JP4368183B2 - Polyion aggregate and biomaterial carrier comprising polyion aggregate - Google Patents

Description

  The present invention relates to a composite material of a synthetic polymer and a biological substance. More specifically, the present invention relates to a block copolymer having an uncharged hydrophilic portion and a cationically charged portion. The present invention relates to a polyion aggregate formed by ion association with a molecule or a biological tissue, and a biological material carrier comprising the aggregate.

  Cationic synthetic polymers and biopolymers such as DNA, proteins, and ion associations with cells are applied in many fields such as genetic engineering, protein engineering, and cell engineering. In particular, cationic polymers have attracted the most attention as synthetic polymers for the construction of gene vectors in gene therapy. As polymers used as the cationic skeleton, for example, polyvinylamines (Patent Document 1), polyethyleneimines (Non-Patent Document 1), and polylysines (Non-Patent Document 2) are disclosed.

  Among these cationic polymers, it has been disclosed that polyethyleneimine that has been used in industrial products such as flocculants and cosmetic additives in the paper industry is the most effective as a DNA carrier (Non-Patent Documents 3 and 4). Research on gene delivery and gene expression using sucrose has increased rapidly. However, commercially available polyethyleneimine is a hyperbranched polymer, and its structure includes tertiary amines and primary amines in addition to secondary amines. Although polyethyleneimine from such a composition forms a polyion complex with DNA, it is disadvantageous for controlling the structure, shape and dimensions of the complex.

  In order to synthesize a polyethyleneimine composed only of a secondary amine, it can be obtained by first synthesizing polyoxazoline and hydrolyzing it. Linear poly (ethyleneimine) can be used to form a complex with DNA, but for more precise control of the structure, shape and dimensions of the complex, a copolymer containing poly (ethyleneimine) It is required to build a body type polymer. As an example, a block copolymer composed of poly (ethyleneimine) and poly (ethylene oxide), that is, a double hydrophilic block copolymer having a so-called cation block has been taken up (Non-Patent Document 5). When such a block copolymer is used, the polyethylenimine moiety that is a cation block is ionically bonded to anionic DNA to form an ion complex that is insoluble in water, but the poly (ethylene oxide) that is a neutral block is A core-corona type ion association micelle is formed so as to cover the outer surface of the ion complex portion. The stability of the ion-associated micelle obtained in this way is extremely high, and the size and shape of the ion-associated micelle can be easily controlled by changing the composition ratio of these blocks, the degree of polymerization, and the like.

  Most of the double hydrophilic block copolymers having cationic blocks contain poly (ethylene oxide) as neutral blocks. This is effective as a gene carrier, but systematic knowledge is not shown about the DNA conveyance in a gene therapy, and the DNA expression mechanism in a cell, and experience accumulation is overwhelming. Therefore, diversity is required for the polymer structure for gene therapy and the complex of the polymer and DNA, and the need for developing various improved complexes is increasing.

  Since poly (ethylene oxide) has characteristics such as non-toxicity and biocompatibility, it has been widely used as a food additive and has recently attracted much interest in medical applications. Although poly (ethylene oxide) has a different structure, poly (ethyloxazoline) has recently attracted attention as a water-soluble polymer exhibiting similar biocompatibility and nontoxicity. For example, poly (ethyloxazoline) can be adsorbed on the lipid membrane surface and used in a drug sustained-release system (Non-patent Document 6).

  Compared to poly (ethylene oxide) for the preparation of vectors for gene therapy, poly (ethyloxazoline) forms a relatively weak hydrogen bond with water molecules, so it is hydrophilic (water soluble) and also potentially hydrophobic Property (lipid solubility). Such characteristics are not found in other water-soluble polymers. Therefore, it is very useful to have a water-soluble polymer such as polyethyloxazoline and another cationic polymer in combination with the copolymer structure and use it as a DNA carrier. A synthesis example of a graft copolymer obtained by using poly (ethyleneimine) as a main chain and introducing poly (ethyloxazoline) as a side chain into the main chain nitrogen has been disclosed (Patent Document 2). However, there is still no synthesis example of a block copolymer having both poly (ethyloxazoline) and a cationic polymer.

