc., waltham, MA, USA) by bioluminescence mode. FIG. 7 shows in vitro images of luciferase in major organs 6 hours after intravenous injection of Luc-SM 102-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, FIG. 9 is a statistical plot of average expression amounts of mLuc mRNA in spleen 6 hours after intravenous injection of Luc-SM 102-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1, table 13 is a ratio of average expression amounts of mLuc mRNA in major organs 6 hours after intravenous injection of Luc-SM 102-lipid nanoparticle formulations at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1.
As shown in fig. 7, 9 and table 14, the expression of LNP in which SM102 participated in preparation in spleen was much lower than that of LNP in which SIM7 participated in preparation in spleen at nitrogen-phosphorus ratios of 1:1, 3:1, 6:1, 9:1, 12:1, 18:1, 24:1, 30:1, 36:1.
TABLE 14 in vivo transfection Performance test of Luc-SM 102-lipid nanoparticle LNP
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (19)
1. A balloon-like lipid nanoparticle with spleen targeting, the nanoparticle comprising:
1) An ionizable cationic lipid;
2) Neutral lipids;
3) A steroid compound;
4) Polyethylene glycol lipids; and
5) A bioactive agent;
The club-shaped lipid nanoparticle is composed of a spherical head part and a long club-shaped tail part.
2. The lipid stick nanoparticle of claim 1, wherein the lipid stick nanoparticle is comprised of a spherical head and one or more long stick tails distributed at its edge.
3. The spherical lipid nanoparticle of claim 1, wherein the ionizable cationic lipid is a compound of formula (I), or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof,
Wherein:
G 1 is selected from the group consisting of: absent, substituted or unsubstituted C 1-C20 alkylene, substituted or unsubstituted C 2-C20 alkenylene, said substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 1 is selected from the group consisting of: H. substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, substituted or unsubstituted 4-8 membered heterocyclyl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted C 6-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted 8-10 membered fused heterocyclyl 、-NRaRb、-NRaRbOH、-N(Ra(OH))(Rb(OH))、-C(O)OR、-OC(O)R、-C(O)NRaRb、-N(Ra)C(O)Rb、CN、-C(O)R、-OR、-O(CH2)mOR、-O(CH2)mNRaRb、-S(O)xR、-S-SR、-C(O)SR、-SC(O)R、-N(Ra)C(O)N(Rb)2、-OC(O)NRaRb、-N(Ra)C(O)ORb, containing 1,2 or 3 heteroatoms selected from N, O or S, said substitution being by 1,2 or 3C 1-C5 alkyl;
R, R a、Rb are each independently selected from the group consisting of: substituted or unsubstituted C 1-C20 alkyl, substituted or unsubstituted C 2-C20 alkenyl, substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
x is an integer from 0 to 2 (e.g., 0,1 or 2);
R 2 is selected from the group consisting of: H. a substituted or unsubstituted C 6-C10 aryl (preferably phenyl), a substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring, anthracycline), a substituted or unsubstituted C 1-C12 alkyl, a substituted or unsubstituted C 2-C12 alkenyl, a substituted or unsubstituted C 2-C12 alkynyl, and optionally substituted with 1, 2 or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is selected from the group consisting of: c 1-C5 alkyl, C 2-C5 alkenyl, C 2-C5 alkynyl;
Q is selected from the group consisting of: n, substituted or unsubstituted C 6-C10 aryl (preferably phenyl), substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring, anthracycline), substituted or unsubstituted C 4-C8 cycloalkyl (preferably cyclopentyl, cyclohexyl), substituted or unsubstituted 4-8 membered heterocyclyl (preferably pyrrole ring, pyrimidine ring) containing 1, 2 or 3 heteroatoms selected from N, O or S; the substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl, br;
Each G 2、G3、G4 is independently selected from the group consisting of: absent, substituted or unsubstituted C 1-C10 alkylene, substituted or unsubstituted C 2-C10 alkenylene, said substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl or Br;
L 1 and L 2 are each independently selected from the group consisting of: is not present in 、-O-、-C(O)-、-OC(O)-、-C(O)O-、-O(CH2)qC(O)O-、-O(CH2)qOC(O)-、-OC(O)O-、-S-、-S-S-、-C(O)NRd-、-NRdC(O)-、-S(＝O)x-、-SC(O)-、-C(O)S-;
R 3 and R 4 are each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-C20 alkyl, substituted or unsubstituted straight or branched C 2-C20 alkenyl, substituted or unsubstituted straight or branched C 2-C20 alkynyl, said substitution means substitution with 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
Each R d is independently selected from the group consisting of: H. c 1-C20 alkylene, C 2-C20 alkenylene, C 3-C8 cycloalkylene, C 3-C8 cycloalkenyl;
m and q are each independently integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).
