Lipid membrane structures for intracellular delivery of siRNA
阅读说明：本技术 用于siRNA细胞内递送的脂质膜结构体 (Lipid membrane structures for intracellular delivery of siRNA ) 是由 原岛秀吉 佐藤悠介 于 2018-06-15 设计创作，主要内容包括：该脂质膜结构体是含有式(I)：(R<Sup>1</Sup>)(R<Sup>2</Sup>)C(OH)-(CH<Sub>2</Sub>)a-(O-CO)b-X的脂质化合物作为脂质成分的脂质膜结构体。式(I)中,a表示3～5的整数；b表示0或1的整数；R<Sup>1</Sup>和R<Sup>2</Sup>分别独立地表示可具有-CO-O-的直链状烃基；X表示5～7元非芳族杂环基或下述的式(B)所示的基团,式(B)中,d表示0～3的整数,R<Sup>3</Sup>和R<Sup>4</Sup>分别独立地表示C<Sub>1-4</Sub>烷基或C<Sub>2-4</Sub>烯基,R<Sup>3</Sup>和R<Sup>4</Sup>可彼此键合形成5～7元非芳族杂环(该环上可被1个或2个的C<Sub>1-4</Sub>烷基或C<Sub>2-4</Sub>烯基取代)。(The lipid membrane structure comprises a compound represented by the formula (I): (R) 1 )(R 2 )C(OH)‑(CH 2 ) Lipid membrane structure comprising a- (O-CO) b-X lipid compound as a lipid component. In the formula (I), a represents an integer of 3-5; b represents 0 or1 is an integer; r 1 And R 2 Each independently represents a linear hydrocarbon group which may have-CO-O-; x represents a 5-7-membered non-aromatic heterocyclic group or a group represented by the following formula (B) wherein d represents an integer of 0 to 3 and R 3 And R 4 Each independently represents C 1‑4 Alkyl or C 2‑4 Alkenyl radical, R 3 And R 4 Can be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring (which may be substituted by 1 or 2C's) 1‑4 Alkyl or C 2‑4 Alkenyl substituted).)
1. A lipid compound represented by the following formula (I) or a salt thereof:
[ chemical formula 1]
(R1)(R2)C(OH)-(CH2)a-(O-CO)b-X…(I)
In the formula (I), a represents an integer of 3-5; b represents an integer of 0 or 1; r1And R2Each independently represents a group represented by the following formula (A):
[ chemical formula 2]
CH3-(CH2)q-(CH＝CH)r-(CH2)s-(CH＝CH)t-(CH2)u-(CO-O)c-(CH2)v-…(A)
In the formula (A), q represents an integer of 1-9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8; c represents 0 or 1; v represents an integer of 4 to 12, except for the case where b and c are 0 at the same time, q is an integer of 3 to 5, r and t are 1, s is 1, and u + v is an integer of 6 to 10;
x represents a 5-7 membered non-aromatic heterocyclic group (wherein the group is bonded to (O-CO) b-through a carbon atom, and the ring may be substituted by 1 or 2C1-4Alkyl or C2-4Alkenyl substituted) or a group represented by the following formula (B):
[ chemical formula 3]
-(CH2)d-N(R3)(R4)…(B)
In the formula (B), d represents an integer of 0 to 3, R3And R4Each independently represents C1-4Alkyl or C2-4Alkenyl (the C)1-4Alkyl or C2-4Alkenyl may be substituted by 1 or 2 phenyl), R3And R4Can be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring (which may be substituted by 1 or 2C's)1-4Alkyl or C2-4Alkenyl substituted).
2. A lipid compound or a salt thereof according to claim 1, wherein r and t are 0, and q + s + u is an integer of 8 to 18, preferably an integer of 10 to 16.
3. A lipid compound or a salt thereof according to claim 1 or 2, wherein r is 1, t is 0, q is an integer of 5 to 9, and s + u is an integer of 5 to 9.
4. A lipid compound or salt thereof according to any of claims 1 to 3 wherein v is an integer of 5 to 12.
5. A lipid compound or salt thereof according to any of claims 1 to 4, wherein a is 4 and b is 0 or 1.
6. A lipid compound or salt thereof according to any of claims 1 to 5, wherein b is 0;
x is a group of the formula (B) wherein d is 0 and R3And R4Each independently represents C1-4Alkyl (R)3Shown as C1-4Alkyl may be substituted by 1 phenyl), or at R3And R4When bonded to each other, form a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group or a 1-piperazinyl group (the 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl group may be substituted by 1C1-4Alkyl substituted).
7. A lipid compound or salt thereof according to any of claims 1 to 5, wherein b is 1;
x is a group represented by the formula (B), wherein d is an integer of 0 to 3, and R is3And R4Each independently represents C1-4Alkyl (R)3Shown as C1-4Alkyl may be substituted by 1 phenyl), or at R3And R4Bonded to each other to form a 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl group (the 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl group may be substituted by 1 or 2 identical or different C' s1-4Alkyl substituted).
8. A lipid compound or salt thereof according to any of claims 1 to 5, wherein b is 1;
x is a 5-7 membered non-aromatic heterocyclic group bonded to (O-CO) b-via a carbon atom, and the 5-7 membered non-aromatic heterocyclic group is pyrrolidinyl, piperidinyl, morpholinyl or piperazinyl which may be substituted by 1 or 2 identical or different C' s1-4Alkyl substituted).
9. The lipid compound or salt thereof according to any of claims 1 to 8, which is used as a lipid component for delivering siRNA to a lipid membrane structure in a cell.
10. A lipid membrane structure comprising the lipid compound or the salt thereof according to in any one of claims 1 to 8 as a lipid component.
11. The lipid membrane structure according to claim 10, which is a liposome.
12. The lipid membrane structure of claim 10 or 11, wherein the siRNA is encapsulated inside.
13. The lipid membrane structure according to claim 12, which is used for knocking down a target gene in a cell.
14. The lipid membrane structure according to claim 13, wherein the cell is an immune cell or a cancer cell.
15. The lipid membrane structure of claim 14, for use in knocking down target genes in dendritic cells in an immunotherapy that is: dendritic cells are isolated/harvested from a patient, and after siRNA is introduced into cells of the dendritic cells in vitro, the dendritic cells with the target gene knocked down are administered to the patient.
Technical Field
The present invention relates to a lipid membrane structure for intracellular delivery of sirna (short interfering rna) and the like. More specifically, the present invention relates to a lipid membrane structure such as a liposome that can easily deliver siRNA or the like into the nucleus of an immune cell, particularly, the cell of a dendritic cell.
This application claims priority from Japanese application No. 2017-117708, 6, 15, 2017, and the contents of which are incorporated herein by reference.