  Many studies have demonstrated that poly (ethyleneimine) is a suitable cationic polymer for gene therapy, but as its cationic polymer and neutral polymer, it is non-toxic, highly biocompatible, and hydrophilic. Development of a copolymer composed of poly (ethyl oxazoline) having both hydrophobicity and construction of a complex of the copolymer and DNA have led to the realization of a biopolymer carrier having excellent properties, particularly a gene This is an important issue for realizing therapeutic vectors.

Japanese Patent Laid-Open No. 2000-135082 Japanese Patent Laid-Open No. 08-120035 O. Boussif et al., Proc. Natl. Acad. Sci. USA, 1995, 92, pp 7297-7301. M.X.Tang et al., Gene Therapy, 1997, Vol. 4, pp823-832. H. Pollard et al., J. Biol. Chem. 1998, 273, pp7507-7511. V. Serguei et al., Bioconjugate Chem. 1998, Vol. 9, pp805-812. Y. Akiyama etal., Macromolecules, 2000, 33, pp5841-5845 M. C. Woodle et al., Bioconjugate Chem. 1994, Vol. 5, pp493-496.

  The problem to be solved by the present invention is a polyion aggregate that has a poly (ethyloxazoline) that is non-toxic, excellent in biocompatibility, has both hydrophilicity and hydrophobicity, and can stably hold a biological substance. And providing a biological material carrier comprising the aggregate.

  A polymer to be used as a carrier for a biological substance such as gene therapy must satisfy two elements, biocompatibility and cationicity. In the present invention, these biocompatibility and cationicity can be satisfied by using poly (ethyleneimine) as the cationic hydrophilic polymer unit. Further, poly (ethyloxazoline) is selected as a nonionic hydrophilic polymer unit, and a block copolymer composed of the two polymer skeletons and an anion capable of binding to the cationic poly (ethyleneimine) unit. A biopolymer having a functional group is mixed in water and ion-bonded with each other, whereby the ion-binding portion becomes water-insoluble, and solid particles of polyion micelles can be formed. The periphery of the solid particles is covered with a nonionic hydrophilic portion, and colloidal particles stable in water can be easily formed. The resulting colloidal particles have DNA incorporated therein, and the outside has water-soluble corona. The part forming the corona is poly (ethyloxazoline) having biocompatibility and further bioadhesiveness.

  Further, by using a block copolymer having a cationic poly (ethyleneimine) block and a nonionic poly (ethyloxazoline) block, the block copolymer and an anionic biological substance, for example, DNA, Colloidal particles are obtained by the ion-bonded aggregate. The colloidal particles are divided into a layer structure of an inner layer and an outer layer. The inner layer is an ionic aggregate of a poly (ethyleneimine) block that is a cationic polymer and an anionic biological substance such as DNA, and the outer layer is a hydrophilic polymer. Is a poly (ethyloxazoline) block. The hydrophilic polymer layer not only functions to increase the physical stability of the colloidal particles, but also serves to prevent contact between a biological substance such as DNA and an external substance.

  That is, the present invention comprises a block copolymer having a cationic poly (ethyleneimine) block, a poly (ethyloxazoline) block, and an anionic biomaterial, and the cationic copolymer in the block copolymer A polyion aggregate in which a poly (ethyleneimine) block and an anionic biological substance are ion-bonded, and a biological material carrier comprising the polyion aggregate are provided.

