WO2012005376A1 - 核酸送達用組成物及び担体組成物、それを用いた医薬組成物、並びに核酸送達方法 - Google Patents
核酸送達用組成物及び担体組成物、それを用いた医薬組成物、並びに核酸送達方法 Download PDFInfo
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- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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Definitions
- the present invention relates to a nucleic acid delivery composition and a carrier composition for delivering a nucleic acid to a target cell or tissue, a pharmaceutical composition using the composition, and a nucleic acid delivery method.
- non-viral carriers have been studied as carriers for delivering nucleic acids to target cells or tissues in nucleic acid therapy.
- Synthetic carriers like drug delivery systems (DDS) that have been studied in conventional medicine, have risks related to toxicity, but are considered to be less toxic than viral vectors, and the size of the nucleic acid to be carried. Since there are no restrictions and precise molecular design of vectors is possible, vigorous development research continues.
- Typical synthetic carriers include cationic lipids and cationic polymers that form ion complexes with negatively charged DNA.
- cationic lipids lipofectin and the like
- poly (L-lysine), DEAE-dextran, polyethyleneimine (for example, see Non-patent Document 2), chitosan (for example, see Non-Patent Document 3), and the like have been studied as cationic polymers.
- these cationic polymers are not only cytotoxic but also insufficient in nucleic acid introduction efficiency and gene expression efficiency.
- the present inventors self-assembled a block copolymer having a cationic polymer segment having a specific amine group in the side chain and a non-electrostatic hydrophilic polymer segment such as polyethylene glycol (PEG) to enclose a nucleic acid.
- PEG polyethylene glycol
- the present inventors can dramatically improve nucleic acid introduction efficiency and gene expression efficiency by mixing a cationic homopolymer having a specific amine group in the side chain with a nucleic acid, as well as animal cells (particularly mammals). It has been reported that toxicity to cells) is also relatively suppressed (see Patent Document 2: International Publication No. 2006/085664 pamphlet). However, for nucleic acid delivery systems comprising such homopolymers, further reduction in cytotoxicity has been sought.
- a specific cationic homopolymer is used in a polyion complex (PIC) type polymer micelle comprising a block copolymer having a specific cationic polymer segment and a non-electrostatic hydrophilic polymer segment.
- PIC polyion complex
- the gist of the present invention is a nucleic acid delivery composition for delivering a nucleic acid to a target cell or tissue, comprising a block copolymer having an uncharged hydrophilic polymer segment and a cationic polymer segment, a cationic polymer,
- the composition for nucleic acid delivery comprising a nucleic acid, wherein the molar percentage (B / H ratio) of the cationic group of the block copolymer to the total cationic group of the block copolymer and the cationic polymer is 25% to 90% It exists in things.
- Another subject matter of the present invention is a carrier composition for delivering a nucleic acid to a target cell or tissue, comprising a block copolymer having an uncharged hydrophilic polymer segment and a cationic polymer segment, and a cationic polymer.
- the molar percentage (B / H ratio) of the cationic groups of the block copolymer to the total cationic groups of the block copolymer and the cationic polymer is 25% to 90%.
- Still another subject matter of the present invention resides in a pharmaceutical composition used for nucleic acid therapy, which comprises the nucleic acid delivery composition or carrier composition described above.
- Yet another subject matter of the present invention resides in a method for delivering a nucleic acid to a target cell or tissue, the method comprising contacting the nucleic acid delivery composition with the target cell or tissue.
- Yet another aspect of the present invention is a method for delivering a nucleic acid to a target cell or tissue, the nucleic acid delivery comprising a block copolymer having an uncharged hydrophilic polymer segment and a cationic polymer segment, and the nucleic acid.
- the block copolymer and the cationic polymer of the nucleic acid delivery composition at the time of contact with the target cell or tissue, the method comprising: contacting the target cell or tissue with the cationic polymer.
- the present invention resides in a method in which the molar percentage (B / H ratio) of the cationic group contained in the block copolymer of the nucleic acid delivery composition to the cationic group is 25% to 90%.
- nucleic acid delivery composition and nucleic acid delivery method of the present invention cytotoxicity is reduced and excellent nucleic acid introduction efficiency is exhibited. Moreover, according to the carrier composition of the present invention, such an excellent nucleic acid delivery composition can be easily obtained.
- the nucleic acid delivery composition and carrier composition of the present invention are suitable, for example, as a pharmaceutical composition for nucleic acid therapy.
- FIGS. 1A and 1B are transmission electron micrographs showing the particle shape at each B / H ratio.
- FIG. 2 is a graph showing the relationship between the B / H ratio and the zeta potential.
- FIG. 3 is a graph showing the relationship between transfection efficiency and the B / H ratio and N / P ratio.
- FIG. 4 is a graph showing the relationship between cytotoxicity, B / H ratio, and N / P ratio.
- FIG. 5 is a graph showing changes in tumor volume over time.
- FIG. 6 is a graph showing the retention in blood of the composition for nucleic acid delivery.
- FIGS. 7A to 7D are CLSM (confocal laser microscope) photographs showing the expression of Venus in tumor tissue. Specifically, FIG. 7 (a) is a control, FIG.
- FIGS. 8A and 8B are CLSM photographs showing the expression status of sFlt-1 in tumor tissue.
- FIG. 8 (a) is the control
- FIG. 8 (b) is the result of the B / H ratio of 70%.
- FIG. 9A is a CLSM photograph in which vascular endothelial cells of the tumor tissue are immunostained
- FIG. 9B is a graph showing the blood vessel density obtained by the image analysis of FIG. 9A.
- FIG. 10 is a graph showing the relationship between transfection efficiency and B / H ratio during pulmonary administration of the composition.
- 11 (a) to 11 (d) are immunostained photographs of lung tissue. Specifically, FIG. 11 (a) shows a B / H ratio of 100%, FIG. 11 (b) shows a B / H ratio of 70%, FIG. 11 (c) shows a B / H ratio of 50%, and FIG. 11 (d) shows a control.
- FIGS. 12A to 12D are graphs showing the expression level of mRNA.
- FIG. 12 (a) is IL-6
- FIG. 12 (b) is TNF- ⁇
- FIG. 12 (c) is Cox-2
- FIG. 12 (d) is the mRNA expression level of IL-10 mRNA. Indicates.
- composition for delivering a nucleic acid to a target cell or tissue comprising a specific block copolymer and a cationic polymer, which will be described later, a nucleic acid, and a block copolymer and a cationic polymer.
- composition composition for nucleic acid delivery of the present invention having a ratio within a specific range described later is provided.
- animal cells can be obtained by using a specific block copolymer and a cationic polymer, which will be described later, in such a ratio that the B / H ratio described below satisfies a specific range.
- a specific block copolymer and a cationic polymer which will be described later, in such a ratio that the B / H ratio described below satisfies a specific range.
- nucleic acid delivery composition having only the advantages of both a conventional block copolymer-only nucleic acid delivery system and a cationic homopolymer-only nucleic acid delivery system.
- Such an effect is a synergistic effect that cannot be predicted from a mere collection of findings regarding conventional nucleic acid delivery systems.
- a composition serving as a carrier for delivering a nucleic acid to a target cell or tissue which contains a specific block copolymer and a cationic polymer described later, a nucleic acid, and a block copolymer,
- a composition (the carrier composition of the present invention) in which the ratio to the cationic polymer is within a specific range described later is also provided.
- the nucleic acid delivery composition of the present invention can be obtained by loading a nucleic acid on such a carrier composition.
- the block copolymer used in the present invention has an uncharged hydrophilic polymer segment and a cationic polymer segment. Only one type of block copolymer may be used, but two or more types may be used in any combination and ratio.
- An uncharged hydrophilic polymer segment is a polymer segment having uncharged and hydrophilic properties.
- “uncharged” means that the segment is neutral as a whole. An example is when the segment has no positive or negative charge. Even if the segment has positive and negative charges in the molecule, the local effective charge density is not high and the charge of the entire segment is neutralized to the extent that it does not interfere with the formation of polymer micelles. In other words, it corresponds to “uncharged”.
- “Hydrophilic” means to be soluble in an aqueous medium.
- the type of uncharged hydrophilic polymer segment is not limited. It may be a segment composed of a single repeating unit or a segment containing two or more repeating units in any combination and ratio.
- Specific examples of the uncharged hydrophilic polymer segment include polyalkylene glycol, poly (2-oxazoline), polysaccharide, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polymethacrylamide, polyacrylic ester, polymethacrylic ester, poly (2-methacryloyloxyethyl phosphorylcholine), peptides / proteins having an isoelectric point of around 7, and derivatives thereof.
- polyalkylene glycol and poly (2-oxazoline) are preferable, and polyalkylene glycol is particularly preferable.
- examples of the polyalkylene glycol include polyethylene glycol and polypropylene glycol, and polyethylene glycol (PEG) is preferable.
- the molecular weight of the uncharged hydrophilic polymer segment is not limited, but it is preferable to have a molecular weight within a predetermined range from the viewpoint of efficiently producing polymer micelles.
- the specific molecular weight range varies depending on the type of the uncharged hydrophilic polymer segment, the combination with the cationic polymer segment, etc., but when polyethylene glycol is used as the uncharged hydrophilic polymer segment, the molecular weight (Mw) is: Preferably it is 500 or more, More preferably, it is 1000 or more, Preferably it is 40000 or less, More preferably, it is the range of 30000 or less.
- the number of repeating units of the uncharged hydrophilic polymer segment is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the uncharged hydrophilic polymer segment satisfies the molecular weight range.
- block micelles can be prevented from associating and precipitating in aqueous solutions, stabilizing them, and efficiently building polymeric micelles that can function as a carrier composition It becomes possible to do.
- the cationic polymer segment is a polymer segment having a cationic group and exhibiting a cationic property (cationic property). However, the cationic polymer segment may have some anionic groups as long as it does not interfere with the formation of polymer micelles.
- the type of cationic polymer segment is not limited. It may be a segment composed of a single repeating unit or a segment containing two or more repeating units in any combination and ratio.
- polyamine or the like is preferable, and poly (amino acid or a derivative thereof) having an amino group in the side chain is particularly preferable.
- Such poly (amino acid or derivative thereof) may be composed of one kind of amino acid or derivative thereof, and may contain two or more kinds of amino acid or derivative thereof in any combination and ratio.
- amino acids or derivatives thereof constituting such poly include, but are not limited to, amino group-containing aspartamide, amino group-containing glutamid, lysine, arginine, histidine and the like. Of these, amino group-containing aspartamide and amino group-containing glutamid are particularly preferable.
- the molecular weight of the cationic polymer segment is not limited, but preferably has a molecular weight within a predetermined range from the viewpoint of efficiently producing polymer micelles.
- the number of repeating units of the cationic polymer segment is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the cationic polymer segment satisfies a predetermined molecular weight range. Specifically, when a polyaspartic acid derivative or polyglutamic acid derivative is used as the cationic polymer segment, the number of repeating units is preferably 5 or more, more preferably 10 or more, and preferably 300 or less, more preferably 200 or less. Range.
- the combination of the uncharged hydrophilic polymer segment and the cationic polymer segment is not limited, and any uncharged hydrophilic polymer segment and any cationic polymer segment can be combined.
- the number of the uncharged hydrophilic polymer segment and the cationic polymer segment is also arbitrary, and each may be one or two or more, and in the case of two or more, they may be the same or different from each other. Usually, it is preferable that one cationic polymer segment is bonded to one uncharged hydrophilic polymer segment. However, from the viewpoint of retaining a large amount of nucleic acid in the polymer micelle, a form in which two or more cationic polymer segments are bonded to one uncharged hydrophilic polymer segment is also suitable.
- the bonding form between the uncharged hydrophilic polymer segment and the cationic polymer segment is not limited, and may be directly bonded or may be bonded via a linking group.
- Examples of the linking group include hydrocarbon groups having a valence corresponding to the total number of uncharged hydrophilic polymer segments and cationic polymer segments.
- the hydrocarbon group as the linking group may be aliphatic, aromatic, or a group in which they are linked. In the case of aliphatic, the hydrocarbon group may be saturated or unsaturated, and may be linear, branched or cyclic.
- the molecular weight of the hydrocarbon group as the linking group is not limited, but is usually 5000 or less, preferably 1000 or less.
- hydrocarbon group as the linking group examples include gallic acid derivatives, 3,5-dihydroxybenzoic acid derivatives, glycerin derivatives, cyclohexane derivatives, L-lysine, etc., and 3,5-dihydroxybenzoic acid derivatives, etc. preferable.
- the linking group is a disulfide group.