Background
As a means for specifically delivering a drug to an affected part, a method of encapsulating a drug in a liposome as a lipid membrane structure has been proposed. In particular, in the field of treatment of malignant tumors, many reports have been made on the effectiveness of liposomes encapsulating antitumor agents. Further, as a lipid membrane structure useful for gene expression, a Multifunctional envelope-type nanostructure (MEND, hereinafter, referred to as "MEND" in the present specification in some cases) has been proposed. This structure is known to be useful as a Drug Delivery System (Drug Delivery System) for selectively delivering a gene or the like into a specific cell, and is useful for gene therapy of a tumor, for example.
As a means for delivering a target substance such as a drug, a nucleic acid, a peptide, a polypeptide, or a sugar to a specific site such as a target organ or a tumor tissue using a lipid membrane structure, many methods of modifying the surface of a lipid membrane structure with a functional molecule have been proposed. When a lipid membrane structure having a drug such as an antitumor agent encapsulated therein reaches a target cell, the lipid membrane structure is taken into the cell by endocytosis, and is contained in an endosome, and then the lipid membrane structure is subjected to enzymatic hydrolysis by lysosomes, and the encapsulated drug is released into the cytoplasm. In order to improve drug release from liposomes taken into endosomes, liposomes (non-patent document 3) and MEND (patent document 4) have been proposed in which the liposome surface is modified with a peptide (GALA: non-patent document 2).
As means for transporting a lipid membrane structure encapsulating a target substance such as a nucleic acid into the nucleus of a target cell, for example, a liposome modified with octapolyarginine on the outer surface (
dendritic cells are antigen presenting cells which are the key components of immune response, and thus are which are important target cells for cancer immunotherapy.an immunocytotherapy (dendritic cell therapy) has been also performed in which dendritic cells are collected from a cancer patient, subjected to antigen introduction or activation in vitro, and then administered to the patient.
In the past, there has been a report of knocking down an immune suppression factor using a lentiviral vector expressing shRNA for introducing RNA into the nucleus of a dendritic cell (
As an artificial delivery system for siRNA introduction, R8/GALA-D-MEND (D-MEND) (non-patent document 8) is reported, D-MEND is a nanocarrier in which MEND is modified with an octameric arginine (R8) peptide as a cell affinity component (element) and a GALA peptide as an endosome escape component (element), and the number of envelope membrane layers of MEND is controlled, D-MEND shows about 70% knockdown in HeLa cell which is a commonly used cancer cell at a low concentration of 12nM, and the activity shows 2-fold or more activity compared with Lipofectamine (LFN2000) 2000(LFN2000) which is a reagent generally used as for introduction by .
However, in the case of transfecting dendritic cells induced from mouse bone marrow cells with D-MEND, it is necessary to make siRNA concentration high (80-120nM) in order to achieve knockdown efficiency of 70-80%, and there is a problem that knockdown efficiency is maintained at about 40% depending on the target factor of siRNA (NPL 9).
To date, a number of cationic lipids have been developed in order to achieve efficient in vivo delivery of sirnas that can inhibit the expression of functional nucleic acids, particularly specific target genes. In particular, the development of a pH-sensitive cationic lipid that is electrically neutral at physiological pH and becomes cationic in a weakly acidic pH environment such as endosome has been remarkable. Jayaraman et al developed DLin-MC3-DMA at factor 7 (F7) knockdown of mouse liver at ED50The amount of siRNA reaches 0.005 mg/kg (non-patent document 10). The inventors have so far developed unique pH-sensitive cationic lipids YSK05 and YSK-C3 as ED in F7 knockdown50Respectively, 0.06 mg siRNA/kg and 0.015mg siRNA/kg (
Furthermore, Dong et al found a unique lipid-like substance cKK-E12 by High-throughput screening (High-throughput screening), in the F7 knockdown, in
In recent years, it has been clarified that many cancer tissues, particularly cancer tissues of human patients, are very rich in interstitial components represented by collagen, which significantly hamper permeability of nanoparticles in cancer tissues, miniaturization of nanoparticles is considered as a very effective strategy for solving the problem, in fact, Cabral et al reported that Lipid Nanoparticles (LNPs) are very difficult to control to be small in technology and reported to be very deficient by controlling the diameter of high-molecular micelles encapsulating a platinum preparation to be as small as about 30nm, and that anti-tumor effects are improved (non-patent document 19), although the same strategy is considered to be very effective in siRNA delivery, in recent years, it has been reported that LNPs (
Disclosure of Invention
Problems to be solved by the invention
The present invention addresses the problem of providing means for efficiently delivering an siRNA or the like into cells, particularly immune cells such as dendritic cells having antigen-presenting ability. More specifically, the present invention addresses the problem of providing an intracellular lipid membrane structure that can efficiently deliver siRNA to various cells including immune cells such as dendritic cells, and a novel compound useful for producing the lipid membrane structure.
In particular, an object of the present invention is to provide a novel compound and a lipid membrane structure which have both excellent delivery efficiency and high safety of siRNA and the like and can overcome reduction in delivery activity of siRNA and the like associated with reduction in particle size of LNP.
Means for solving the problems
First, the present inventors have intensively studied means for efficiently delivering siRNA into cells in order to achieve efficient knockdown of target genes in immune cells, particularly dendritic cells having antigen presenting ability. As a result, the following were found: in the formation of a lipid membrane structure such as MEND, a very high endosome escape property can be achieved by using a lipid compound such as YSK12, which has two unsaturated bonds in two fatty acid chains and has a pKa increased by extending the carbon chain of a hydrophilic portion, as a lipid component. Moreover, it has also been found that: in a lipid membrane structure prepared from a lipid composition containing the lipid compound, target gene knockdown by siRNA can be performed extremely efficiently (
The present inventors have intensively studied a novel compound capable of imparting biodegradability, excellent endosomal escape ability, and LNP stabilization ability by extending two hydrocarbon chains of an appropriate length from a tertiary hydroxyl site of YSK12 and bonding medium-to long-chain fatty acids via an ester bond, in order to provide a novel compound and a lipid membrane structure having both more excellent delivery efficiency and high safety of a target substance (hereinafter, sometimes referred to as "substance to be delivered") to be delivered to a cell such as siRNA and the like based on the structure of YSK12 at step .