  The polyion aggregate of the present invention is a block having a poly (ethyleneimine) block useful as a biological material carrier, and a poly (ethyloxazoline) block that is nontoxic and excellent in biocompatibility, and has both hydrophilicity and hydrophobicity. By using the copolymer as a carrier polymer, colloidal particles stable in water can be easily formed. In addition, since the outer layer of the obtained colloidal particles is made of poly (ethyloxazoline), the colloidal particles have high physical stability and can prevent contact with external substances such as DNA incorporated therein. Thus, even when injected into a living body, the poly (ethyloxazoline) layer can protect biological substances such as DNA from degrading enzymes in the body, and can safely carry genes to target cells.

  The polyion aggregate of the present invention comprises a block copolymer having a cationic poly (ethyleneimine) block and a poly (ethyloxazoline) block, and an anionic biological substance. A cationic poly (ethyleneimine) block and an anionic biological substance are ion-bonded.

[Block copolymer]
The cationic poly (ethyleneimine) block possessed by the block copolymer is a cationic block composed of ethyleneimine and a unit in which hydrogen of the ethyleneimine is substituted with an alkyl group, and has hydrophilicity. It is. That is, the main chain nitrogen may be a secondary amine, a protonated form of a tertiary amine, or a quaternary ammonium form. Among these, it is more preferable to use a polyethyleneimine, which is a secondary amine having a good ionic bond strength with an anionic biological material, particularly a nucleic acid (DNA, RNA) and capable of forming a hydrogen bond.

  In addition, the poly (ethyloxazoline) block of the block copolymer has good hydrophilicity in the ethyloxazoline structure constituting the block and also has a potential hydrophobicity. Furthermore, since poly (ethyloxazoline) is non-toxic and has excellent biocompatibility and bioadhesiveness, the block copolymer of the present invention is suitable as a polymer for biomaterial carriers including gene therapy. Can be used for

  The block copolymer has a cationic poly (ethyleneimine) block and a nonionic poly (ethyloxazoline) block, and the cationic poly (ethyleneimine) block and an anionic biological substance By making the ion binding portion insoluble in water, a polyion aggregate having a hydrophilic portion and a hydrophobic portion in the structure can be formed. Therefore, the block copolymer can form stable colloidal particles regardless of whether they are linear or star-shaped, and the particle diameter of the colloidal particles can be controlled by controlling the composition ratio. Since it is easy, it is extremely effective for obtaining colloidal particles of polyion aggregates incorporating biological substances therein.

  Among the above block copolymers, in particular, the following formula (i)

（式中ｐは１０〜５００００の整数を表す。）
で表されるポリ（エチレンイミン）ブロックと、下記式（ii）

(In the formula, p represents an integer of 10 to 50,000.)
And a poly (ethyleneimine) block represented by the following formula (ii)

（式中ｑは１０〜５０００の整数を表す。）
で表されるポリ（エチルオキサゾリン）ブロックとを有し、前記式（i）で表されるポリ（エチレンイミン）ブロックがカチオン化されてなるブロック共重合体は、特に生体物質キャリアー用途に好適に使用することができる。

(Wherein q represents an integer of 10 to 5000)
And a block copolymer obtained by cationizing the poly (ethyleneimine) block represented by the formula (i) is particularly suitable for a biological material carrier. Can be used.

  The cationic poly (ethyleneimine) block and the nonionic poly (ethyloxazoline) block constituting the block copolymer are a cationic poly (ethyleneimine) block A and a poly (ethyloxazoline) block. Is expressed as B, A-B-A, B-A-B, A-B-A-B-... The number of blocks in one molecular chain of the coalescence is preferably a diblock or a triblock that easily forms colloidal particles, and each block is preferably linear.

  Examples of the block copolymer include structures represented by the following formula (1) or the following formula (2).

X- (AB) n or X- (BA) n (1)
(In the formula (1), X is a residue of a polymerization initiator having a valence of 1 or more, A is a cationic poly (ethyleneimine) block, B is a nonionic poly (ethyloxazoline) block, and n is a valence of X. And an integer of at least 1.