- the disulfide group is used to link one uncharged hydrophilic polymer segment and one cationic polymer segment.
- the disulfide group is cleaved by the environment where the polymer micelle is placed or by external action, and the morphology and properties of the polymer micelle Can be changed. If this is utilized, it becomes possible to promote site-specific release of a drug (this aspect will be described later) encapsulated in a polymer micelle by cleaving a disulfide group at a specific site in the living body. Conceivable.
- the ratio of the uncharged hydrophilic polymer segment to the cationic polymer segment is also arbitrary, but from the viewpoint of efficiently producing the polymer micelle, the molecular weight of the uncharged hydrophilic polymer segment contained in the polymer micelle
- the ratio is preferably within a predetermined range. Since the specific ratio is preferably determined in consideration of the amount of nucleic acid, it will be described later in the [Nucleic acid] column.
- block copolymer Preferable specific examples of the block copolymer of the present invention include a polyethylene glycol (PEG) segment as an uncharged hydrophilic polymer segment, and a poly (amino acid or derivative thereof) segment as a cationic polymer segment. Examples thereof include block copolymers shown in (IV).
- PEG polyethylene glycol
- IV block copolymers
- R 1 represents a hydrogen atom or an unsubstituted or substituted linear or branched C 1-12 alkyl group
- R 2 represents a methylene group or an ethylene group
- R 3 represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group
- R 4 is the same as R 5 or is an initiator residue
- Each R 5 independently represents a hydroxyl group, an oxybenzyl group or a —NH— (CH 2 ) a —X group
- Each X is independently a bulky amine compound residue having a pKa value of 7.4 or less, Represents an amine compound residue containing one or more of primary, secondary, tertiary amine or quaternary ammonium salt, or a non-amine compound residue
- L 1 and L 2 each independently represent a linking group
- a is an integer of 1 to 5
- m is an integer from 5 to 20,000
- n is an integer of 2 to
- R 1 represents a hydrogen atom or an unsubstituted or substituted linear or branched C 1-12 alkyl group
- R 2 represents a methylene group or an ethylene group
- R 3 represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group
- R 4 is the same as R 5 or is an initiator residue
- Each R 5 independently represents a hydroxyl group, an oxybenzyl group or a —NH— (CH 2 ) a —X group
- Each X is independently a bulky amine compound residue having a pKa value of 7.4 or less, Represents an amine compound residue containing one or more of primary, secondary, tertiary amine or quaternary ammonium salt, or a non-amine compound residue
- L 1 and L 2 each independently represent a linking group
- a is an integer of 1 to 5
- Each R 6 is independently a hydrogen atom or a protecting group
- m is an integer
- R 1 represents a hydrogen atom or an unsubstituted or substituted linear or branched C 1-12 alkyl group.
- Examples of the C 1-12 alkyl include methyl, ethyl, n-propyl, iso-propyl, Examples include n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl, undecyl and the like.
- examples of the substituent include acetalized formyl group, cyano group, formyl group, carboxyl group, amino group, C 1-6 alkoxycarbonyl group, C 2-7 acylamide group, tri A —C 1-6 alkylsiloxy group (wherein the three alkyl groups may be the same or different), a siloxy group or a silylamino group may be mentioned.
- the substituent When the substituent is an acetalized formyl group, it can be converted to another formyl group (—CHO: aldehyde group) by hydrolysis under mild acidic conditions.
- a formyl group, carboxyl group or amino group When such a formyl group, carboxyl group or amino group is present in the vicinity of the outer edge of the polymer micelle, it can be used to covalently bond other proteins to the polymer micelle via these groups.
- proteins include antibodies or fragments having specific binding properties thereof (F (ab ′) 2, F (ab), etc.), or other functional or target-directing properties to polymer micelles. And the protein to be obtained.
- Examples of a method for producing a PEG segment having such a functional group at one end include a method for producing a PEG segment of a block copolymer described in WO96 / 32434, WO96 / 33233, and WO97 / 06202.
- R 2 represents a methylene group or an ethylene group.
- the repeating unit containing R 2 of the poly (amino acid or derivative thereof) segment corresponds to an aspartic acid derivative unit when R 2 is a methylene group, and corresponds to a glutamic acid derivative unit when R 2 is an ethylene group.
- the aspartic acid derivative unit and the glutamic acid derivative unit may each be present independently forming a block, and randomly It may be mixed.
- R 3 represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group.
- the protecting group include a C 1-6 alkylcarbonyl group, and an acetyl group is preferable.
- the hydrophobic group include derivatives such as benzene, naphthalene, anthracene, and pyrene.
- the polymerizable group include a methacryloyl group and an acryloyl group. If the copolymer of the general formula (I) or (III) has such a polymerizable group, the copolymer can be used as a so-called macromer. For example, after forming a polymer micelle, if necessary, another comonomer can be used and crosslinked via these polymerizable groups.
- R 4 is a hydroxyl group, an oxybenzyl group, a —NH— (CH 2 ) a —X group, or an initiator residue, like R 5 .
- R 4 is an initiator residue
- the block copolymers of the general formulas (I) to (IV) are polymerized with the second method described later (that is, the protected amino acid NCA is polymerized using a low molecular initiator.
- R 4 is a structure derived from the initiator used when the poly (amino acid or its derivative) segment is synthesized and then combined with the PEG segment.
- a specific example of an initiator residue is —NH—R 9 .
- R 9 is an unsubstituted or substituted linear or branched C 1-20 alkyl group.
- R 5 each independently represents a hydroxyl group, an oxybenzyl group, or a —NH— (CH 2 ) a —X group, most of which (usually 85% or more, preferably 95% or more, more preferably 98% or more). , Particularly preferably 100%) is preferably a —NH— (CH 2 ) a —X group.
- X is not limited as long as the block copolymer satisfies the conditions of the present invention (or in accordance with the object of the present invention), but is usually selected from residues classified into the following groups A to E.
- Group A Bulky amine compound residue having a pKa value of 7.4 or less:
- X 2 is a hydrogen atom or a C 1-6 alkyl group.
- Group B Amine compound residue containing both primary amine and secondary amine, tertiary amine or quaternary ammonium salt:
- X 3 is an amino C 1-6 alkyl group
- R 7 is a hydrogen atom or a methyl group
- d and e are each independently an integer of 1 to 5.
- Group C amine compound residue containing only primary amine:
- f is an integer from 0 to 15.
- Group D Amine compound residue containing only secondary amine, tertiary amine or quaternary ammonium salt and not included in Group A:
- group D d and e are each independently an integer of 1 to 5, and R 8 is a protecting group such as a Z group, a Boc group, an acetyl group, or a trifluoroacetyl group.
- Group E non-amine compound residues: In group E, g is an integer of 0 to 15.
- X may contain only one residue selected from the residues of group A and group B, but group C is group A. And at least one residue selected from the residues of the group D must be included at the same time, and the group D must include at least one residue selected from the residues of the groups B and C at the same time.
- the group E can be included in order to change the physical properties of the copolymer, but the above conditions must be satisfied in the portion excluding the group E.
- Each R 6 independently represents a hydrogen atom or a protecting group, but most of them (usually 85% or more, preferably 95% or more, more preferably 98% or more, particularly preferably 100%) are hydrogen atoms. It is preferable.
- the protecting group include a Z group, a Boc group, an acetyl group, and a trifluoroacetyl group that are commonly used as a protecting group for an amino group.
- L 1 and L 2 each independently represent a linking group.
- the type of L 1 and L 2 is not limited, but L 1 is preferably a group represented by — (CH 2 ) b —NH— (where b is an integer of 1 to 15), and L 2 Is preferably a group represented by — (CH 2 ) c —CO— (where c is an integer of 1 to 15).
- m is usually an integer of 5 or more, preferably 10 or more, more preferably 40 or more, and usually 20,000 or less, preferably 3,000 or less, more preferably 2,000 or less, particularly preferably 1,000 or less. It is.
- n is an integer of usually 5 or more, preferably 10 or more, more preferably 40 or more, and usually 5,000 or less, preferably 1,000 or less, more preferably 500 or less, particularly preferably 300 or less.
- x is usually an integer of 0 or more, preferably 1 or more, more preferably 10 or more, and usually 5,000 or less. However, x ⁇ n. y and z are each independently an integer of usually 0 or more, preferably 1 or more, and usually 5,000 or less. However, y + z ⁇ n. Particularly preferably, 10 ⁇ y ⁇ n ⁇ 10 and 10 ⁇ z ⁇ n ⁇ 10.
- each repeating unit may form a block for each type and is mixed randomly. You may do it.
- the cationic group possessed by the poly (amino acid or derivative thereof) segment may be a free cationic group or may form a salt.
- the counter ion forming the salt is not limited, but Cl ⁇ , Br ⁇ , I ⁇ , (1 / 2SO 4 ) ⁇ , NO 3 ⁇ , (1 / 2CO 3 ) ⁇ , ( 1 / 3PO 4 ) ⁇ , CH 3 COO ⁇ , CF 3 COO ⁇ , CH 3 SO 3 ⁇ , CF 3 SO 3 — and the like.
- the method for producing the block copolymers of the general formulas (I) to (IV) is not limited, but examples thereof include the two methods described below.
- a PEG derivative having an amino group at the terminal is used, and an N-carboxylic acid anhydride of a protected amino acid such as ⁇ -benzyl-L-aspartate or N ⁇ -ZL-lysine is introduced from the amino terminal.
- a method of synthesizing a block copolymer by polymerizing (NCA), and then converting the protected amino acid side chain of the obtained poly (amino acid derivative) segment into a desired amino acid side chain can be mentioned.
- the resulting block copolymer has the general formula (I) or (III).
- a method of synthesizing a poly (amino acid or derivative thereof) segment having a desired amino acid side chain and then binding it to a PEG segment can also be mentioned.
- the structure of the obtained block copolymer is any one of the general formulas (I) to (IV).
- the method is arbitrary.
- a polyaspartic acid structure And an ester-amide exchange reaction by aminolysis of a poly ( ⁇ -benzyl-L-aspartate) moiety described in Japanese Patent No. 2777530 and the like.
- the benzyl ester may be hydrolyzed by catalytic reduction, acid, alkali, etc., converted to polyaspartic acid or polyglutamic acid, and then combined with a compound having these residues using a condensing agent or the like.
- a condensing agent or the like can be mentioned.
- a protective group, a hydrophobic group, a polymerizable group, etc. are introduced later to the terminal (R 1 , R 3 , R 5 , R 6 ) of the block copolymer.
- the method is arbitrary, but examples include methods used in ordinary synthesis, such as a method using an acid halide, a method using an acid anhydride, and a method using an active ester.
- the cationic polymer is a polymer having a cationic group and exhibiting a cationic property (cationic property).
- the cationic polymer may have some anionic groups as long as the formation of polymer micelles is not hindered.
- the type of cationic polymer is not limited. It may be a polymer composed of a single repeating unit or a segment containing two or more repeating units in any combination and ratio.
- the cationic polymer is preferably a polyamine or the like, and particularly preferably a polyamino acid having an amino group in the side chain or a derivative thereof.
- examples of the polyamino acid having an amino group in the side chain or a derivative thereof include polyaspartamide, polyglutamide, polylysine, polyarginine, polyhistidine, and derivatives thereof. Particularly preferred are polyaspartamide derivatives and polyglutamide derivatives.
- the molecular weight of the cationic polymer is not limited, but preferably has a molecular weight within a predetermined range from the viewpoint of efficiently producing a homogeneous polymer micelle.
- the number of repeating units of the cationic polymer is not limited, but is usually determined according to the type of the repeating unit so that the molecular weight of the cationic polymer satisfies a predetermined molecular weight range. Specifically, when a polyaspartic acid derivative or polyglutamic acid derivative is used as the cationic polymer, the number of repeating units is preferably 5 or more, more preferably 10 or more, and preferably 300 or less, more preferably 200 or less. Range.
- Specific examples of cationic polymer include poly (amino acids or derivatives thereof) represented by the following formulas (I ′) to (IV ′).
- R 2 , R 3 , R 4 , R 5 , n and x represent the same definitions as the groups having the same symbols in the formulas (I) and (II).
- R 2 , R 3 , R 4 , R 5 , R 6 , n, x, y and z are groups having the same signs in the formulas (III) and (IV)). Represents the same definition as
- R 5 is a —NH— (CH 2 ) a —X group
- X is usually a group of residues classified into the groups A to E described above.
- the group B is preferable, and the following amine compound residues are particularly preferable.