That is, according to the present invention, there is provided a lipid compound represented by the following formula (I) or a salt thereof:
[ chemical formula 1]
(R1)(R2)C(OH)-(CH2)a-(O-CO)b-X…(I)
In the formula (I), a represents an integer of 3-5; b represents an integer of 0 or 1; r1And R2Each independently represents a group represented by the following formula (A):
[ chemical formula 2]
CH3-(CH2)q-(CH＝CH)r-(CH2)s-(CH＝CH)t-(CH2)u-(CO-O)c-(CH2)v-…(A)
In the formula (A), q represents an integer of 1-9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8; c represents 0 or 1; v represents an integer of 4 to 12, except for the case where b and c are 0 at the same time, q is an integer of 3 to 5, r and t are 1, s is 1, and u + v is an integer of 6 to 10;
x represents a 5-7 membered non-aromatic heterocyclic group (wherein the group is bonded to (O-CO) b-through a carbon atom, and the ring may be substituted by 1 or 2C1-4Alkyl or C2-4Alkenyl substituted) or a group represented by the following formula (B):
[ chemical formula 3]
-(CH2)d-N(R3)(R4)…(B)
In the formula (B), d represents an integer of 0 to 3, R3And R4Each independently represents C1-4Alkyl or C2-4Alkenyl (the C)1-4Alkyl or C2-4Alkenyl may be substituted by 1 or 2 phenyl), R3And R4Can be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring (which may be substituted by 1 or 2C's)1-4Alkyl or C2-4Alkenyl substituted).
According to a preferred aspect of the above invention, there is provided: the lipid compound or a salt thereof, wherein in the formula (A), r and t are 0, and q + s + u is an integer of 8 to 18, preferably an integer of 10 to 16; the lipid compound or a salt thereof, wherein r is 1, t is 0, q is an integer of 5 to 9, preferably 6 to 8, and s + u is an integer of 5 to 9, preferably 6 to 8; the lipid compound or a salt thereof, wherein v is an integer of 5 to 12, preferably an integer of 6 to 10. More preferably, there is provided: the lipid compound or a salt thereof, wherein in the formula (A), r and t are 0, q + s + u is an integer of 8 to 18, preferably 10 to 16, and v is an integer of 5 to 12, preferably 6 to 10; the lipid compound or a salt thereof, wherein r is 1, t is 0, q is an integer of 5 to 9, preferably 6 to 8, s + u is an integer of 5 to 9, preferably 6 to 8, and v is an integer of 5 to 12, preferably 6 to 10.
According to a preferred embodiment of the present invention, there is provided the lipid compound or a salt thereof described above, wherein in the formula (I), a is 4 and b is 0 or 1. More preferably, the lipid compound or a salt thereof is provided, wherein in the formula (I), a is 4; b is 0 or 1; r1And R2Each independently represents a group represented by the formula (A), wherein r and t are 0, q + s + u is an integer of 8 to 18, preferably 10 to 16, and v is an integer of 5 to 12, preferably 6 to 10, or a group wherein r is 1, t is 0, q is an integer of 5 to 9, preferably 6 to 8, s + u is an integer of 5 to 9, preferably 6 to 8, and v is an integer of 5 to 12, preferably 6 to 10.
Further preferred embodiments provide the lipid compound or a salt thereof, wherein in the formula (I), B is 0, X is a group represented by the formula (B) (wherein d is 0, and R is3And R4Each independently represents C1-4Alkyl (R)3Shown as C1-4Alkyl may be substituted by 1 phenyl), or at R3And R4When bonded to each other, form a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group or a 1-piperazinyl group (the 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl group may be substituted by 1C1-4Alkyl substitution); the lipid compound or a salt thereof, wherein B is 1, X is a group represented by the formula (B) (wherein d is an integer of 1 to 3, and R is3And R4Each independently represents C1-4Alkyl (R)3Shown as C1-4Alkyl may be substituted by 1 phenyl), or at R3And R4When bonded to each other, form a 1-pyrrolidinyl group, a 1-piperidinyl group, a 1-morpholinyl group or a 1-piperazinyl group (the 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl group may be 1 or 2 identical orDifferent C1-4Alkyl substitution); the lipid compound or a salt thereof, wherein the 5-to 7-membered non-aromatic heterocyclic group represented by X (the group is bonded to (O-CO) b-via a carbon atom) is a pyrrolidinyl group, a piperidinyl group, a morpholinyl group or a piperazinyl group (the pyrrolidinyl group, the piperidinyl group, the morpholinyl group or the piperazinyl group may be substituted by 1 or 2 identical or different C's)1-4Alkyl substituted).
From another points of view, the present invention provides a lipid compound represented by the above formula (I) or a salt thereof, which is used as a lipid component of a lipid membrane structure for delivering a substance to be delivered such as siRNA into a cell, according to a preferred embodiment of the present invention, there is provided the above lipid compound wherein the cell is an immune cell or a cancer cell, more preferably a dendritic cell, a monocyte, a macrophage or a cancer cell, the above lipid compound or a salt thereof wherein the lipid membrane structure is a liposome, and the above lipid compound or a salt thereof wherein the lipid membrane structure is a Multifunctional Envelope Nanostructure (MEND).
From the viewpoint of further , according to the present invention, there is provided a lipid membrane structure containing a lipid compound represented by the above formula (I) as a lipid component, such as a liposome, further, according to a preferred embodiment of the present invention, the lipid membrane structure is a lipid membrane structure for delivering a substance, preferably siRNA, into a cell, and a substance to be delivered such as siRNA or the like is encapsulated inside.
In addition, according to another preferable embodiments, there are provided a lipid membrane structure comprising 1 or 2 or more kinds of compounds selected from the group consisting of the lipid compound of the above formula (I), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), cholesterol (Chol), 1, 2-dimyristoyl-sn-glycerol and
Further, according to the present invention, there is provided a method for delivering a substance to be delivered such as siRNA or the like into a cell, preferably an immune cell, particularly preferably a dendritic cell, the method comprising the steps of: the substance to be delivered is encapsulated inside, and the lipid membrane structure described above containing the lipid compound represented by the above formula (I) as a lipid component is brought into contact with a cell. The method may be carried out in vivo in mammalian organisms including humans, or in vitro using cells isolated/collected from the organism.
For example, in the case of using dendritic cells, dendritic cell therapy can be performed by administering dendritic cells in which a target gene is knocked down to a patient after introducing a substance to be delivered into the cells by the above-described method for dendritic cells isolated/collected from the patient. Thus, according to the present invention, there is provided an immunotherapy method comprising: dendritic cells are isolated/collected from a patient, and after introducing a substance to be delivered into the cells of the dendritic cells in vitro, the dendritic cells with the target gene knocked down are administered to the patient. Further, according to the present invention, there is also provided the above lipid membrane structure for use in knocking down a target gene in a dendritic cell in an immunotherapy which is: dendritic cells are isolated/collected from a patient, and after introducing a substance to be delivered into the cells of the dendritic cells in vitro, the dendritic cells with the target gene knocked down are administered to the patient.