4. The spherical-rod-like lipid nanoparticle of claim 3, wherein G 1 is C 1-C20 alkylene;
R 1 is selected from the group consisting of: substituted or unsubstituted C 3-C8 cycloalkyl, substituted or unsubstituted C 3-C8 cycloalkenyl, substituted or unsubstituted 4-8 membered heterocyclyl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted C 6-C10 aryl, 5-10 membered heteroaryl containing 1,2 or 3 heteroatoms selected from N, O or S, substituted or unsubstituted 8-10 membered fused heterocyclyl 、-NRaRb、-NRaRbOH、-N(Ra(OH))(Rb(OH))、-O(CH2)mNRaRb, containing 1,2 or 3 heteroatoms selected from N, O or S, said substitution referring to substitution with 1,2 or 3 groups selected from the group consisting of: c 1-C5 alkyl substitution;
R 2 is selected from the group consisting of: benzene rings, naphthalene rings, and optionally substituted with 1, 2, or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
Q is selected from the group consisting of: n, benzene ring;
Each G 2、G3、G4 is independently selected from the group consisting of: absent, C 1-C10 alkylene;
l 1 and L 2 are each independently selected from the group consisting of: absence, -O-, -C (O) -, -OC (O) -, -C (O) O-, -O (CH 2)qC(O)O-、-O(CH2)q OC (O) -, -OC (O) O-;
R 3 and R 4 are each independently a linear or branched C 1-C20 alkyl group;
R a、Rb are each independently selected from the group consisting of: c 1-C20 alkyl, C 1-C20 alkylene;
r c is C 1-C5 alkyl;
Each m and q is an integer independently selected from the group consisting of: 1.2, 3, 4, 5, 6, 7, 8.
5. The lipid stick nanoparticle of claim 4, wherein the compound is of formula (IA) or (IB):
wherein ring a is selected from the group consisting of: benzene ring, naphthalene ring, anthracene ring, cyclopentyl, cyclohexyl, pyrrole ring, pyrimidine ring;
R 2 is selected from the group consisting of: benzene ring, naphthalene ring, anthracene ring, C 1-C6 alkyl, and optionally substituted with 1,2, or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is C 1-C5 alkyl.
6. The lipid stick nanoparticle of claim 5, wherein the compound is a compound of formula (IA'),
Wherein,
P is an integer selected from the group consisting of: 2.3, 4 and 5;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
L 1 and L 2 are each independently selected from the group consisting of: absence of, -O-, -O (CH 2)qC(O)O-、-O(CH2)q OC (O) -;
q is an integer selected from the group consisting of: 4. 5, 6, 7, 8;
R 3 and R 4 are each independently a linear or branched C 8-C16 alkyl group;
R a、Rb is independently selected from the group consisting of: c 1-C3 alkyl, C 1-C3 alkylene.
7. The lipid nanoparticle in the form of a sphere stick according to claim 6, wherein the compound is a compound of formula (IB'),
Wherein,
P is an integer selected from the group consisting of: 2.3, 4 and 5;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
Each G 2、G3、G4 is independently C 1-C10 alkylene;
l 1 and L 2 are each independently selected from the group consisting of: absence, -O-, -C (O) -, -OC (O) -, -C (O) O-, -OC (O) O-;
R 3 and R 4 are each independently a linear or branched C 8-C18 alkyl group;
R a、Rb is independently selected from the group consisting of: c 1-C3 alkyl, C 1-C3 alkylene.