- R 7 is a hydrogen atom or a methyl group, and d and e are each independently an integer of 1 to 5)
- the ratio of the block copolymer to the cationic polymer of the nucleic acid delivery composition and the carrier composition of the present invention is the molar percentage of the cationic group of the block copolymer to the total cationic group of the block copolymer and the cationic polymer (this specification). This is sometimes expressed as “B / H ratio” in the book.) Specifically, it is represented by the following formula.
- the B / H ratio is usually more than 25%, preferably 30% or more, more preferably 40% or more, still more preferably 50% or more, and usually 90% or less, preferably 85% or less, More preferably, the range is 80% or less.
- the reason why such an effect can be obtained by keeping the B / H ratio within the above range is not clear, but by making the B / H ratio a range equal to or more than the lower limit, the zeta electrons are compared. While maintaining the target value close to 0 and sufficiently suppressing toxicity, the particle shape is maintained in a spherical shape or a substantially spherical shape by keeping the B / H ratio in the range below the upper limit value. As a result of improving the stability of the particles in the blood and in the blood and thereby sufficiently exhibiting the nucleic acid introduction efficiency, it is presumed that both low cytotoxicity and high nucleic acid introduction efficiency are compatible.
- nucleic acid delivery composition and the carrier composition of the present invention contain two or more block copolymers and / or two or more cationic polymers, the entire two or more block copolymers and / or The B / H ratio of the whole of two or more kinds of cationic polymers only needs to satisfy the above range.
- nucleic acid used in the nucleic acid delivery composition of the present invention is not limited. That is, examples of the nucleic acid include DNA, RNA, natural or non-natural nucleic acid analogs (for example, peptide nucleic acids), modified nucleic acids, modified nucleic acids, and the like. In addition, the nucleic acid may be single-stranded or double-stranded, and the presence or absence of protein coding or the presence or absence of other functions is not limited.
- the nucleic acid is preferably a functional nucleic acid that can exert some action on a living body, tissue, cell or the like when delivered to the living body.
- Functional nucleic acids include plasmid DNA, siRNA, miRNA (microRNA), antisense RNA, antisense DNA, decoy nucleic acid, ribozyme, DNA enzyme, various suppressor genes (such as cancer suppressor genes), functional modified nucleic acids / modifications Nucleic acids (for example, nucleic acids in which the phosphate portion of the nucleic acid is modified to phosphorothioate, methylphosphonate, phosphate triester, phosphoramidate, etc., or for applications such as stabilization of polymeric micelles, such as cholesterol and vitamin E And a nucleic acid to which a hydrophobic functional group is bonded. These are selected according to the use of the composition for nucleic acid delivery.
- the plasmid DNA may be any DNA that can exhibit a desired function in the target cell / tissue.
- Various plasmid DNAs are known, and those skilled in the art can select a desired plasmid DNA according to the use of the nucleic acid delivery composition.
- siRNA may be any as long as it can suppress the expression of a target gene using RNA interference (RNAi).
- RNAi RNA interference
- Preferred target genes for RNA interference include cancer (tumor) genes, anti-apoptotic genes, cell cycle-related genes, proliferation signal genes, and the like.
- the base length of siRNA is not limited, but is usually less than 30 bases, preferably 19 to 21 bases.
- Nucleic acids may be used alone or in combinations of two or more in any ratio. Since the nucleic acid molecule becomes a polyanion, it can be bound (associated) with the side chain of the polycation part of the block copolymer by electrostatic interaction.
- the ratio of the nucleic acid to the block copolymer and the cationic polymer is the molar ratio of the [cationic group possessed by the block copolymer and the cationic polymer] to the [phosphate group possessed by the nucleic acid] (this is referred to as “N / P ratio” in this specification). May be displayed.)
- the N / P ratio is not limited, but is usually 2 or more, preferably 4 or more, more preferably 6 or more, and usually 200 or less, preferably 100 or less, preferably 50 or less. It is a range.
- the nucleic acid delivery composition of the present invention is superior in nucleic acid introduction efficiency as compared with the conventional PIC polymer micelle nucleic acid delivery composition. With a low N / P ratio), it is possible to efficiently deliver a nucleic acid and allow gene expression.
- nucleic acid delivery composition and carrier composition of the present invention in addition to the block copolymer, the cationic polymer and the nucleic acid, other components are added so long as the formation of the polymer micelle is not prevented or the stability is not lowered. Can be added. Although there is no restriction
- the uncharged or charged polymer includes any uncharged or charged polymer other than the above-described block copolymer and cationic polymer.
- Examples of the chargeable nanoparticles include metal-based nanoparticles having a charge on the surface.
- the said other component may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.
- the amount of the other component used is not limited, but it is preferable to suppress it to such an extent that the formation of polymer micelles is not hindered. Specifically, it is usually 30% or less, preferably 20% or less, more preferably 10% or less, based on the total weight of the composition of the present invention.
- the carrier composition of the present invention is usually prepared by mixing the block copolymer and the cationic polymer, and other components used as necessary. Specifically, a first aqueous solution containing the block copolymer and a second aqueous solution containing the cationic polymer are prepared. The first and second aqueous solutions may be purified by filtration if desired.
- the concentration of the block copolymer in the first aqueous solution and the concentration of the cationic polymer in the second aqueous solution are not limited, the ratio of the block copolymer to the cationic polymer, the block copolymer and the cationic polymer to the aqueous solution. It is appropriately determined in consideration of conditions such as solubility and polymer micelle formation efficiency. If the solvent of the 1st and 2nd aqueous solution is an aqueous solvent, the kind will not be limited.
- Water is preferable, but a solvent in which other components are mixed with water, such as physiological saline, an aqueous buffer, a mixed solvent of water and a water-soluble organic solvent, or the like is also used as long as the formation of polymer micelles is not hindered. be able to.
- aqueous buffer include 10 mM HEPES buffer.
- the pH of the first and second aqueous solutions can be appropriately adjusted within a range that does not hinder the formation of polymer micelles, but is preferably pH 5 or more, more preferably pH 6.5 or more, and preferably Is pH 9 or less, more preferably pH 7.5 or less.
- the pH can be easily adjusted by using a buffer as a solvent. Adjusting and using the pH of the first and second aqueous solutions is advantageous for maintaining the charged state of the block copolymer and the cationic polymer and efficiently forming polymer micelles.
- the salt concentrations of the first and second aqueous solutions can be appropriately adjusted within a range that does not hinder the formation of polymer micelles, but are preferably 5 mM or more, more preferably 10 mM or more, and preferably 300 mM or less, more preferably 150 mM or less.
- the mixing method of the first and second aqueous solutions is not limited.
- the second aqueous solution may be added to the first aqueous solution, and the first aqueous solution may be added to the second aqueous solution.
- the temperature at the time of mixing the first and second aqueous solutions is not limited as long as it does not interfere with the formation of polymer micelles, but should be set in consideration of the solubility according to the temperature of the block copolymer and the cationic polymer. Is preferred. Specifically, it is usually 0 ° C. or higher, preferably 60 ° C. or lower, more preferably 50 ° C. or lower.
- the carrier composition containing the formed polymer micelle may be immediately used for a desired use, but in order to equilibrate the system, a time for allowing the mixed solution to stand may be provided.
- the time for which the mixed solution is allowed to stand varies depending on conditions such as the formation efficiency of polymer micelles, but is preferably 50 hours or less, more preferably 30 hours or less.
- a crosslinking agent is not used as described above, it may be preferable not to provide a standing time because the diameter of the formed polymer micelle tends to increase with time.
- the other components may be added and mixed during or after the mixing of the first and second aqueous solutions.
- the other components may be added and mixed as they are, but an aqueous solution containing the other components may be prepared and mixed.
- Preparation conditions such as an aqueous solvent, pH, temperature, and ionic strength in the preparation of the aqueous solution of the other components are the same as those described above for the first and second aqueous solutions.
- operations such as dialysis, dilution, concentration, and stirring may be added as appropriate.
- the nucleic acid delivery composition of the present invention usually comprises mixing the nucleic acid with (i) the block copolymer and the cationic polymer, and other components used as necessary, or (ii) It is prepared by mixing with the carrier composition of the present invention prepared in advance.
- a nucleic acid is further added to the carrier composition obtained by mixing the first aqueous solution (an aqueous solution of a block copolymer) and the second aqueous solution (an aqueous solution of a cationic polymer). What is necessary is just to mix in addition. Nucleic acid may be added and mixed immediately after the preparation of the carrier composition by mixing the first and second aqueous solutions, but the mixture is allowed to stand to equilibrate the system before adding more nucleic acid. You may mix.
- the nucleic acid may be added and mixed as it is.
- an aqueous solution containing the nucleic acid (third aqueous solution) may be prepared and added and mixed. Good. Preparation conditions such as an aqueous solvent, pH, temperature, and ionic strength in the preparation of the third aqueous solution are the same as those described above for the first and second aqueous solutions. Furthermore, operations such as dialysis, dilution, concentration, and stirring may be added as appropriate.
- the shape of the nucleic acid delivery composition and the carrier composition of the present invention is not limited, but is usually spherical or substantially spherical.
- the particle size of the nucleic acid delivery composition and the carrier composition of the present invention is the kind and amount ratio of the block copolymer and the cationic polymer, the presence or absence of other components, the surrounding environment of the nucleic acid delivery composition and the carrier composition (aqueous medium) However, it is preferably 10 nm or more, more preferably 50 nm or more, and preferably 200 nm or less, more preferably 150 nm or less.
- the particle size of the nucleic acid delivery composition and the carrier composition tends to increase with time under the presence of salts such as physiological environment and physiological saline, but the particle size increases by introducing a cross-linking agent. Can be prevented.
- the intraparticle structure of the nucleic acid delivery composition and the carrier composition of the present invention is not clear, but it is estimated as follows in consideration of the fact that the zeta potential is close to 0 as described later.
- the hydrophilic segment of the block copolymer is densely present around the outer shell of the particle, and the cationic polymer segment and the cationic polymer of the block copolymer are mainly present on the inner side of the particle. It is considered to have a micelle structure.
- the hydrophilic segment of the block copolymer is densely present around the outer shell of the particle, while the cationic polymer segment and the cationic polymer of the block copolymer are electrostatically bonded to the nucleic acid, It is considered to have a structure of a PIC type polymer micelle that exists mainly in an encapsulated / supported state inside the particle.
- the nucleic acid delivery composition of the present invention can be used to deliver nucleic acids to target cells or tissues in vitro or in vivo. According to the nucleic acid delivery composition of the present invention, it is possible to easily deliver a nucleic acid that has been difficult to deliver into a target cell in a stable state, and to suppress cytotoxicity. Is possible. In addition, it can be used as a means for efficiently introducing the nucleic acid encapsulated and supported inside the particles of the nucleic acid delivery composition into the target cell by utilizing the change in pH inside and outside the cell. Furthermore, when a gene encoding a protein is used as a nucleic acid, and the gene is delivered and expressed in a cell or tissue, high gene expression efficiency can be obtained by using the nucleic acid delivery composition of the present invention. Become.
- the nucleic acid delivery composition may be brought into contact with the target cell or tissue.
- the target cell or tissue is cultured in the presence of the nucleic acid delivery composition of the present invention, or the target cell or tissue is cultured.
- the nucleic acid delivery composition may be added to the culture.
- the nucleic acid delivery composition of the present invention is obtained by an administration method commonly used in the art such as gene therapy. What is necessary is just to administer to the individual (or the individual to be treated) which needs introduction of the nucleic acid concerned.
- Examples of such individuals include, but are not limited to, humans, mice, rats, rabbits, dogs, cats, monkeys, cows, horses, pigs, birds and the like.
- Examples of the administration method include direct introduction or transplantation in the vicinity of or into the target cell or tissue, intravenous injection, intraarterial injection, intramuscular injection, oral administration, transpulmonary administration, and the like.
- Each condition such as the dose, the number of administrations, and the administration period can be appropriately set according to the type and condition of the test animal.
- the lung is highly sensitive to foreign substances, and inflammation may be caused by conventional drug delivery using DDS, and transpulmonary administration was extremely difficult.
- the composition for nucleic acid delivery of the present invention Since it is possible to obtain high gene expression efficiency while keeping toxicity low, it can be suitably used for transpulmonary administration.
- the carrier composition of the present invention can be used for the same applications as the nucleic acid delivery composition by supporting the nucleic acid to obtain the nucleic acid delivery composition of the present invention.
- the technique for supporting the nucleic acid is as described in [Method for preparing nucleic acid delivery composition].
- composition of the present invention comprising the nucleic acid delivery composition or carrier composition of the present invention.