Effects of the invention
The lipid compound of the present invention can provide a lipid membrane structure which combines excellent delivery efficiency and high safety of a substance to be delivered such as siRNA and which can overcome a decrease in delivery activity of siRNA or the like accompanied by a decrease in particle size of LNP. The lipid membrane structure can be imparted with biodegradability, excellent endosome escape ability, and LNP stabilization ability. The lipid membrane structure provided by the present invention can be efficiently transported into any cell, such as an immune cell containing a dendritic cell, into which a substance to be delivered, such as siRNA, is difficult to introduce, and can efficiently escape from an endosome. Thus, the lipid membrane structure can efficiently release the encapsulated substance to be delivered in the cell, and the target gene is knocked down by the substance to be delivered. Therefore, the lipid membrane structure of the present invention can be used for effective immunotherapy using a substance such as siRNA, preferably dendritic cell therapy, in cancer treatment. Further, when a lipid membrane structure such as a liposome is prepared using the lipid compound provided by the present invention as a lipid component, a very high endosome escape property can be achieved, and a substance to be delivered such as siRNA can be efficiently delivered from the lipid membrane structure containing the lipid compound to the cytoplasm.
Drawings
FIG. 1 is a schematic diagram of the order of preparation of LNP by the alcohol dilution method.
FIG. 2 is a graph showing the pKa of each LNP in the lipid compounds having different hydrophilic site-containing chemical structures in example 2.
FIG. 3A is a graph showing the in vivo knockdown activity of F7 in each LNP of lipid compounds having different chemical structures and containing hydrophilic sites in example 2.
FIG. 3B is a graph showing in vitro knockdown activity of each LNP of the lipid compounds having different respective chemical structures and containing hydrophilic sites in example 2.
FIG. 3C is a graph showing the hemolytic activity of each LNP of the lipid compounds having different chemical structures each containing a hydrophilic site in example 2.
FIG. 4A is a graph showing the pKa of LNPs containing lipid compounds (CL4 series) having different hydrophobic scaffold structures in example 3.
FIG. 4B is a graph showing the pKa of LNPs containing lipid compounds (CL15 series) different in each hydrophobic scaffold structure in example 3.
FIG. 5 is a graph showing the in vitro knockdown activity of the hydrophobic scaffold structures of the individual different CL 15-LNPs of example 3.
FIG. 6A is a graph showing the in vivo knockdown activity of F7 in LNPs containing lipid compounds (CL4 series) each having a different hydrophobic scaffold structure in example 3.
FIG. 6B is a graph showing the in vivo knockdown activity of F7 in LNPs containing lipid compounds (CL15 series) each having a different hydrophobic scaffold structure in example 3.
FIG. 7A is a graph showing the results of optimizing the lipid profile of the formulation of example 3 for the in vivo F7 knockdown activity of CL4H6-LNP as an indicator.
FIG. 7B is a graph showing the results of optimizing the lipid/siRNA charge ratio for the formulation of example 3, using the in vivo F7 knockdown activity of CL4H6-LNP as an indicator.
FIG. 8 is a graph showing the dose-dependent dependence of the in vivo F7 knockdown efficiency with the best composition of CL4H6-LNP in example 3.
FIG. 9A is a graph showing the results of evaluating the safety of CL4H6-LNP in example 4, and ALT/AST values in plasma after 24 hours of administration.
FIG. 9B is a graph showing the body weight change of mice from immediately before administration to 24 hours after administration as a result of evaluating the safety of CL4H6-LNP in example 4.
FIG. 10 is a graph showing the in vitro knockdown activity of CL15-LNP controlled to have an average particle size of about 35nm in example 4.
FIG. 11 is a graph showing the pKa of LNPs containing lipid compounds (CL4H series) having different hydrophobic scaffold structures in example 5.
FIG. 12 is a graph showing the in vivo F7 knock-down activity of LNPs containing lipid compounds (CL4H series) with different respective hydrophobic scaffold structures in example 5.
FIG. 13A is a graph showing the results of measurement of the amount of siRNA in the
FIG. 13B is a graph showing the results of measurement of the amount of siRNA in the liver (ng/g liver) 24 times after administration in example 6, mice administered with CL4H6-LNP, YSK05-LNP, and YSK13-C3-LNP loaded with siRNA against F7.
[ FIG. 13C]Is the amount of siRNA in the
FIG. 14 is a fluorescent staining image of blood vessels (FITC), lipids (DiI), and siRNA (Cy5) of the
FIG. 15A is a graph showing the chronological changes in the relative amounts of F7 protein in plasma (the amount of F7 protein in plasma of LNP-naive mice (NT) on each day of recovery is set to 100) in the mice that were administered with CL4H6-LNP, YSK05-LNP, and YSK13-C3-LNP loaded with siRNA against F7 in example 6.
[ FIG. 15B]ED, which is obtained by knocking down F7 with the passage of time (Durability) (day) after administration of LNP in comparison with the amount of F7 protein in plasma, in example 6, mice administered with CL4H6-LNP, YSK05-LNP, and YSK13-C3-LNP loaded with siRNA against F750The relationship of (a) to (b).
FIG. 16A is a graph showing the chronological changes in the content of each cationic lipid in the liver of mice in example 6 to which CL4H6-LNP, YSK05-LNP, and YSK13-C3-LNP loaded with siRNA against F7 were administered.
FIG. 16B is a graph showing the chronological changes in the content of each cationic lipid in the spleen of mice in example 6 to which CL4H6-LNP, YSK05-LNP, and YSK13-C3-LNP loaded with siRNA against F7 were administered.
FIG. 17 is a graph showing the chronological changes in the amount of PEG-DSG-modified CL4H6-LNP in the blood relative to the amount of PEG-DSG-modified CL4H6-LNP (the amount (ID) of PEG-DSG-modified CL4H6-LNP administered to mice is 100%) in the mice administered with PEG-DSG-modified CL4H6-LNP in example 7.
FIG. 18A is a graph showing the measurement results of the relative PLK1 expression amount in the
FIG. 18B is a graph showing the results of measurement of the rate of body weight change (%) from before administration to 24 hours after administration in OSRC2 cell-subcutaneously transplanted mice in example 7, which were administered with PEG-DSG modified CL4H6-LNP and PEG-DSG modified YSK05-LNP loaded with siRNA against
FIG. 19 is a graph showing the results of measuring the relative CD45 expression level of macrophages induced by ICR mouse bone marrow cells transfected with siRNA against CD45 in example 8 (the CD45 expression level of tumor-associated macrophages (NT) to which no siRNA was administered was set at 100%) after culturing the macrophages for 24 hours.