[X- (AB) n] mY, or [X- (BA) n] mY (2)
(In formula (2), X is a monovalent or higher polymerization initiation compound residue, Y is a monovalent or higher terminal compound residue, A is a poly (ethyleneimine) block, B is a poly (ethyloxazoline) block, and n is An integer of at least 1 within the range of valence of X, and m is an integer of at least 1 within the range of valence of Y.)

  The monovalent or higher polymerization initiating compound in the above formulas (1) and (2) is an initiator for cationic ring-opening living polymerization, and may be either a low molecular compound or a high molecular compound. Such a polymerization initiator is preferably one having a valence of 1 to 12, and is bonded to a cationic poly (ethyleneimine) block or a nonionic poly (ethyloxazoline) block depending on the valence. Therefore, when the valence of the polymerization initiator is 1 and 2, it becomes a linear block copolymer, and if the valence is higher than that, a star-shaped block copolymer, particularly a hexavalent benzene skeleton. Then, it becomes a typical star shape.

  As a polymerization initiator for such low molecular weight compounds, a compound having a functional group such as an alkyl chloride group, an alkyl bromide group, an alkyl iodide group, a toluenesulfonyloxy group, or a trifluoromethylsulfonyloxy group in the molecule is used. be able to. Specifically, for example, methylbenzene chloride, methylbenzene bromide, methylbenzene iodide, methylbenzene toluenesulfonate, methylbenzene trifluoromethylsulfonate, methane bromide, iodide methane, methane toluenesulfonate or toluenesulfone. Monohydric acids such as acid anhydride, trifluoromethylsulfonic acid anhydride, 5- (4-bromomethylphenyl) -10,15,20-tri (phenyl) porphyrin, bromomethylpyrene, dibromomethylbenzene, diiodination Divalent compounds such as methylbenzene, trivalent compounds such as tribromomethylbenzene, tetravalent compounds such as tetrabromomethylbenzene, tetra (4-chloromethylphenyl) porphyrin, tetrabromoethoxyphthalocyanine, hexabromomethyl Such as benzene It includes the value more than that.

  Among these, alkyl bromide, alkyl iodide, alkyl toluenesulfonate, and alkyl trifluoromethylsulfonate have high polymerization initiation efficiency, and it is particularly preferable to use alkyl bromide and alkyl toluenesulfonate.

  As a polymerization initiator for a polymer compound, for example, a compound in which a bromine atom or an iodine atom is bonded to a terminal carbon atom of poly (ethylene glycol), a compound in which a toluenesulfonyl group is bonded to a terminal oxygen atom, or the like can be used. . In that case, the molecular weight of poly (ethylene glycol) may be 800 to 10,000, and particularly preferably 1500 to 5000.

  The terminal compound having a valence of 1 or more in the above formula is one that terminates the terminal of a substantially cationic poly (ethyleneimine) block or nonionic poly (ethyloxazoline) block, preferably 1 to 12 It is bonded at the end of the ionic hydrophilic polymer block A or the nonionic hydrophilic polymer block B depending on the valence. Therefore, when the valence of the compound is 1 and 2, it becomes a linear water-soluble block copolymer, and if it has a valence higher than that, the water-soluble block copolymer that is a star or a combination thereof is used. It becomes a polymer.

  Specific examples of such terminal compounds include 5- (4-aminophenyl) -10,15,20-tri (phenyl) porphyrin, tetra (4-aminophenyl) porphyrin, aminopyrene, and 5- (4-hydroxyphenyl). ) -10,15,20-tri (phenyl) porphyrin, tetra (4-hydroxyphenyl) porphyrin, tetra (3,5-dihydroxyphenyl) porphyrin, aminomethylpyrene, tetraaminophthalocyanine, and the like. , May have a substituent.

  The block copolymer used in the present invention has a polymerization initiator residue, but does not necessarily have the above-mentioned terminal compound residue, in which case the group dissociated from the polymerization initiator or Hydrogen is bonded.