- the pharmaceutical composition of the present invention can be used, for example, for a therapy (gene therapy) for delivering / introducing a desired nucleic acid by targeting a cell or tissue causing various diseases.
- the individual to be administered with the pharmaceutical composition of the present invention is the same as that described above for the nucleic acid delivery composition.
- the disease to be treated by the pharmaceutical composition of the present invention is not limited, but cancer (for example, lung cancer, pancreatic cancer, brain wound, liver cancer, breast cancer, colon cancer, neuroblastoma, bladder cancer, etc.) ), Circulatory diseases, motor organ diseases, central system diseases, and the like.
- the pharmaceutical composition of the present invention may contain, in addition to the nucleic acid delivery composition or carrier composition of the present invention, other components generally used in the manufacture of drugs.
- other components include excipients, extenders, fillers, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, Preservatives, flavoring agents, soothing agents, stabilizers, tonicity agents and the like can be mentioned.
- Such other components may be used alone or in combination of two or more in any ratio. The details of the types and amounts of these other components can be appropriately determined by those skilled in the art depending on the purpose, application, method of use, etc. of the pharmaceutical composition.
- the form of the pharmaceutical composition of the present invention is also arbitrary, but usually an intravenous injection (including infusion) is adopted, and is provided in the state of, for example, a unit dose ampoule or a multi-dose container.
- the method of using the pharmaceutical composition of the present invention is also arbitrary. Any pharmaceutical composition containing a nucleic acid delivery composition can be administered as it is. In the case of a pharmaceutical composition containing a carrier composition and not containing a nucleic acid, it may be mixed with the nucleic acid before use and loaded on the carrier composition before being administered.
- the present invention also provides a method for delivering a nucleic acid to a target cell or tissue in vitro or in vivo (the nucleic acid delivery method of the present invention).
- the nucleic acid delivery method of the present invention includes the following methods (1) and (2).
- nucleic acid delivery composition or pharmaceutical composition of the present invention (1) Method using the nucleic acid delivery composition or pharmaceutical composition of the present invention
- the nucleic acid delivery composition or pharmaceutical composition of the present invention described above is used, and this is applied to target cells or in vitro or in vivo.
- the nucleic acid is delivered to the target cell or tissue. Details of the nucleic acid delivery composition and the pharmaceutical composition, the method of contacting the target cell or tissue, and the like are as described above.
- nucleic acid can be introduced with high efficiency while suppressing cytotoxicity.
- the coexistence ratio between the block copolymer and the cationic polymer of the block copolymer nucleic acid composition in the target cell or tissue is adjusted so that the B / H ratio satisfies the specific range.
- nucleic acid can be introduced with high efficiency while suppressing cytotoxicity as in the above method (1) (method of delivering nucleic acid using the nucleic acid delivery composition of the present invention). Is possible.
- the details of the method (2) are as follows.
- the details of the block copolymer and the nucleic acid constituting the block copolymer nucleic acid composition are as described above for the nucleic acid delivery composition of the present invention.
- the use ratio of the block copolymer and the nucleic acid is also arbitrary, and may be appropriately selected according to the purpose and conditions of nucleic acid delivery. However, in consideration of the coexistence ratio of the cationic polymer coexisting in situ, the N / P It is preferable to use at such a ratio that the range of the ratio is satisfied.
- the block copolymer nucleic acid composition can also be prepared by mixing each component in accordance with the preparation of the nucleic acid delivery composition of the present invention.
- the details of the cationic polymer are also as described above for the nucleic acid delivery composition of the present invention.
- the above-described method of bringing the nucleic acid delivery composition of the present invention into contact with the target cell or tissue can be used.
- a preferred contact method for achieving such concentration control in vitro a method of adding in advance to a culture medium before culturing, a method of adding to a culture medium or culture after culturing can be mentioned.
- preferred contact methods for achieving such control in vivo include local administration, blood administration, and the like.
- the order in which the block copolymer nucleic acid composition and the cationic polymer are brought into contact with the target cell or tissue is not limited and is arbitrary. That is, the block copolymer nucleic acid composition and the cationic polymer may be contacted with the target cell or tissue at the same time, but one of them is first contacted with the target cell or tissue, and then the other is contacted with the target cell or tissue. May be. When these are brought into contact with the target cell or tissue at the same time, they may be brought into contact with the target cell or tissue individually, or both may be mixed and then brought into contact with the target cell or tissue. When these are individually contacted with a target cell or tissue, the contact technique may be the same or different.
- the nucleic acid delivery method of the present invention can be used for various applications.
- a target is a treatment for delivering / introducing a desired nucleic acid by targeting a cell or tissue causing various diseases ( It can be suitably used for gene therapy.
- the block copolymer includes a polyethylene glycol (hereinafter referred to as “PEG”) segment and a poly (aspartic acid-diethylenetriamine derivative) (hereinafter referred to as “PAsp (DET)”) segment. (Hereinafter referred to as “PEG-PAsp (DET)”) was used.
- n represents the degree of polymerization of PAsp (DET) and is about 61.
- c represents the number of repeating ethylene groups as a linking group, and is about 3.
- the molecular weight of the PEG segment of PEG-PAsp (DET) was about 12000, and the molecular weight of the PAsp (DET) segment was about 14,000.
- PEG-PAsp (DET) was prepared by the following procedures (1) and (2).
- PEG-PBLA PEG-polybenzyl-L-aspartate
- PEG-PAsp (DET) was synthesized using the aminolysis reaction of PEG-PBLA. 50 mg of dried PEG-PBLA was dissolved in 2 mL of NMP (N-methylpyrrolidone) and cooled to 5 ° C. and 0 ° C., respectively. In a separate container, a solution obtained by diluting 50-fold molar amount of DET with an equal amount of NMP with respect to the benzyl ester group of PEG-PBLA was prepared and cooled to the same temperature as the PEG-PBLA solution.
- NMP N-methylpyrrolidone
- the PEG-PBLA solution was slowly added dropwise thereto, and after the reaction for 1 hour, the reaction solution was well cooled in 5N HCl (preferably about 1N dilute acid) while controlling the solution temperature to 5 ° C. or lower. It was dripped. Thereafter, dialysis was performed against an aqueous HCl solution of about 0.01 N at 4 ° C. (pH 2), and excess acid was removed by dialysis against pure water. The finally obtained aqueous polymer solution was lyophilized to recover PEG-PAsp (DET) as a salt. The quantitative aminolysis reaction was confirmed by 1 H NMR measurement.
- Homo-PAsp (Cationic polymer) ⁇ Structure of Homo-PAsp (DET)>
- the cationic polymer the following poly (aspartic acid-diethylenetriamine derivative) homopolymer (hereinafter referred to as “Homo-PAsp (DET)”) was used.
- n represents the degree of polymerization of PAsp (DET) and is about 54.
- the molecular weight of the Homo-PAsp (DET) was about 10,000.
- Homo-PAsp (DET) was prepared according to the above ⁇ Method for producing PEG-PAsp (DET)>. That is, polybenzyl-L-aspartate (PBLA) was obtained in the same manner as in step (1) of ⁇ Method for producing PEG-PAsp (DET)> except that n-butylamine was used instead of PEG. Produced. Mw / Mn by GPC was 1.06, and the degree of polymerization by 1 H NMR measurement was 65.
- PBLA polybenzyl-L-aspartate
- step (1) of the ⁇ Preparation of PEG-PAsp (DET)> the same procedure was performed except that 50 mg of PBLA obtained above was used instead of PEG-PBLA, thereby performing Homo-PAsp (DET). ) was prepared.
- nucleic acid As the nucleic acid, a plasmid (Luc pDNA) encoding luciferase was used. This plasmid was obtained from Riken Cell Bank, introduced into Escherichia coli, amplified by culture, and used after purification using NucleoBond Xtra Maxi (manufactured by Nippon Genetics).
- composition for nucleic acid delivery was prepared by the following procedure.
- solution when simply indicated as “solution”, it means a solution in 10 mM HEPES buffer.
- N / P ratios are described below in 10 mM HEPES buffer.
- composition prepared by the above procedure (nucleic acid delivery) with a B / H ratio of 100% (block copolymer alone), 75%, 50%, 25% or 0% (cationic polymer alone) and an N / P ratio of 3. Composition) was used. About each composition (composition for nucleic acid delivery), the nucleic acid was dye
- FIG. 1A and 1B show transmission electron micrographs obtained for each composition.
- Each photograph in FIG. 1 (b) is an enlarged photograph of a representative particle in the corresponding photograph in FIG. 1 (a).
- the particle size was relatively large and the particle shape was relatively elongated.
- the B / H ratio decreased, the particle size gradually decreased. It can be seen that the shape approaches a spherical shape.
- the relationship between the obtained zeta potential and the value of the B / H ratio is shown in the graph of FIG.
- the zeta potential was close to 0 mV, whereas when the B / H ratio was lower than 25%, the zeta potential rapidly increased. From this, in the range where the B / H ratio is 25% or more, the uncharged hydrophilic polymer segment of the block copolymer is present outside the particle, and the cationic polymer segment of the block copolymer is electrostatically bonded to the nucleic acid to It is presumed that PIC type polymer micelle particles existing inside are formed.
- the B / H ratio is 100% (block copolymer alone), 80%, 70%, 60%, 50%, 25% or 0% (cationic polymer alone), and the N / P ratio is 4, 6, 8, 12 16, the composition prepared in the above procedure (composition for nucleic acid delivery) was used.
- the transfection efficiency and cytotoxicity of each composition (composition for nucleic acid delivery) were measured by the following procedure.
- HUVEC normal human umbilical vein endothelial cells
- composition for nucleic acid delivery composition for nucleic acid delivery
- the cells were washed with PBS, and 400 ⁇ L of fresh EBM-2 was added to each well and further cultured for 24 hours.
- cell counting kit Cell Counting Kit-8: manufactured by Dojindo Laboratories
- the number of viable cells in each well was counted according to the instruction manual, and used as an index of cytotoxicity.
- Cell lysate (Cell Culture Lysis Buffer: Promega) is added at 200 ⁇ L / well and the photoluminescence intensity is measured using a luciferase assay kit (Luciferase Assay System Kit: Promega) and an LB940 instrument (Mithras). Thus, luciferase activity was determined and used as an index of transfection efficiency.
- the amount of protein in the cell lysate was determined with a MicroBCA (registered trademark) protein assay reagent kit (MicroBCA (registered trademark) Protein Assay Reagent Kit: manufactured by Thermo Scientific).
- FIG. 3 shows a graph showing the relationship between the transfection efficiency obtained for each composition (composition for nucleic acid delivery) and the B / H ratio and N / P ratio.
- the composition with a B / H ratio of 100% showed very low transfection efficiency, whereas the composition with a B / H ratio of 80% or higher showed a clear improvement in transfection efficiency.
- the transfection efficiency was also improved as the N / P ratio increased.
- composition for nucleic acid delivery composition for nucleic acid delivery
- B / H ratio of 25% or less
- the composition with a B / H ratio exceeding 25% had an increased number of viable cells.
- Example Group II Examination of in vivo characteristics of nucleic acid delivery composition
- PEG-C3-PAsp (DET) PEG-C6-PAsp (DET)
- C3 and C6 are respectively displayed in FIG.
- Each polymer was synthesized according to the method described in Example Group I ⁇ Method for producing PEG-PAsp (DET)>.
- sFlt-1 Human sFlt-1 cDNA (2.4 kb) excised from the pVL1393 baculovirus vector (pDNA), and was provided by Professor Shibuya, Tokyo Medical and Dental University. After purification by agarose gel electrophoresis, it is inserted into the pCAcc vector using Rapid DNA Ligation Kit (Roche), amplified in E. coli (DH5 ⁇ ) culture, and about 10 colonies formed on the culture plate are separated.
- pDNA pVL1393 baculovirus vector
- each pDNA is examined by agarose electrophoresis, etc., pDNA into which the desired sequence is inserted is selected, and the amplified product is purified using NucleoBond (registered trademark) Xtra (manufactured by Nippon Genetics). Used.
- Plasma (Venus pDNA) encoding fluorescent protein (Venus) Venus pDNA was obtained from Riken Cell Bank, introduced into Escherichia coli, amplified by culture, and purified after using NucleoBond Xtra Maxi (manufactured by Nippon Genetics).
- Cy5-labeled plasmid encoding luciferase This is a plasmid in which the Luc pDNA is labeled with Cy5.
- Cy5 was labeled according to the procedure described in the instruction manual.