FIG. 20 is a graph showing the results of measurement of the relative CD45 expression amount (%) of tumor-associated
FIG. 21 is a graph showing the chronological changes in the rate of body weight change (%) on
Detailed Description
Hereinafter, embodiments of the present invention will be specifically described. In the present specification, "X1 to X2(X1 and X2 satisfy the real number of X1 < X2)" means "X1 or more and X2 or less".
A lipid compound according to an embodiment of the present invention (a lipid compound of the present invention) is represented by the following formula (I).
[ chemical formula 4]
(R1)(R2)C(OH)-(CH2)a-(O-CO)b-X…(I)
In the formula (I), a represents an integer of 3 to 5, preferably 4.
b represents an integer of 0 or 1. In the case where b is 0, the absence of an-O-CO-group means a single bond.
In the formula (I), R1And R2Each independently represents a group represented by the following formula (A).
[ chemical formula 5]
CH3-(CH2)q-(CH＝CH)r-(CH2)s-(CH＝CH)t-(CH2)u-(CO-O)c-(CH2)v-…(A)
In the formula (A), q represents an integer of 1-9; r represents 0 or 1; s represents an integer of 1 to 3; t represents 0 or 1; u represents an integer of 1 to 8; v represents an integer of 4 to 12. c represents 0 or 1. Preferably, b is 0 and c is 1, or b is 1 and c is 0.
In preferable embodiments, r and t are 0, and q + s + u is an integer of 8 to 18, preferably 10 to 16, in preferable embodiments, r is 1, t is 0, q is an integer of 5 to 9, preferably 6 to 8, and s + u is an integer of 5 to 9, preferably 6 to 8, in preferable embodiments, v is an integer of 4 to 12, preferably 6 to 10, more preferably 6, and further preferably a is 4, and b is 0 or 1.
Wherein, in the case where b and c are both 0, q is an integer of 3 to 5, r and t are 1, s is 1, and u + v is an integer of 6 to 10.
In the formula (I), X represents a 5-7-membered non-aromatic heterocyclic group or a group represented by the following formula (B).
[ chemical formula 6]
-(CH2)d-N(R3)(R4)…(B)
X is a 5-7 membered non-aromatic heterocyclic group bonded to (O-CO) b-via a carbon atom, which ring may be substituted by 1 or 2C1-4Alkyl (having 1 to 4 carbon atoms)Alkyl) or C2-4Alkenyl (alkenyl having 2 to 4 carbon atoms) is substituted. Examples of the hetero atom contained in the 5-to 7-membered non-aromatic heterocyclic group include: nitrogen atom, oxygen atom, sulfur atom, or the like. The ring-constituting hetero atom may be 1 or may contain 2 or more hetero atoms which may be the same or different. The heterocyclic group may have 1 or 2 or more double bonds in the heterocyclic ring, but the heterocyclic ring is not an aromatic ring. Saturated heterocycles are sometimes preferred. In addition, in the substituent in which 1 or 2 hydrogen atoms in the 5-to 7-membered non-aromatic heterocyclic group are substituted, as C1-4Examples of the alkyl group include: methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, tert-butyl, etc., as C2-4Alkenyl groups include: vinyl group (ethenyl group), propenyl group, butenyl group, and the like.
In the formula (B), d represents an integer of 0 to 3, R3And R4Each independently represents C1-4Alkyl or C2-4An alkenyl group. As C1-4Alkyl and C2-4Examples of the alkenyl group include the same groups as those listed above. R3And R4Shown as C1-4Alkyl or C2-4Alkenyl groups may be substituted with 1 or 2 phenyl groups, respectively. In addition, R3And R4Can be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring. The 5-to 7-membered non-aromatic heterocyclic ring may be substituted by 1 or 2C1-4Alkyl or C2-4And (3) alkenyl substitution.
According to another preferred embodiments, in the lipid compound represented by formula (I), B is 0 and X represents a group represented by formula (B). in the case of this embodiment, d is preferably 0 and R is preferably3And R4Each independently may represent C1-4Alkyl (R)3Shown as C1-4Alkyl groups may be substituted with 1 phenyl group), and may be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring. At R3And R4In the case of bonding to one another, 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl is preferably formed, which 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl may be substituted by 1C1-4Alkyl substitution.
According to a further preferred embodiments, B is 1 and X represents a radical of the formula (B). in the case of this embodiment, d is preferably 1 to3 is an integer of R3And R4Each independently may represent C1-4Alkyl (R)3Shown as C1-4Alkyl groups may be substituted with 1 phenyl group), and may be bonded to each other to form a 5-to 7-membered non-aromatic heterocyclic ring. At R3And R4In the case of bonding to one another, 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl is preferably formed, which 1-pyrrolidinyl, 1-piperidinyl, 1-morpholinyl or 1-piperazinyl can be substituted by 1 or 2 identical or different C' s1-4Alkyl substitution.
In addition, according to another preferable embodiments, b is 1, X is a 5-7 membered non-aromatic heterocyclic group (the group is bonded to (O-CO) b-through a carbon atom), and the 5-7 membered non-aromatic heterocyclic group is preferably pyrrolidinyl, piperidinyl, morpholinyl or piperazinyl which may be substituted by 1 or 2C's which may be the same or different1-4Alkyl substitution.
The lipid compound represented by the formula (I) may be present as an acid addition salt, and the kind of the acid constituting the salt is not particularly limited, and may be any kinds of inorganic acids, organic acids, and the like1And R2In some cases, optical isomers may exist, and optical isomers in a pure form, a mixture of arbitrary optically active forms, racemates, and the like are also included in the scope of the present invention.
Among the compounds of formula (I), particularly preferred compounds are those wherein R is1And R2The same, a is a compound of 4. The compounds of formula (I), including the compounds, can be readily prepared by the methods specifically shown in the examples of this specification. With reference to the production method of this example, one skilled in the art can easily produce any of the compounds of formula (I) included in the range by appropriately selecting the starting compounds, reagents, reaction conditions, and the likeProvided is a compound. The pKa of the compound of formula (I) is not particularly limited, and may be selected, for example, from about 4.0 to 9.0, preferably from about 4.5 to 8.5, and the type of each substituent is preferably selected so as to give a pKa in this range. Uptake of lipid membrane structures such as liposomes into cells by endocytosis is affected by the pKa of the lipid membrane structures. The pKa of the lipid membrane structure that is easily taken up by endocytosis varies depending on the type of cell. Therefore, it is preferable to adjust the pKa of the compound of formula (I) so that the pKa of the lipid membrane structure is in a range that is easily taken into the target cell.
Examples of the lipid constituting the lipid membrane structure of the present invention include: phospholipids, glycolipids, sterols (sterols), saturated or unsaturated fatty acid esters, or saturated or unsaturated fatty acids, and the like.