8. A club-like lipid nanoparticle according to claim 3, wherein the ionizable cationic lipid is selected from the group consisting of:
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9. A spherical lipid nanoparticle according to claim 3, wherein G 1 is a substituted or unsubstituted C 1-C10 alkylene group, preferably a substituted or unsubstituted C 1-C4 alkylene group, more preferably a substituted or unsubstituted ethylene group, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 1 is selected from the group consisting of: -NR aRb、-NRaRbOH、-N(Ra(OH))(Rb (OH));
R a、Rb are each independently selected from the group consisting of: substituted or unsubstituted C 1-C10 alkyl, preferably substituted or unsubstituted C 1-C4 alkyl, more preferably substituted or unsubstituted methylene or ethylene, said substitution being by 1, 2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
R 2 is selected from the group consisting of: a substituted or unsubstituted C 6-C10 aryl (preferably phenyl), a substituted or unsubstituted 8-14 membered fused aryl (preferably naphthalene ring), and optionally substituted with 1, 2 or 3 groups selected from the group consisting of: F. cl, br, I, R c、ORc;
R c is selected from the group consisting of: c 1-C5 alkyl;
Q is selected from the group consisting of: n, substituted or unsubstituted C 6-C10 aryl (preferably phenyl); the substitution means substitution with 1, 2 or 3 groups independently selected from the group consisting of: c 1-C5 alkyl, F, cl, br;
Each G 2、G3、G4 is independently selected from the group consisting of: an absent, substituted or unsubstituted C 1-C8 alkylene group;
L 1 and L 2 are each independently selected from the group consisting of: absence of, -O-, -C (O) O-, -O (CH 2)q C (O) O-;
R 3 and R 4 are each independently a substituted or unsubstituted, linear or branched C 1-C16 alkyl group, said substitution being by 1,2 or 3 groups independently selected from the group consisting of: F. cl or Br substitution;
q is an integer from 1 to 8 (e.g., 1,2,3, 4, 5, 6, 7, 8).
10. The spherical-rod-like lipid nanoparticle of claim 9, wherein the ionizable cationic lipid is selected from the group consisting of:
Preferably, the ionizable cationic lipid is selected from the following compounds:
11. The spherical rod-like lipid nanoparticle according to claim 1, wherein the mole percentage of the ionizable cationic lipid is 40% -70%, preferably 45% -55%; the mole percentage of the neutral lipid is 5% -15%, preferably 8% -12%; the molar percentage of the steroid is 30% -50%, preferably 35% -45%; the polyethylene glycol lipid is 1-5%, preferably 1-3%; or alternatively
The mole ratio of the ionizable cationic lipid to the neutral lipid to the steroid to the polyethylene glycol lipid is (40-70): 5-15): 30-50): 1-5; preferably (45-55): (8-12): (35-45): (1-3); more preferably 50:10:38.5:1.5.
12. The spherical-rod-shaped lipid nanoparticle of claim 1, wherein the bioactive agent is a therapeutic agent; including, but not limited to, nucleic acids, antibodies, protein drugs (including, but not limited to, antibodies), antibody drug conjugates, small molecule compounds; nucleic acids are preferred.
13. The spherical-rod lipid nanoparticle of claim 12, wherein the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotides, DNA, plasmids, ribosomal RNA (rRNA), micrornas (miRNA), transfer RNAs (tRNA), small interfering RNAs (siRNA), and micronuclear RNAs (snRNA); mRNA is preferred.
14. The spherical-rod-shaped lipid nanoparticle according to claim 1, wherein the particle size of the nanoparticle is 100nm to 240nm, preferably 105nm to 200nm, more preferably 110nm to 160nm.
15. The club-like lipid nanoparticle of claim 1, wherein the ratio of nitrogen to phosphorus of the ionizable cationic lipid to the therapeutic agent is from 9:1 to 36:1, such as 9:1, 12:1, 18:1, 24:1, 30:1, 36:1; preferably, the nitrogen to phosphorus ratio is 12:1, 18:1, 24:1, 30:1; more preferably, the nitrogen to phosphorus ratio is 12:1, 18:1.
16. A pharmaceutical composition comprising the lipid nanoparticle of any one of claims 1-15 and a pharmaceutically acceptable excipient.
17. Use of the lipid nanoparticle of any one of claims 1-15 or the pharmaceutical composition of claim 16 in the manufacture of a medicament for the prevention or treatment of infectious diseases, tumors and autoimmune diseases.
18. The use of claim 17, wherein the medicament is a nucleic acid medicament capable of targeting the spleen.
19. A method of preparing the lipid nanoparticle of any one of claims 1-15, comprising the steps of:
s1: providing a mixture of an ionizable cationic lipid, optionally a neutral lipid, a steroid, a polyethylene glycol lipid, and an organic solvent;
S2: providing an aqueous solution of a bioactive agent;
S3: mixing the mixed solution and the aqueous solution in the steps S1 and S2 at a certain flow rate ratio (preferably 1:3) and a volume ratio (preferably 1:3) by a microfluidic system, and diluting the mixed solution (preferably 5 times) by water to obtain an intermediate solution;
s4: carrying out ultrafiltration concentration on the intermediate product solution to obtain concentrated solution;
S5: and (3) passing the concentrated solution through a filter membrane to obtain the lipid nanoparticle.
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