- BxPC3 subcutaneously inoculated mouse (animal) 8-week-old mouse Balb / c (female, obtained from Charles River Laboratories) as it is (hereinafter may be referred to as “Balb / c mouse”) or 5-week-old nude mouse Balb / c (female, Charles River Laboratory) was obtained by inoculating the human pancreatic cancer BxPC3 subcutaneously for 2 to 3 weeks until the tumor volume grew to about 45 mm 3 (hereinafter sometimes referred to as “BxPC3 subcutaneously inoculated mouse”).
- PEG-C3-PAsp (DET) or PEG-C6-PAsp (DET) is used as the block copolymer
- sFlt-1 pDNA is used as the nucleic acid
- the B / H ratio is 100% (block copolymer alone), 70%, or 50 % Of each composition (composition for nucleic acid delivery) (2OD, 200 ⁇ L) was systemically administered by intravenous injection to the aforementioned BxPC3 subcutaneously inoculated mice (6 mice per composition). Administration to each animal was performed on the 0th, 4th and 8th days after the start of the experiment.
- nucleic acid per mouse was 20 ⁇ g.
- the tumor suppression effect by nucleic acid delivery was examined by measuring the tumor volume over time after the start of the experiment.
- administration and tumor volume measurement were performed in the same manner as described above using 10 mM HEPES buffer.
- FIG. 5 shows a graph showing the change over time in the tumor volume obtained for each B / H ratio.
- PEG-C3-PAsp shown as “C3” in the figure
- PEG-C6-PAsp shown as “C6” in the figure
- the control HPES buffer
- the tumor suppressive effect was seen compared with the case of). The effect is superior to a composition having a B / H ratio of 50% or 70% compared to a composition having a B / H ratio of 100%, and particularly remarkable in a composition having a B / H ratio of 70%. Tumor suppressive effect was observed.
- compositions obtained using PEG-C3-PAsp (DET) as a block copolymer, Cy5-pDNA as a nucleic acid, and a B / H ratio of 100% (block copolymer alone), 70%, or 50%
- the composition for delivery) (2OD, 200 ⁇ L) was systemically administered by intravenous injection to the aforementioned BxPC3 subcutaneously inoculated mice (8 mice for each composition). The dose was determined so that the dose of nucleic acid per mouse was 20 ⁇ g.
- FIG. 6 shows a graph showing the blood retention index (ratio of fluorescence intensity 20 minutes after administration to fluorescence intensity immediately after administration) obtained for each B / H ratio.
- the fluorescence intensity derived from the nucleic acid was maintained at the same level as that of the composition having a B / H ratio of 100% even 20 minutes after administration.
- the fluorescence intensity derived from the nucleic acid was lowered.
- FIGS. 7 (a) to (d) CLSM photographs of tumor tissue sections obtained for each B / H ratio are shown in FIGS. 7 (a) to (d).
- blue represents the cell nucleus
- red represents the vascular endothelial cell
- green represents the Venus expression site.
- Almost no Venus expression was seen in the control (HEPES buffer) (FIG. 7 (a)) and compositions with B / H ratios of 100% (FIG. 7 (b)) and 50% (FIG. 7 (c)).
- the composition having a B / H ratio of 70% FIG. 7 (d)
- Venus expression was also observed in cells distant from the blood vessel.
- PECAM-1 which is a vascular endothelial cell marker
- a fluorescently labeled secondary antibody were used.
- cell nuclei and vascular endothelial cells in the same section were also immunostained.
- the section after staining was observed with a confocal laser microscope (CLSM), and the expression level and expression site of sFlt-1 were examined, which was used as an index of nucleic acid expression effectiveness by nucleic acid delivery.
- CLSM confocal laser microscope
- administration, immunostaining, and CLSM observation were performed in the same manner as described above using 10 mM HEPES buffer instead of the nucleic acid delivery composition.
- FIG. 8 (a) A CLSM photograph of the tumor tissue section obtained for the control (HEPES buffer) is shown in FIG. 8 (a), and a CLSM photograph of the tumor tissue section obtained for the composition for nucleic acid delivery having a B / H ratio of 70% is shown in FIG. 8 (b). ).
- blue represents the cell nucleus
- red represents the vascular endothelial cell
- green represents the sFlt-1 expression site.
- the control HPES buffer
- almost no sFlt-1 expression was observed, whereas in the nucleic acid delivery composition having a B / H ratio of 70%, significant sFlt-1 expression was observed even in cells distant from the blood vessel. It was.
- vascular endothelial cells were immunostained.
- PECAM-1 which is a vascular endothelial cell marker
- a fluorescently labeled secondary antibody were used.
- the stained sections were observed with a confocal laser microscope (CLSM), and seven CLSM photographs were taken for each section.
- CLSM photograph was subjected to image analysis with LSM510 (manufactured by Carl Zeiss), the green light emission pixel ratio (corresponding to the vascular endothelial cell ratio) was measured, the average value of each section was obtained as the blood vessel density, and blood vessels by nucleic acid delivery It was used as an index of the inhibitory effect on newborn.
- composition for nucleic acid delivery obtained with a B / H ratio of 70% Administration
- immunostaining and CLSM observation were performed in the same manner as described above.
- administration, immunostaining, and CLSM observation were performed in the same manner as described above using 10 mM HEPES buffer.
- FIG. 9A shows an example of an immunostained CLSM photograph of a tumor tissue obtained for each B / H ratio
- FIG. 9B shows a graph showing the blood vessel density obtained by image analysis of the immunostained CLSM photograph.
- the composition using Luc ⁇ pDNA (B / H ratio 70%) showed no increase or decrease in blood vessel density, while the composition using sFlt-1 pDNA was particularly
- the blood vessel density was significantly reduced with a composition having a B / H ratio of 70%, and a remarkable angiogenesis inhibitory effect was observed.
- PEG-PAsp (DET) is used as block copolymer, Luc pDNA is used as nucleic acid, and B / H ratio is 100% (block copolymer alone), 75%, 50%, 25% or 0% (cationic polymer alone)
- Each of the obtained compositions (4OD, 50 ⁇ L) was transpulmonary administered to the Balb / c mice (five animals per composition).
- Transpulmonary administration was performed by using a microspray (microsprayer Model IA-1C-R, manufactured by Penn Century) and spraying each of the above compositions directly into the bronchi of a mouse. 24 hours after administration, 30 mg of cells in the lung were collected, and the cells were gently washed with PBS.
- linear polyethyleneimine Linear Polyethylenimine: LPEI, Exgen 500, manufactured by Fermentas
- LPEI Linear Polyethylenimine
- Exgen 500 manufactured by Fermentas
- FIG. 10 Medium “LPEI”.