Examples of the phospholipid and phospholipid derivative include: phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol phosphate, 1, 2-dimyristoyl-1, 2-deoxyphosphatidylcholine, plasmalogen, phosphatidic acid, and the like, and these may be used 1 kind or 2 or more kinds in combination. The fatty acid residue in these phospholipids is not particularly limited, and examples thereof include: examples of the saturated or unsaturated fatty acid residue having 12 to 20 carbon atoms include: acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid. Further, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can be used.
Examples of glycolipids include: glyceroglycolipids (e.g., sulfoxyribosylglycerides (sulfoxylosylglycerides), diglycosyl diglycerides, digalactosyldiglyceride, galactosyl diglyceride, glycosyl diglycerides), glycosphingolipids (e.g., galactosylceresides, lactosylcerebrosides, gangliosides), and the like.
Examples of sterols (sterols) include: sterols derived from animals (e.g., cholesterol, cholesteryl succinate, lanosterol, dihydrolanosterol, streptosterol, dihydrocholesterol), sterols derived from plants (phytosterol) (e.g., stigmasterol, sitosterol, campesterol, brassicasterol), sterols derived from microorganisms (e.g., zymosterol, ergosterol), and the like.
Examples of the saturated or unsaturated fatty acid include: c12-20 saturated or unsaturated fatty acids such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.
Examples of the saturated or unsaturated fatty acid ester include: glycerin fatty acid ester in which 1 or 2 hydroxyl groups of glycerin form ester bonds with fatty acid. Examples of the fatty acid residue in the glycerin fatty acid ester include: an acyl group derived from a C12-20 saturated or unsaturated fatty acid such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid. Specifically, there may be mentioned: dimyristoyl glycerol (DMG), distearoyl glycerol (DSG), and the like.
The form of the lipid membrane structure is not particularly limited, and examples of the form of dispersion in an aqueous solvent (aqueous solvent, water-based solvent) include: unilamellar liposomes, multilamellar liposomes, O/W emulsions, W/O/W emulsions, spherical micelles, rope micelles, irregular (amorphous) lamellar structures, and the like. A preferable embodiment of the lipid membrane structure of the present invention includes a liposome. Hereinafter, a liposome is described as a preferred embodiment of the lipid membrane structure of the present invention, but the lipid membrane structure of the present invention is not limited to a liposome.
The lipid membrane structure of the present invention is a lipid membrane structure for delivering a substance to be delivered such as siRNA into a cell, and is characterized in that the substance to be delivered is encapsulated inside and that the lipid structure contains a lipid compound represented by the above formula (I) as a lipid component. The type of cell (target cell) to which a substance to be delivered is delivered by the lipid membrane structure of the present invention is not particularly limited. The lipid membrane structure of the present invention can deliver a substance to be delivered to various cells constituting an animal such as immune cells, endothelial cells, epithelial cells, fibroblasts, hepatocytes (liver parenchymal cells), pancreatic cells, nerve cells, smooth muscle cells, and cardiac muscle cells, or a wide variety of cells such as cancer cells that are cancerated by these cells, and stem cells that have the ability to differentiate. The target cell may be a cell existing in an animal body, or may be a cell cultured in vitro such as a cultured cell or a primary cultured cell. Examples of the immune cell include: dendritic cells, macrophages, lymphocytes (T cells, B cells, NK cells), granulocytes, monocytes, and the like. Preferred examples of the cells to be delivered by the lipid membrane structure of the present invention include: immune cells and cancer cells, particularly preferred are: dendritic cells, monocytes, macrophages and cancer cells.
Hereinafter, siRNA will be described as a preferred example of a substance to be delivered (a substance to be delivered), but the substance to be delivered is not limited to siRNA. For example, in addition to nucleic acids such as microrna, mRNA, and plasmid, the lipid membrane structure of the present invention may be encapsulated with an active ingredient of any drug such as an antitumor agent, an anti-inflammatory agent, an antibacterial agent, and an antiviral agent, or any substance such as a saccharide, a peptide, a low-molecular compound, and a metal compound.
The siRNA (small interfering RNA) is a low-molecular double-stranded RNA consisting of 21 to 23 base pairs, is involved in RNA interference (RNAi), and inhibits the expression of a gene sequence-specifically by destruction of mRNA, and it has been reported that the synthesized siRNA causes RNA interference in human cells, and is expected to be applied to the fields of use as a drug or treatment of cancer, etc. since the RNA interference using the siRNA can knock down a gene, the type of siRNA that can be used in the present invention is not particularly limited, and any siRNA may be used as long as RNA interference can be caused, &ttttranslation & ">tt and/ttt &g, a double-stranded RNA of 21 to 23 base pairs may be used, and the 3 'portion of the RNA strand adopts a structure in which 2 bases are protruded, each strand has a phosphate group at the 5' end and a structure in which a 3 'end has a hydroxyl group is substituted with a phosphate group, and a phosphodiester bond portion is substituted with a hydroxyl group at the 2' end of a ribose backbone.
siRNA can be delivered into cells, preferably immune cells or cancer cells, particularly preferably dendritic cells, monocytes, macrophages or cancer cells, using the lipid membrane structure of the present invention. The method may be carried out in vivo in mammalian organisms including humans, or in vitro using cells isolated/collected from the organism. For example, in the case of using dendritic cells, dendritic cell therapy can be performed by administering dendritic cells in which a target gene is knocked down to a patient after introducing siRNA into the cells using the lipid membrane structure of the present invention to dendritic cells isolated/collected from the patient. Without being bound by any particular theory, the double-stranded siRNA delivered into cells by the lipid membrane structure of the present invention is dissociated into single strands by the action of an enzyme called helicase, forms a complex (RISC) with Argonaute protein or the like exhibiting endonuclease activity to a target mRNA, and can knock down a target gene by RNA interference.
The lipid component of the lipid membrane structure of the present invention may be a lipid compound of formula (I) alone, but it is generally preferable to form the lipid membrane structure by combining 1 or 2 or more of the lipids described above with the lipid compound of formula (I) , the combination and the blending ratio of the plurality of lipids are not particularly limited, and as specifically shown in the examples, the type and blending ratio of the lipids used, for example, as an index of the knockdown activity against a target gene or the like may be optimized, for example, as the lipid component, the combination of the compound of formula (I), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), cholesterol (Chol), 1, 2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol 2000 dimyristoyl glycerol (PEG-DMG2000) may have a content of the compound of formula (I) of about 80 to 90 mol%, preferably about 85 mol%, PEG-DMG2000 of about 1 to 2 mol%, preferably about 1 mol%, and/or the mole ratio of the compound of formula (I) of about 85 mol% and 0/15 to 0/15% of the lipid component may be deactivated.