- a solution prepared by dissolving 10 mg of Luc pDNA in 50 ⁇ l of 10 mM HEPES buffer solvent was administered, lung cells were collected, and luciferase activity was determined by the same procedure (“nucleic acid alone” in FIG. 10).
- a graph showing the transfection efficiency of each composition is shown in FIG.
- FIGS. 11 (a) to 11 (d) Optical micrographs obtained for each composition are shown in FIGS. 11 (a) to 11 (d).
- FIGS. 11 (a) to 11 (d) In an individual administered with a composition having a B / H ratio of 0% (cationic polymer alone) (FIG. 11 (a)), inflammation was induced in the lung tissue (the part circled in the figure), while B In the lung tissue of an individual administered with a composition having a 50% / H ratio (FIG. 11 (b)) and an individual administered with a composition having a B / H ratio of 100% (block copolymer alone) (FIG. 11 (c)), There was no significant change compared to the lung tissue of the control individual (FIG. 11 (d)), and little inflammation was observed.
- the expression level of each mRNA was measured by quantitative PCR using TaqMan Gene Expression Assays and ABI Prism 7500 Sequence Detector (Applied Biosystems). Moreover, the measured mRNA expression level was displayed as a ratio value (relative expression level) to the value measured in the non-administration group. As a control, 50 ⁇ l of 10 mM HEPES buffer was used for intrapulmonary administration and measurement of mRNA expression level of each inflammatory cytokine by the same method as described above. The relative expression levels of the obtained IL-6, TNF- ⁇ , Cox-2, and IL-10 mRNA are shown in FIGS. 12 (a) to 12 (d), respectively.
- nucleic acid delivery composition and nucleic acid delivery method there are provided an excellent nucleic acid delivery composition and nucleic acid delivery method, and a carrier composition thereof that exhibit high nucleic acid introduction efficiency and at the same time, are significantly suppressed in cytotoxicity.
- a nucleic acid delivery composition and carrier composition can be suitably used, for example, as a pharmaceutical composition for nucleic acid therapy, and its industrial value is extremely high.
Abstract
Description
合成担体は、従来の医療で検討されてきた薬剤送達系(DDS)と同様、毒性等に関するリスクは存在するものの、ウイルスベクターに比べれば毒性が少ないと考えられることや、担持させる核酸のサイズに制限がなく、ベクターの精密な分子設計が可能であることから、精力的な開発研究が続けられている。
カチオン性脂質(リポフェクチン等)については、インビトロ(in vitro)ではある程度の成果が得られている(例えば非特許文献1参照)が、インビボ(in vivo)では必ずしも所期の効果が得られていない。
一方、カチオン性ポリマーとしては、ポリ(L-リシン)、DEAE-デキストラン、ポリエチレンイミン(例えば非特許文献2参照)、キトサン(例えば非特許文献3参照)等が検討されている。しかし、これらのカチオン性ポリマーは細胞毒性を有する上に、核酸導入効率・遺伝子発現効率も不十分であった。
本発明の別の主旨は、標的細胞又は組織に核酸を送達するための担体組成物であって、非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとを有するブロックコポリマーと、カチオン性ポリマーとを含んでなり、ブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、ブロックコポリマーが有するカチオン基のモル百分率(B/H比)が、25%~90%である、担体組成物に存する。
本発明の更に別の主旨は、核酸治療に用いられる医薬組成物であって、前記の核酸送達用組成物又は担体組成物を含んでなる医薬組成物に存する。
本発明の更に別の主旨は、標的細胞又は組織に核酸を送達するための方法であって、前記の核酸送達用組成物を標的細胞又は組織に接触させることを含んでなる方法に存する。
本発明の更に別の主旨は、標的細胞又は組織に核酸を送達するための方法であって、非荷電親水性ポリマーセグメント及びカチオン性ポリマーセグメントを有するブロックコポリマーと、核酸とを含んでなる核酸送達用組成物、並びに、カチオン性ポリマーを、標的細胞又は組織に接触させることを含んでなるとともに、標的細胞又は組織への接触時における、核酸送達用組成物のブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、核酸送達用組成物のブロックコポリマーが有するカチオン基のモル百分率(B/H比)を、25%~90%とする方法に存する。
本発明で使用されるブロックコポリマーは、非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとを有する。ブロックコポリマーは、1種のみを使用してもよいが、2種以上を任意の組合せ及び比率で使用してもよい。
非荷電親水性ポリマーセグメントは、非荷電且つ親水性の性質を有するポリマーセグメントである。ここで「非荷電」とは、セグメントが全体として中性であることをいう。例としてはセグメントが正・負の電荷を有さない場合が挙げられる。また、セグメントが正・負の荷電を分子内に有する場合であっても、局所的な実効電荷密度が高くなく、高分子ミセルの形成を妨げない程度にセグメント全体の荷電が中和されていれば、やはり「非荷電」に該当する。また、「親水性」とは水性媒体に対して溶解性を示すことをいう。
カチオン性ポリマーセグメントは、カチオン基を有し、カチオン性(陽イオン性)を示すポリマーセグメントである。但し、カチオン性ポリマーセグメントは、高分子ミセルの形成を妨げない範囲で、多少のアニオン基を有していてもよい。
非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとの組み合わせは制限されず、任意の非荷電親水性ポリマーセグメントと任意のカチオン性ポリマーセグメントとを組み合わせることが可能である。
非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとの結合形態も制限されず、直接結合していてもよいが、連結基を介して結合していてもよい。
連結基の例としては、非荷電親水性ポリマーセグメント及びカチオン性ポリマーセグメントの総個数に対応する価数を有する炭化水素基が挙げられる。連結基としての炭化水素基は脂肪族でも芳香族でもそれらが連結したものでもよく、脂肪族の場合には飽和でも不飽和でもよく、また、直鎖でも分岐でも環状でもよい。連結基としての炭化水素基の分子量は、制限されるものではないが、通常5000以下、好ましくは1000以下である。連結基としての炭化水素基の例としては、没食子酸誘導体、3,5-ジヒドロキシ安息香酸誘導体、グリセリン誘導体、シクロヘキサン誘導体、L-リシン等が挙げられるが、3,5-ジヒドロキシ安息香酸誘導体等が好ましい。
本発明のブロックコポリマーの好ましい具体例としては、非荷電親水性ポリマーセグメントとしてポリエチレングリコール(PEG)セグメントを有し、カチオン性ポリマーセグメントとしてポリ(アミノ酸又はその誘導体)セグメントを有する、下記式(I)~(IV)に示すブロックコポリマーが挙げられる。
R1は、水素原子、又は、未置換若しくは置換の直鎖若しくは分枝のC1-12アルキル基を表し、
R2は、メチレン基又はエチレン基を表し、
R3は、水素原子、保護基、疎水性基又は重合性基を表し、
R4は、R5と同一であるか、又は開始剤残基であり、
R5は各々独立に、水酸基、オキシベンジル基又は-NH-(CH2)a-X基を表し、
Xは各々独立に、
pKa値が7.4以下の嵩高いアミン化合物残基、
一級、二級、三級アミン若しくは四級アンモニウム塩のうち一種又は二種以上を含むアミン化合物残基、又は
アミンでない化合物残基を表し、
L1及びL2は各々独立に、連結基を表し、
aは、1~5の整数であり、
mは、5~20,000の整数であり、
nは、2~5,000の整数であり、
xは、0~5,000の整数であり、
但し、xはnより大きくない。)
R1は、水素原子、又は、未置換若しくは置換の直鎖若しくは分枝のC1-12アルキル基を表し、
R2は、メチレン基又はエチレン基を表し、
R3は、水素原子、保護基、疎水性基又は重合性基を表し、
R4は、R5と同一であるか、又は開始剤残基であり、
R5は各々独立に、水酸基、オキシベンジル基又は-NH-(CH2)a-X基を表し、
Xは各々独立に、
pKa値が7.4以下の嵩高いアミン化合物残基、
一級、二級、三級アミン若しくは四級アンモニウム塩のうち一種又は二種以上を含むアミン化合物残基、又は
アミンでない化合物残基を表し、
L1及びL2は各々独立に、連結基を表し、
aは、1~5の整数であり、
R6は各々独立に、水素原子又は保護基であり、
mは、5~20,000の整数であり、
nは、2~5,000の整数であり、
yは、0~5,000の整数であり、
zは、0~5,000の整数であり、
但し、y+zはnより大きくない。)
R1は、水素原子、又は、未置換若しくは置換の直鎖若しくは分枝のC1-12アルキル基を表すが、C1-12アルキルとしては、メチル、エチル、n-プロピル、iso-プロピル、n-ブチル、sec-ブチル、tert-ブチル、n-ペンチル、n-ヘキシル、デシル、ウンデシル等が挙げられる。C1-12アルキル基が置換される場合の置換基としては、アセタール化ホルミル基、シアノ基、ホルミル基、カルボキシル基、アミノ基、C1-6アルコキシカルボニル基、C2-7アシルアミド基、トリ-C1-6アルキルシロキシ基(ここで3つのアルキル基は同一でも異なっていてもよい)、シロキシ基又はシリルアミノ基が挙げられる。
Xは、ブロックコポリマーが本発明の条件を満たす(又は本発明の目的に沿う)限り制限されないが、通常は以下のA群~E群に分類される残基の中から選択される。
nは、通常5以上、好ましくは10以上、より好ましくは40以上、また、通常5,000以下、好ましくは1,000以下、より好ましくは500以下、特に好ましくは300以下の整数である。
xは、通常0以上、好ましくは1以上、より好ましくは10以上、また、通常5,000以下の整数である。但し、x≦nである。
y及びzは、各々独立に、通常0以上、好ましくは1以上、また、通常5,000以下の整数である。但し、y+z≦nである。特に好ましくは、10≦y≦n-10、10≦z≦n-10である。
また、一般式(I)~(IV)において、ポリ(アミノ酸又はその誘導体)セグメントが有するカチオン性基は、遊離カチオン性基であってもよいが、塩を形成していてもよい。この場合、塩を形成する対イオンとしては、制限されるものではないが、Cl-、Br-、I-、(1/2SO4)-、NO3 -、(1/2CO3)-、(1/3PO4)-、CH3COO-、CF3COO-、CH3SO3 -、CF3SO3 -等が挙げられる。
第1の方法としては、末端にアミノ基を有するPEG誘導体を用い、そのアミノ末端から、β-ベンジル-L-アスパルテート、Nε-Z-L-リシン等の保護アミノ酸のN-カルボン酸無水物(NCA)を重合させてブロックコポリマーを合成し、その後得られたポリ(アミノ酸誘導体)セグメントの保護アミノ酸側鎖を、所望のアミノ酸側鎖に変換する方法が挙げられる。この場合、得られるブロックコポリマーの構造は、一般式(I)又は(III)となる。
第2の方法としては、所望のアミノ酸側鎖を有するポリ(アミノ酸又はその誘導体)セグメントを合成してから、これをPEGセグメントと結合させる方法も挙げられる。この場合、得られるブロックコポリマーの構造は、一般式(I)~(IV)の何れかとなる。
また、第1及び第2の何れの方法を用いる場合も、ブロックコポリマーの末端(R1、R3、R5、R6)に保護基、疎水性基、重合性基等を後から導入する場合、その手法は任意であるが、酸ハロゲン化物を用いる方法、酸無水物を用いる方法、活性エステルを用いる方法等、通常の合成で用いられる手法が挙げられる。
カチオン性ポリマーは、カチオン基を有し、カチオン性(陽イオン性)を示すポリマーである。但し、カチオン性ポリマーは、高分子ミセルの形成を妨げない範囲で、多少のアニオン基を有していてもよい。
前記条件を満たすカチオン性ポリマーを用いることにより、ブロックコポリマーの水性溶液中での会合・沈殿を防止して安定化し、担体組成物として機能し得る高分子ミセルを効率的に構築することが可能となる。
本発明のカチオン性ポリマーの好ましい具体例としては、下記式(I’)~(IV’)に示すポリ(アミノ酸又はその誘導体)が挙げられる。
本発明の核酸送達用組成物及び担体組成物が有するブロックコポリマーとカチオン性ポリマーとの比率は、ブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、ブロックコポリマーが有するカチオン基のモル百分率(本明細書ではこれを「B/H比」と表示する場合がある。)によって表される。具体的には以下の式で表される。
本発明の核酸送達用組成物に使用される核酸は制限されない。即ち、核酸としては、DNA、RNA、天然又は非天然の核酸類縁体(例えばペプチド核酸等)、改変核酸、修飾核酸等が挙げられるが、何れであってもよい。また、核酸は一本鎖でも二本鎖でもよく、タンパク質のコード化の有無やその他の機能の有無も制限されない。
なお、核酸分子はポリアニオンとなるため、前記ブロックコポリマーのポリカチオン部分の側鎖と、静電的相互作用により結合(会合)することができる。
ブロックコポリマー及びカチオン性ポリマーに対する核酸の比率は、[核酸が有するリン酸基]に対する[ブロックコポリマー及びカチオン性ポリマーが有するカチオン性基]のモル比(本明細書ではこれを「N/P比」と表示する場合がある。)で表される。
本発明の核酸送達用組成物及び担体組成物を製造する際に、ブロックコポリマー、カチオン性ポリマー及び核酸に加え、高分子ミセルの形成を妨げない、或いは安定性を下げない範囲で、その他の成分を添加することができる。その他の成分に特に制限はないが、具体例としては、非荷電又は荷電性の重合体、荷電性ナノ粒子等が挙げられる。
荷電性ナノ粒子としては、表面に架電を有する金属系ナノ粒子等が挙げられる。
前記その他の成分の使用量も制限されないが、高分子ミセルの形成を妨げない程度に抑えることが好ましい。具体的には、本発明の組成物の総重量に対して、通常30%以下、好ましくは20%以下、より好ましくは10%以下とすることが望ましい。
本発明の担体組成物は、通常は、前記のブロックコポリマー及びカチオン性ポリマー、並びに必要に応じて用いられるその他の成分を、混合することにより調製される。
具体的には、前記のブロックコポリマーを含んでなる第1の水性溶液と、前記のカチオン性ポリマーを含んでなる第2の水性溶液とを用意する。第1及び第2の水性溶液は、所望により濾過して精製してもよい。
第1及び第2の水性溶液の溶媒は、水性溶媒であれば、その種類は限定されない。好ましくは水であるが、高分子ミセルの形成を妨げない範囲で、水に他の成分を混合した溶媒、例えば生理食塩水、水性緩衝液、水と水溶性有機溶媒との混合溶媒等も用いることができる。水性緩衝液としては10mM HEPES緩衝液等が挙げられる。
第1及び第2の水性溶液の混合方法も限定されない。第1の水性溶液に第2の水性溶液を加えてもよく、第2の水性溶液に第1の水性溶液を加えてもよい。また、容器に第1及び第2の水性溶液を同時に入れて混合してもよい。得られた第1及び第2の水性溶液の混合液を、適宜攪拌してもよい。
また、更に透析、希釈、濃縮、撹拌等の操作を適宜付加してもよい。
本発明の核酸送達用組成物は、通常は、前記の核酸を、(i)前記のブロックコポリマー及びカチオン性ポリマー、並びに必要に応じて用いられるその他の成分と混合するか、或いは、(ii)予め調製された本発明の担体組成物と混合することにより調製される。
また、更に透析、希釈、濃縮、撹拌等の操作を適宜付加してもよい。
本発明の核酸送達用組成物及び担体組成物の形状は、限定されないが、通常は球状又は略球状である。
本発明の核酸送達用組成物及び担体組成物の粒径は、ブロックコポリマー及びカチオン性ポリマーの種類及び量比、その他の成分の有無、核酸送達用組成物及び担体組成物の周辺環境(水性媒体の種類)等に応じて異なるが、好ましくは10nm以上、より好ましくは50nm以上、また、好ましくは200nm以下、より好ましくは150nm以下である。
なお、生理環境や生理食塩水中等の塩存在条件下では、核酸送達用組成物及び担体組成物の粒径は経時的に増大する傾向があるが、架橋剤を導入することにより粒径の増大を防止することが出来る。
担体組成物においては、ブロックコポリマーの親水性セグメントが粒子外殻周辺に密集して存在するとともに、ブロックコポリマーのカチオン性ポリマーセグメント及びカチオン性ポリマーが主に粒子内部側に存在する、PIC型高分子ミセルの構造を有しているものと考えられる。
本発明の核酸送達用組成物は、インビトロ又はインビボにおいて、核酸を標的細胞又は組織に送達するために使用できる。本発明の核酸送達用組成物によれば、標的細胞内へ安定したまま送達することが困難であった核酸を、容易に安定化した状態で送達することができるとともに、細胞毒性も抑制することが可能となる。また、細胞内外のpHの変化を利用し、核酸送達用組成物の粒子内部に内包・担持された核酸を、標的細胞内に効率的に導入する手段として使用することができる。更に、核酸としてタンパク質をコードする遺伝子を用い、これを細胞又は組織に送達して発現させる場合には、本発明の核酸送達用組成物を用いることにより、高い遺伝子発現効率を得ることが可能となる。
インビトロで本発明の核酸送達用組成物と標的細胞又は組織との接触を達成するには、本発明の核酸送達用組成物の存在下で標的細胞又は組織を培養するか、又は標的細胞又は組織の培養物中に核酸送達用組成物を添加すればよい。
なお、特に肺は外来の異物に対する感受性が高く、従来のDDSを用いた薬物送達では炎症が惹起される場合があり、経肺投与は極めて困難であったが、本発明の核酸送達用組成物によれば、毒性を低く抑えつつ高い遺伝子発現効率を得ることが可能であるため、経肺投与にも好適に使用することができる。
本発明によれば、本発明の核酸送達用組成物又は担体組成物を含んでなる医薬組成物(本発明の医薬組成物)も提供される。本発明の医薬組成物は、例えば、各種疾患の原因となる細胞又は組織を標的として、所望の核酸を送達・導入する治療(遺伝子治療)に用いることができる。
本発明の医薬組成物の使用方法も任意である。核酸送達用組成物を含む医薬組成物であればそのまま投与することが可能である。担体組成物を含み、核酸を含まない医薬組成物の場合には、使用前に核酸と混合し、担体組成物に核酸を担持させてから、投与に供すればよい。
また、本発明によれば、インビトロ又はインビボにおいて、核酸を標的細胞又は組織に送達する方法(本発明の核酸送達方法)が提供される。本発明の核酸送達方法には、以下の(1)(2)の方法が包含される。
本方法では、先に説明した本発明の核酸送達用組成物又は医薬組成物を用い、これをインビトロ又はインビボにおいて標的細胞又は組織と接触させることにより、標的細胞又は組織に核酸を送達する。核酸送達用組成物及び医薬組成物の詳細、標的細胞又は組織への接触方法等については、前述したとおりである。本方法によれば、細胞毒性を低く抑えつつ、高い効率で核酸導入を行うことが出来る点も、前述したとおりである。
本方法では、本発明の核酸送達用組成物又は医薬組成物からカチオン性ポリマーを除いた組成物(即ち、ブロックコポリマーと核酸を含む組成物。以降の記載では「ブロックコポリマー系核酸組成物」という場合がある。)を用い、これをインビトロ又はインビボにおいて標的細胞又は組織と接触させるとともに、別途カチオン性ポリマーを標的細胞又は組織に接触させ、現位置(in situ)でブロックコポリマー系核酸組成物と共存させる方法である。更に、標的細胞又は組織におけるブロックコポリマー系核酸組成物のブロックコポリマーとカチオン性ポリマーとの共存比を、B/H比が前記特定の範囲を満たすような比に調整する。本方法(2)によっても、前記方法(1)(本発明の核酸送達用組成物を用いて核酸送達を行う方法)と同様、細胞毒性を低く抑えつつ、高い効率で核酸導入を行うことが可能である。
ブロックコポリマー系核酸組成物を構成するブロックコポリマー及び核酸の詳細については、本発明の核酸送達用組成物について前述したとおりである。ブロックコポリマーと核酸との使用比率も任意であり、核酸送達の目的や条件等に応じて適宜選択すればよいが、原位置で共存するカチオン性ポリマーの共存比率を勘案して、前記N/P比の範囲が満たされるような比率で使用することが好ましい。ブロックコポリマー系核酸組成物の調製についても、本発明の核酸送達用組成物の調製に準じて、各成分を混合することにより行うことが可能である。
カチオン性ポリマーの詳細についても、本発明の核酸送達用組成物について前述したとおりである。
(ブロックコポリマー)
<PEG-PAsp(DET)の構造>
ブロックコポリマーとしては、ポリエチレングリコール(以降の記載では「PEG」と表示する)セグメントとポリ(アスパラギン酸-ジエチレントリアミン誘導体)(以降の記載では「PAsp(DET)」と表示する)セグメントとを有する、以下に示すブロックコポリマー(以降の記載では「PEG-PAsp(DET)」と表示する)を用いた。
mはPEGの重合度を表し、約270である。
nはPAsp(DET)の重合度を表し、約61である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。
cは連結基であるエチレン基の繰り返し数を表し、約3である。)
PEG-PAsp(DET)のPEGセグメントの分子量は約12000、PAsp(DET)セグメントの分子量は約14000であった。
PEG-PAsp(DET)は、以下の(1)(2)の手順で作製した。
NCA(アミノ酸無水物)化合物を少量のDMF(ジメチルホルムアミド)に溶解し、そこに塩化メチレンを加えた。重合開始剤として、ジクロロメタンに溶解した片末端に1級アミノ基を有するPEGを加え、35℃で2日間攪拌した。これらの操作は、乾燥アルゴン雰囲気下で行った。生成したPEG-PBLAは、n-ヘキサン/酢酸エチル(6/4)の混合溶液中に滴下して沈殿させ、濾過後減圧乾燥することによって回収した。生成したPEG-PBLAの分子量分布を、PEGスタンダードの検量線を用いてゲル濾過クロマトグラフィー(GPC)解析したところ、Mw(重量平均分子量)/Mn(数平均分子量)は1.05であった。また、1H NMR測定により重合度を求めたところ、65であった。
PEG-PAsp(DET)はPEG-PBLAのアミノリシス反応を利用して合成した。乾燥させたPEG-PBLA50mgをNMP(N-メチルピロリドン)2mL中に溶解させ、それぞれ5℃及び0℃に冷却した。別容器にPEG-PBLAのベンジルエステル基に対して50倍モル量のDETを等量のNMPで希釈した溶液を調製し、前記PEG-PBLA溶液と同じ温度に冷却した。そこに前記PEG-PBLA溶液をゆっくりと滴下し、1時間の反応の後、反応溶液をよく冷却した5N HCl(好ましくは1N程度の希酸)に溶液温度が5℃以下になるよう制御しながら滴下した。その後、0.01N程度のHCl水溶液に対して4℃に保ったまま透析を行い(pH2)、さらに純水に対して透析を行うことで過剰の酸を取り除いた。最終的に得られたポリマー水溶液を凍結乾燥し、PEG-PAsp(DET)を塩として回収した。定量的なアミノリシス反応は1H NMR測定により確認した。
<Homo-PAsp(DET)の構造>
カチオン性ポリマーとしては、以下に示すポリ(アスパラギン酸-ジエチレントリアミン誘導体)ホモポリマー(以降の記載では「Homo-PAsp(DET)」と表示する)を用いた。
nはPAsp(DET)の重合度を表し、約54である。
a、bは何れも0より大きく、1未満の数である。但しa+b=1である。)
なお、前記Homo-PAsp(DET)の分子量は約10000であった。
Homo-PAsp(DET)は、前記<PEG-PAsp(DET)の製法>に準じて調製した。
即ち、前記<PEG-PAsp(DET)の製法>の工程(1)において、PEGの代わりにn-ブチルアミンを用いた他は同様の操作を行うことにより、ポリベンジル-L-アスパルテート(PBLA)を作製した。GPCによるMw/Mnは1.06、1H NMR測定による重合度は65であった。
次いで、前記<PEG-PAsp(DET)の製法>の工程(1)において、PEG-PBLAの代わりに上で得られたPBLA50mgを用いた他は同様の操作を行うことにより、Homo-PAsp(DET)を調製した。
核酸としては、ルシフェラーゼをコードするプラスミド(Luc pDNA)を用いた。このプラスミドは、理研セルバンクより入手し、大腸菌に導入し、培養によって増幅し、NucleoBond Xtra Maxi(日本ジェネティクス社製)を用いて精製した後に使用した。
前記のブロックコポリマー(PEG-PAsp(DET))、カチオン性ポリマー(Homo-PAsp(DET))及び核酸(Luc pDNA)を用い、以下の手順で組成物(核酸送達用組成物)を調製した。なお、以下の記載において単に「溶液」と表示する場合は、10mM HEPESバッファー中溶液を指すものとする。
1mg/mLブロックコポリマー溶液と、1mg/mLカチオン性ポリマー溶液とを、B/H比が後述の種々の値を満たすように混合した後、10mM HEPESバッファー中でN/P比が後述の種々の値を満たすように所定量のpDNAと混合することにより、組成物(核酸送達用組成物)を得た。
B/H比を100%(ブロックコポリマー単独)、75%、50%、25%又は0%(カチオン性ポリマー単独)とし、N/P比を3として、上記手順で調製した組成物(核酸送達用組成物)を用いた。各組成物(核酸送達用組成物)について、酢酸ウラニル水溶液を用いて核酸を染色した後、粒子形状を透過型電子顕微鏡で観察した。
B/H比を100%(ブロックコポリマー単独)、75%、50%、25%又は0%(カチオン性ポリマー単独)とし、N/P比を3として、上記手順で調製した組成物(核酸送達用組成物)を用いた。各組成物(核酸送達用組成物)のゼータ電位を以下の手順により測定した。
各組成物(核酸送達用組成物)を折り畳みキャピラリーセル(folded capillary cells)(Malvern Instruments, Ltd.)に注入し、Malvern Instruments, Ltd.製Nano ZSを用いて測定に供した。得られた結果から、以下のスモルコフスキー(Smoluchowski)方程式によりゼータ電位を算出した。
ζ=4πηυ/e ・・・スモルコフスキー(Smoluchowski)方程式
(式中、ζはゼータ電位を表し、ηは溶媒の粘度を表し、υは電気泳動移動度を表し、eは溶媒の誘電率を表す。)
B/H比を100%(ブロックコポリマー単独)、80%、70%、60%、50%、25%又は0%(カチオン性ポリマー単独)とし、N/P比を4、6、8、12、16として、上記手順で調製した組成物(核酸送達用組成物)を用いた。各組成物(核酸送達用組成物)のトランスフェクション効率及び細胞毒性を、以下の手順で測定した。
HUVEC(正常ヒト臍帯静脈内皮細胞)を24ウェルプレートに対し、各ウェル20,000細胞の細胞密度となるように、400μLのEBM-2とともに播種し、24時間培養した。その後、各組成物(核酸送達用組成物)を各ウェルに30μLずつ加え、更に24時間培養した。細胞をPBSで洗浄し、新たなEBM-2を各ウェルに400μLずつ加え、更に24時間培養した。その後、セルカウンティングキット(Cell Counting Kit-8:同仁化学研究所製)を用い、使用説明書に従って、各ウェル内の生存細胞数を計数することにより、細胞毒性の指標とした。
(ブロックコポリマー)
ブロックコポリマーとしては、実施例群Iの式に示すPEG-PAsp(DET)において、c=3又はc=6のものを用いた(以降の記載ではそれぞれ「PEG-C3-PAsp(DET)」及び「PEG-C6-PAsp(DET)」と表示する。また、図5中ではそれぞれ「C3」及び「C6」と表示する。)。各ポリマーは実施例群Iの<PEG-PAsp(DET)の製法>に記載の手法に準じて合成した。
カチオン性ポリマーとしては、実施例群Iと同じHomo-PAsp(DET)を用いた。
核酸としては、以下の何れかを用いた。
・sFlt-1をコードするプラスミド(sFlt-1 pDNA)
sFlt-1はpVL1393バキュロウィルスベクター(pDNA)から切り出されたHuman sFlt-1 cDNA(2.4 kb)であり、東京医科歯科大渋谷教授から供与されたものである。アガロースゲル電気泳動で精製した後、Rapid DNA Ligation Kit(ロシュ社製)を用いてpCAccベクターに挿入し、大腸菌(DH5α)培養で増幅させ、培養プレート上に形成された10程度のコロニーをそれぞれ取り分けて更に培養し、それぞれのpDNAをアガロース電気泳動などで調べ、所望の配列が挿入されたpDNAを選別し、増幅したものを、NucleoBond(登録商標) Xtra(日本ジェネティクス社製)を用いて精製して使用した。