The particle size of the lipid membrane structure of the present invention is not particularly limited, but is preferably about 60 to 140nm, more preferably about 80 to 120nm, and about 20 to 50nm, more preferably about , in view of knock-out efficiency, the polydispersity index (PDI) is about 0.05 to 0.1, preferably about 0.06 to 0.08, and further about , preferably about 0.07, and the Z-potential may be about 5.5mV to 6.0mV, preferably about 5.8 mV.
The lipid membrane structure of the present invention may be subjected to appropriate surface modification and the like as required.
For example, in order to promote the intranuclear transport of the lipid membrane structure of the present invention, the lipid membrane structure may be surface-modified with an oligosaccharide compound of 3 or more sugars, for example. The kind of the oligosaccharide compound having 3 or more saccharides is not particularly limited, and for example, an oligosaccharide compound having 3 to about 10 saccharide units bonded to each other can be used, and an oligosaccharide compound having 3 to about 6 saccharide units bonded to each other can be preferably used.
More specifically, examples of the oligosaccharide compound include Cellotriose (Cellotriose: -D-glucopyranosyl- (1 → 4) - -, D-glucopyranosyl- (1 → 4) -D-glucose), potato trisaccharide (Chacotriose: 0-L-rhamnopyranose- (1 → 2) - [ 1-L-rhamnopyranose- (1 → 4) ] -D-glucose), Isomaltotriose (Gentianose: 6-D-fructopyranosyl 1-D-glucopyranosyl- (1 → 6) -2-D-glucopyranoside), xylopyranose (Isomaltotriose: 3-D-glucopyranosyl- (1 → 6) -4-D-galactopyranosyl- (1 → 6) -D → pyranosyl → 6), xylopyranose- (1 → 4 → 6) -2-D-glucopyranosyl-glucopyranose) - ((5-D → 4 → 7-glucopyranosyl- (1 → 6) -glucopyranosyl-galactose → 4 → 6) -glucopyranosyl-D → pyranosyl-glucose → 4 → 6 → pyranosyl-glucopyranosyl-D → 4 → 6 → 4 → 2-glucopyranosyl-D → 4 → 6 → 4 → 6-glucopyranosyl → 4 → 6-glucopyranosyl → 4 → pyranosyl-glucopyranosyl → 4 → pyranoglucose → 4 → pyranoglucose → 4 → 6-glucopyranosyl-glucopyranose → 4 →.
More specifically, isomaltotriose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and the like can be suitably used, and among these, maltotriose, maltotetraose, maltopentaose, and maltohexaose in which glucose is bonded to α 1-4 can be suitably used in , maltotriose or maltotetraose is particularly preferable, and maltotriose is most preferable, and the amount of surface modification of the lipid membrane structure by the oligosaccharide compound is not particularly limited, and is, for example, about 1 to 30 mol%, preferably about 2 to 20 mol%, and more preferably about 5 to 10 mol% based on the total lipid mass.
The method of surface-modifying the lipid membrane structure with an oligosaccharide compound is not particularly limited, and for example, a liposome obtained by surface-modifying the lipid membrane structure with a monosaccharide such as galactose or mannose is known (patent document 3), and therefore the surface modification method described in the publication can be adopted. The entire disclosure of the above publication is included in the disclosure of the present specification by reference. This means is a method of modifying the surface of the lipid membrane structure by bonding a monosaccharide compound to a polyalkylene glycolated lipid, and is preferable because the surface of the lipid membrane structure can be modified simultaneously with the polyalkylene glycol.
The surface of the lipid membrane structure is modified with a hydrophilic polymer such as polyalkylene glycol, thereby improving the stability such as the blood retention of the liposome, and the like, and this means is described in, for example, Japanese patent application laid-open No. H1-249717, Japanese patent application laid-open No. H2-149512, Japanese patent application laid-open No. H4-346918, Japanese patent application laid-open No. 2004-10481 and the like, polyalkylene glycol is preferable as the hydrophilic polymer, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol and the like are used as the polyalkylene glycol, and the molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and about 1,000 to 5,000 is preferable in the step .
The surface modification of the lipid membrane structure with the polyalkylene glycol can be easily performed by, for example, constructing the lipid membrane structure by using the polyalkylene glycol-modified lipid as the lipid membrane-constituting lipid. For example, in the case of modification by polyethylene glycol, stearylated polyethylene glycol (e.g., stearic acid PEG45(STR-PEG45), etc.) may be used. Furthermore, N- [ carbonyl-methoxypolyethylene glycol-2000 ] -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- [ carbonyl-methoxypolyethylene glycol-5000 ] -1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- [ carbonyl-methoxypolyethylene glycol-750 ] -1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [ carbonyl-methoxypolyethylene glycol-2000 ] -1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [ carbonyl-methoxypolyethylene glycol-5000 ] -1, polyethylene glycol derivatives such as 2-distearoyl-sn-glycero-3-phosphoethanolamine, but polyalkylene glycolated lipids are not limited thereto.
In addition, by bonding the oligosaccharide compound to the polyalkylene glycol, surface modification by the polyalkylene glycol and the oligosaccharide compound can be achieved simultaneously. However, the method of surface-modifying the lipid membrane structure with a polyalkylene glycol or an oligosaccharide compound is not limited to the above-described method, and for example, a lipidated compound such as a stearylated polyalkylene glycol or oligosaccharide compound may be used as the constituent lipid of the lipid membrane structure to perform surface modification.
In the production of the lipid membrane structure of the present invention, as a lipid derivative for improving the retention in blood, glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerol phospholipid derivatives, and the like can be used, for example. In addition, as the hydrophilic polymer for improving the retention in blood, besides polyalkylene glycol, dextran, pullulan, polysucrose, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, and the like may be used for surface modification.
The lipid membrane structure of the present invention may contain 1 or 2 or more species selected from the group consisting of: membrane stabilizers such as sterols, or glycerol or fatty acid esters thereof; antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene; a charged (charged) species; and membrane polypeptides and the like. Examples of the charged substance imparting a positive charge include: saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane; or a cationic polymer. Examples of the charged substance imparting a negative charge include: dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and the like. Examples of the membrane polypeptide include: membrane-external polypeptides and membrane-internal polypeptides, and the like. The amount of these substances to be blended is not particularly limited, and may be appropriately selected depending on the purpose.