Venus pDNAは理研セルバンクより入手し、大腸菌に導入した後に培養によって増幅させ、NucleoBond Xtra Maxi(日本ジェネティクス社製)を用いて精製した後に使用した。
このプラスミドは、実施例群Iと同様に入手・調製した。
上記Luc pDNAにCy5標識を付したプラスミドである。Mirus社から購入したLabel IT Nucleic Acid Labeling Kit を使用し、取扱説明書に記載の手順に従ってCy5をラベルした。
8週齢マウスBalb/c(メス、チャールズリバーラボラトリー社より入手)をそのまま(以降「Balb/cマウス」と表示する場合がある)、又は5週齢ヌードマウスBalb/c(メス、チャールズリバーラボラトリー社より入手)にヒト膵臓ガンBxPC3を皮下接種し、腫瘍体積がおよそ45mm3に成長するまで2~3週間飼育したもの(以降「BxPC3皮下接種マウス」と表示する場合がある)を使用した。
前記のブロックコポリマー(PEG-C3-PAsp(DET)又はPEG-C6-PAsp(DET))、カチオン性ポリマー(Homo-PAsp(DET))及び核酸((sFlt-1 pDNA、Cy5-pDNA、Venus pDNA又はLuc pDNA)を用い、B/H比を以下の各項目に記載の値に種々変更し、N/P比を8として、実施例群Iの(核酸送達用組成物の調製)に記載の手順に従って、組成物(核酸送達用組成物)を調製した。
ブロックコポリマーとしてPEG-C3-PAsp(DET)又はPEG-C6-PAsp(DET)を用い、核酸としてsFlt-1 pDNAを用い、B/H比を100%(ブロックコポリマー単独)、70%、又は50%として得られた各組成物(核酸送達用組成物)(2OD、200μL)を、前記のBxPC3皮下接種マウス(各組成物あたり6頭ずつ)に対して、経静脈注射により全身投与した。各動物に対する投与は、実験開始後0日目、4日目及び8日目に行った。1回の投与量は、マウス1頭あたりの核酸投与量が20μgとなるように決定した。実験開始後の腫瘍体積を経時的に測定することにより、核酸送達による腫瘍抑制効果を調べた。
また、対照実験として、10mM HEPESバッファーを用いて、上記と同様の手法で投与及び腫瘍体積測定を行った。
ブロックコポリマーとしてPEG-C3-PAsp(DET)を用い、核酸としてCy5-pDNAを用い、B/H比を100%(ブロックコポリマー単独)、70%、又は50%として得られた各組成物(核酸送達用組成物)(2OD、200μL)を、前記のBxPC3皮下接種マウス(各組成物あたり8頭ずつ)に対して、経静脈注射により全身投与した。投与量は、マウス1頭あたりの核酸投与量が20μgとなるように決定した。投与後に経時的に採血を行い、採取した血液サンプル中の蛍光強度をIVIS(登録商標)イメージングシステム(Caliper社(Xenogen社)製)で測定した。投与直後の血液サンプル中の蛍光強度に対する、投与20分後の血液サンプル中の蛍光強度の比率を、血中滞留性の指標として求めた。
ブロックコポリマーとしてPEG-C3-PAsp(DET)を用い、核酸としてVenus pDNAを用い、B/H比を100%(ブロックコポリマー単独)、70%、又は50%として得られた各組成物(核酸送達用組成物)(2OD、200μL)を、前記のBxPC3皮下接種マウス(各組成物あたり1頭ずつ)に対して、経静脈注射により全身投与した。投与量は、マウス1頭あたりの核酸投与量が20μgとなるように決定した。投与から2日後に腫瘍を切除し、10μm厚の切片を作製した。この切片を共焦点レーザー顕微鏡(CLSM)で観察し、Venusの発現量及び発現部位を調べることにより、核酸送達による核酸発現有効性の指標とした。なお、Venusの発現部位の基準として、同切片の細胞核及び血管内皮細胞を免疫染色し、その部位も観察した。
また、対照実験として、10mM HEPESバッファーを用いて、上記と同様の手法で投与及びCLSM観察を行った。
ブロックコポリマーとしてPEG-C3-PAsp(DET)を用い、核酸としてsFlt-1 pDNAを用い、B/H比を70%として得られた各組成物(核酸送達用組成物)(2OD、200μL)を、前記のBxPC3皮下接種マウス(各組成物あたり1頭ずつ)に対して、経静脈注射により全身投与した。投与量は、マウス1頭あたりの核酸投与量が20μgとなるように決定した。投与から2日後に腫瘍を切除し、10μm厚の切片を作製し、血管内皮細胞を免疫染色した。免疫染色には血管内皮細胞マーカーであるPECAM-1と、蛍光標識した二次抗体を使用した。また、sFlt-1の発現部位の基準として、同切片の細胞核及び血管内皮細胞も免疫染色した。染色後の切片を共焦点レーザー顕微鏡(CLSM)で観察し、sFlt-1の発現量及び発現部位を調べることにより、核酸送達による核酸発現有効性の指標とした。
また、対照実験として、核酸送達用組成物の代わりに10mM HEPESバッファーを用い、上記と同様の手法で投与、免疫染色及びCLSM観察を行った。
ブロックコポリマーとしてPEG-C3-PAsp(DET)を用い、核酸としてsFlt-1 pDNAを用い、B/H比を100%(ブロックコポリマー単独)、70%、又は50%として得られた各組成物(核酸送達用組成物)(2OD、200μL)を、前記のBxPC3皮下接種マウス(各組成物あたり3頭ずつ)に対して、経静脈注射により全身投与した。各動物に対する投与は2回、実験開始後0日目及び4日目に行った。投与量は、マウス1頭あたりの核酸投与量が20μgとなるように決定した。投与から6日後に腫瘍を切除し、10μm厚の切片を作製し、血管内皮細胞を免疫染色した。免疫染色には血管内皮細胞マーカーであるPECAM-1と、蛍光標識した二次抗体を使用した。染色後の切片を共焦点レーザー顕微鏡(CLSM)で観察し、各切片あたり7枚のCLSM写真を撮像した。得られたCLSM写真をLSM510(カールツァイス社製)で画像解析して、緑色発光画素比率(血管内皮細胞比率に相当)を計測し、各切片の平均値を血管密度として求め、核酸送達による血管新生の抑制効果の指標とした。
更に、対照実験として、10mM HEPESバッファーを用いて、上記と同様の手法で投与、免疫染色及びCLSM観察を行った。
ブロックコポリマーとしてPEG-PAsp(DET)を用い、核酸としてLuc pDNAを用い、B/H比を100%(ブロックコポリマー単独)、75%、50%、25%又は0%(カチオン性ポリマー単独)として得られた各組成物(4OD、50μL)を、前記のBalb/cマウス(各組成物あたり5頭ずつ)に対して経肺投与した。経肺投与は、マイクロスプレー(microsprayer Model IA-1C-R, Penn Century社製)を用い、前記各組成物ををマウスの気管支内に直接噴射することにより行った。
投与から24時間後、肺内の細胞30mgを採取し、細胞をPBSで穏やかに洗浄した。細胞溶解液(Cell Culture Lysis Buffer:Promega社製)を200μLずつ加え、ルシフェラーゼアッセイキット(Luciferase Assay System Kit:Promega社製)及びLB940機器(Mithras社製)を用いてフォトルミネッセンス強度を測定することにより、ルシフェラーゼ活性を決定し、トランスフェクション効率の指標とした。なお、細胞溶解液中のタンパク質量は、MicroBCA(登録商標)タンパク質アッセイ試薬キット(MicroBCA(登録商標) Protein Assay Reagent Kit:Thermo Scientific社製)により決定した。
また、市販の遺伝子導入試薬である直鎖状ポリエチレンイミン(Linear Polyethylenimine:LPEI, Exgen 500, Fermentas社製)についても、同様の手順で投与、肺細胞採取、ルシフェラーゼ活性の決定を行った(図10中「LPEI」)。
また、対照として、Luc pDNA 10mgを10mM HEPESバッファー溶媒50μlに溶解させた溶液についても、同様の手順で投与、肺細胞採取、ルシフェラーゼ活性の決定を行った(図10中「核酸単独」)。
各組成物のトランスフェクション効率を表すグラフを図10に示す。B/H比100%(ブロックコポリマー単独)及び、B/H比0%(カチオン性ポリマー単独)の組成物に比べて、B/H比が75%及び50%の各組成物では、トランスフェクション効率の明らかな向上が見られた。
ブロックコポリマーとしてPEG-PAsp(DET)を用い、核酸としてLuc pDNAを用い、B/H比を100%(ブロックコポリマー単独)、50%又は0%(カチオン性ポリマー単独)として得られた各組成物(4OD、50μL)を用い、上記と同様に各組成物の肺内投与を行ったBalb/cマウス(各組成物あたり1頭ずつ)について、投与から4時間後に肺組織を採取し、ヘマトキシリンとエオジンによって組織染色し、光学(明視野?)顕微鏡写真によって炎症の有無を観察した。
また、対照として、肺内投与を行わなかったBalb/cマウスについても、同様の手順により炎症の有無を観察した。
各組成物について得られた光学顕微鏡写真を図11(a)~(d)に示す。B/H比0%(カチオン性ポリマー単独)の組成物を投与した個体(図11(a))では、肺組織に炎症が惹起された(図中丸で囲んだ箇所)のに対して、B/H比50%の組成物を投与した個体(図11(b))及びB/H比100%(ブロックコポリマー単独)の組成物を投与した個体(図11(c))の肺組織では、対照個体(図11(d))の肺組織と比べて大きな変化はなく、炎症はほとんど見られなかった。
ブロックコポリマーとしてPEG-PAsp(DET)を用い、核酸としてLuc pDNAを用い、B/H比を100%(ブロックコポリマー単独)、50%又は0%(カチオン性ポリマー単独)として得られた各組成物(4OD、50μL)を用い、上記と同様に各組成物の肺内投与を行ったBalb/cマウス(各組成物あたり5頭ずつ)について、投与から4時間後に肺内の細胞30mgを採取し、炎症性サイトカインであるIL-6、TNF-α、Cox-2及びIL-10のmRNAの発現量を測定した。各mRNAの発現量の測定は、TaqMan Gene Expression Assays及びABI Prism 7500 Sequence Detector(Applied Biosystems社)を用いて定量PCRにより行った。また、測定されたmRNA発現量は、非投与群において測定された値に対する比の値(相対発現量)として表示した。
また、対照として、10mM HEPESバッファー50μlを用いて、上記と同様の手法で肺内投与及び各炎症性サイトカインのmRNA発現量の測定を行った。
得られたIL-6、TNF-α、Cox-2及びIL-10のmRNAの相対発現量を、それぞれ図12(a)~(d)に示す。B/H比0%(カチオン性ポリマー単独)の組成物を投与した個体では、いずれの炎症性サイトカインについても、対照群と比べて発現量が大幅に増加したのに対して、B/H比50%の組成物及びB/H比100%(ブロックコポリマー単独)の組成物を投与した個体では、いずれの炎症性サイトカインについても、その発現量は低く保たれていた。
Claims (14)
- 標的細胞又は組織に核酸を送達するための核酸送達用組成物であって、
非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとを有するブロックコポリマーと、カチオン性ポリマーと、核酸とを含んでなり、
ブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、ブロックコポリマーが有するカチオン基のモル百分率(B/H比)が、25%~90%である、核酸送達用組成物。 - 核酸が有するリン酸基に対する、ブロックコポリマー及びカチオン性ポリマーが有する総カチオン性基のモル比(N/P比)が、2~200である、請求項1に記載の核酸送達用組成物。
- 粒子状である、請求項1又は2に記載の核酸送達用組成物。
- ブロックコポリマーの非荷電親水性ポリマーセグメントが、ポリアルキレングリコールセグメントである、請求項1~3の何れか一項に記載の核酸送達用組成物。
- ブロックコポリマーのカチオン性ポリマーセグメントが、側鎖にアミノ基を有するポリ(アミノ酸又はその誘導体)からなるセグメントである、請求項1~4の何れか一項に記載の核酸送達用組成物。
- カチオン性ポリマーが、側鎖にアミノ基を有するポリ(アミノ酸又はその誘導体)である、請求項1~5の何れか一項に記載の核酸送達用組成物。
- 標的細胞又は組織に核酸を送達するための担体組成物であって、
非荷電親水性ポリマーセグメントとカチオン性ポリマーセグメントとを有するブロックコポリマーと、カチオン性ポリマーとを含んでなり、
ブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、ブロックコポリマーが有するカチオン基のモル百分率(B/H比)が、25%~90%である、担体組成物。 - 粒子状である、請求項7に記載の担体組成物。
- ブロックコポリマーの非荷電親水性ポリマーセグメントが、ポリアルキレングリコールセグメントである、請求項7又は8に記載の担体組成物。
- ブロックコポリマーのカチオン性ポリマーセグメントが、側鎖にアミノ基を有するポリ(アミノ酸又はその誘導体)からなるセグメントである、請求項7~9の何れか一項に記載の担体組成物。
- カチオン性ポリマーが、側鎖にアミノ基を有するポリ(アミノ酸又はその誘導体)である、請求項7~10の何れか一項に記載の担体組成物。
- 核酸治療に用いられる医薬組成物であって、請求項1~6の何れか一項に記載の核酸送達用組成物、又は、請求項7~11の何れか一項に記載の担体組成物を含んでなる医薬組成物。
- 標的細胞又は組織に核酸を送達するための方法であって、請求項1~6の何れか一項に記載の核酸送達用組成物を、標的細胞又は組織に接触させることを含んでなる方法。
- 標的細胞又は組織に核酸を送達するための方法であって、
非荷電親水性ポリマーセグメント及びカチオン性ポリマーセグメントを有するブロックコポリマーと、核酸とを含んでなる核酸送達用組成物、並びに、カチオン性ポリマーを、標的細胞又は組織に接触させることを含んでなるとともに、
標的細胞又は組織への接触時における、核酸送達用組成物のブロックコポリマー及びカチオン性ポリマーが有する総カチオン基に対する、核酸送達用組成物のブロックコポリマーが有するカチオン基のモル百分率(B/H比)を、25%~90%とする方法。
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Also Published As
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US9314529B2 (en) | 2016-04-19 |
CA2804815C (en) | 2016-06-07 |
JPWO2012005376A1 (ja) | 2013-09-05 |
JP5826174B2 (ja) | 2015-12-02 |
CN102971002B (zh) | 2019-04-05 |
US20130109743A1 (en) | 2013-05-02 |
EP2591792B1 (en) | 2016-12-21 |
EP2591792A1 (en) | 2013-05-15 |
CN102971002A (zh) | 2013-03-13 |
DK2591792T3 (en) | 2017-04-03 |
EP2591792A4 (en) | 2014-12-24 |
CA2804815A1 (en) | 2012-01-12 |
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