The lipid membrane structure of the present invention can impart any 1 or 2 or more functions such as a temperature-change-sensitive function, a membrane-permeation function, a gene expression function, and a pH-sensitive function. By appropriately adding these functions, for example, the retention of a lipid membrane structure such as a nucleic acid containing a gene in blood can be improved, and the capture rate of reticuloendothelial tissue such as liver or spleen can be reduced. Further, the lipid membrane structure after being taken into a target cell by endocytosis can efficiently escape from endosomes and be transported into the nucleus, and a high gene expression activity can be achieved in the nucleus.
Examples of the temperature change-sensitive lipid derivative that can impart a temperature change-sensitive function include dipalmitoylphosphatidylcholine and the like. Examples of the pH-sensitive lipid derivative that can impart a pH-sensitive function include dioleoyl phosphatidylethanolamine.
The lipid membrane structure of the present invention may be modified with a substance such as an antibody that specifically binds to a receptor or an antigen on the cell surface. By this modification, the efficiency of delivery of the substance into the nucleus of the cell can be improved. For example, a monoclonal antibody against a biological component specifically expressed in a target tissue or organ is preferably disposed on the surface of the lipid membrane structure. This method is described in, for example,
The surface of the lipid membrane structure of the present invention may be modified with a polypeptide containing a plurality of consecutive arginine residues (hereinafter referred to as "polyarginine"). As the polyarginine, a polypeptide containing 4 to 20 consecutive arginine residues can be preferably used, a polypeptide consisting of only 4 to 20 consecutive arginine residues can be more preferably used, and octapolyarginine or the like can be particularly preferably used. The intracellular delivery efficiency of a target substance encapsulated in a liposome can be improved by modifying the surface of a lipid membrane structure such as a liposome with polyarginine such as octapolyarginine (
Examples of such compounds include compounds in which O, O ' -N-didodecanoyl-N- (α -trimethylaminoacetyl) -diethanolamine chloride, O ' -N-ditetradecanoyl-N- (α -trimethylaminoacetyl) -diethanolamine chloride, O ' -N-dihexadecanoyl-N- (α -trimethylaminoacetyl) -diethanolamine chloride, O ' -N-dioctadecyl (ジオクタデセノイル) -N- (α -trimethylaminoacetyl) -diethanolamine chloride, O ', O "-tridecanoyl-N- (ω -trimethylaminodecanoyl) aminomethane bromide and N- [ α -trimethylaminoacetyl ] -didodecyl-D-glutamate ammonium, dimethyldioctadecyl glutamate ammonium, 2, 3-dioleoxy-N- [2- (spermine carboxamide) ethyl) -N, N-dimethyl-1-propane trifluoroacetate, 1, 2-dimyristoxy-propyl-N- [3- (dimethylcarbamoyl) ethyl) -N, N- (β -dimethylamino) ethyl-carbamoyl ] amine bromide and/or compounds in which the nucleic acid membrane is filled with the function.
As MENDs, for example, MENDs having a structure in which a complex of a nucleic acid such as plasmid DNA and a cationic polymer such as protamine is used as a core and the core is encapsulated inside a lipid-coated membrane in a liposome form, and further, it is reported that a peptide for adjusting pH responsiveness or membrane permeability may be disposed on the lipid-coated membrane of MENDs as necessary, and the outer surface of the lipid-coated membrane may be modified with an alkylene glycol such as polyethylene glycol, and MENDs designed to encapsulate a condensed DNA and a cationic polymer inside the lipid-coated membrane of MENDs so that gene expression can be efficiently achieved are also known.
The form of the lipid membrane structure is not particularly limited, and examples thereof include: a form in which the aqueous dispersion is dispersed in an aqueous solvent (for example, water, physiological saline, phosphate buffered physiological saline, or the like), a form in which the aqueous dispersion is freeze-dried, or the like.
examples are produced by dissolving all lipid components in an organic solvent such as chloroform, drying the solution under reduced pressure by an evaporator or spray-drying the solution by a spray dryer to form a lipid membrane, adding an aqueous solvent to the dried mixture, and emulsifying the mixture by an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like, and also by a method known as a method for producing liposomes, for example, a reverse phase evaporation method, and the like, and when the size of the lipid membrane structure is to be controlled, the size of the lipid membrane structure in a dispersed state is extruded (extrusion filtration) under high pressure by using a membrane filter or the like having a system, and for example, in the case of liposomes, the average particle diameter is about 60 to 140nm, preferably about 80 to 120nm, and as another preferred embodiments, the average particle diameter is measured by dlnas method (dlnas) from the viewpoint of knocking out the diameter of the efficiency reduction, for example, the average particle diameter is preferably about 20 to 50 nm.
The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include: buffers such as phosphate buffer, citrate buffer, and phosphate-buffered saline, physiological saline, and media for cell culture. These aqueous solvents (dispersion media) can stably disperse the lipid membrane structure, and monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose, disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose, trisaccharides such as raffinose and melezitose, and polysaccharides such as cyclodextrin; examples of the sugar include sugars (aqueous solutions) such as erythritol, xylitol, sorbitol, mannitol, and maltitol, and polyhydric alcohols (aqueous solutions) such as glycerol, diglycerol, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, and 1, 3-butanediol. In order to stably store the lipid membrane structure dispersed in the aqueous solvent for a long period of time, it is desirable to exclude the electrolyte from the aqueous solvent as much as possible from the viewpoint of physical stability such as suppression of aggregation. In addition, from the viewpoint of chemical stability of lipids, it is desirable to set the pH of the aqueous solvent to a weakly acidic to neutral pH (about pH3.0 to 8.0) and/or to remove dissolved oxygen by nitrogen bubbling or the like.
When the aqueous dispersion of the lipid membrane structure obtained is freeze-dried or spray-dried, for example, when monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose, and xylose are used; disaccharides such as lactose, sucrose, cellobiose, trehalose, and maltose; trisaccharides such as raffinose and melezitose; polysaccharides such as cyclodextrin; sugars (aqueous solutions) such as erythritol, xylitol, sorbitol, mannitol, maltitol, and the like can improve stability. When the aqueous dispersion is frozen, for example, if the above-mentioned saccharide or a polyhydric alcohol (aqueous solution) such as glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, or 1, 3-butanediol is used, stability can be improved.
Inside the lipid membrane structure of the present invention, other substances may be encapsulated within a range that does not hinder the function of siRNA. The type of substance to be encapsulated is not particularly limited, and any substance such as saccharides, peptides, nucleic acids, low-molecular compounds, and metal compounds may be encapsulated in addition to the active ingredient of any drug such as an antitumor agent, an anti-inflammatory agent, an antibacterial agent, and an antiviral agent. Examples of the nucleic acid include: more specifically, examples of the nucleic acid containing a gene include: genes incorporated into plasmids, and the like, but are not limited to this particular protocol.
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