  The block copolymer used in the present invention is preferable when the mass average molecular weight is in the range of 1,000 to 1,000,000, particularly in the range of 5,000 to 100,000, since colloidal stability is improved. The polymerization degree of the cationic poly (ethyleneimine) block is preferably in the range of 10 to 50000, particularly preferably in the range of 20 to 500, and the polymerization degree of the nonionic poly (ethyloxazoline) block is 10 It is preferable to be in the range of ˜5000, particularly in the range of 20 to 500. By setting the degree of polymerization of each block within the above range, it is possible to improve the ease of handling of the copolymer and the association efficiency between the cationic poly (ethyleneimine) block and the anionic biological substance.

  The ratio of the cationic poly (ethyleneimine) block to the nonionic poly (ethyloxazoline) block is 5 in terms of the molar ratio of structural units forming the polymer (ethyleneimine units) / (ethyloxazoline units). It is preferable to set it as the range of / 1-1 / 5. By setting the molar ratio of the two in this range, the characteristics of the poly (ethyloxazoline) block can be fully expressed without impairing the characteristics of the excellent biomaterial carrier of the poly (ethyleneimine) block.

[Method for producing block copolymer]
As a method for synthesizing the block copolymer used in the present invention, a poly (N-formylethyleneimine) block or a poly (methyloxazoline) block, which is a precursor copolymer, and a poly (ethyloxazoline) block are used. Preferentially hydrolyzing a poly (N-formylethyleneimine) block or poly (methyloxazoline) block of a block copolymer to be constructed (hereinafter, the block copolymer is abbreviated as a precursor copolymer). Is obtained.

  The precursor copolymer was obtained by subjecting 2-oxazoline or 2-methyl-2-oxazoline to cation ring-opening living polymerization in the presence of the above-described polymerization initiating compound, and further to 2-ethyl-2- It can be obtained by polymerizing oxazoline. Moreover, it can also be set as a multiblock precursor copolymer by repeating polymerization of 2-oxazoline or 2-methyl-2-oxazoline and polymerization of 2-ethyl-2-oxazoline alternately.

  When the valence of the polymerization initiating compound used here is 1 or 2, a linear block copolymer is obtained, and when the valence is higher than that, a star-shaped block copolymer is obtained.

  As the solvent that can be used in the cationic ring-opening living polymerization, a known and commonly used aprotic inert solvent, aprotic polar solvent, or the like can be used.

  The target block copolymer can be obtained by preferentially hydrolyzing the poly (N-formylethyleneimine) block or poly (N-acetylethyleneimine) block in the obtained precursor copolymer. The hydrolysis reaction needs to be performed in an emulsion state.

  Poly (N-formylethyleneimine) block or poly (methyloxazoline) block and poly (ethyloxazoline) block are both water-soluble, but poly (N-formylethyleneimine) block and poly (methyloxazoline) Compared to the block, the poly (ethyloxazoline) block is more soluble in organic solvents, so it dissolves poly (ethyloxazoline) in the aqueous solution of the precursor copolymer but is incompatible with water. When the organic solvent is mixed and stirred, the (N-formylethyleneimine) block or poly (methyloxazoline) block dissolves in the aqueous phase, and the poly (ethyloxazoline) block dissolves in the organic solvent phase to form an emulsion. . In this case, the precursor copolymer acts as an emulsifier. The emulsion may be either O / W type or W / O type.

  As the organic solvent used for forming the emulsion, methylene chloride, chloroform, chlorobenzene, nitrobenzene, methoxybenzene, toluene, and a mixed solvent thereof can be used.

  After forming the emulsion, an acid or alkali is added to the aqueous phase as a hydrolysis catalyst to preferentially hydrolyze the poly (N-formylethyleneimine) block or the poly (N-acetylethyleneimine) block. Usual inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid can be used as the acid, and ordinary inorganic alkalis such as sodium hydroxide, potassium hydroxide and ammonia can be used as the alkali.

  For example, when an O / W type emulsion is used as a reaction field, the acid or alkali concentration in the aqueous phase constitutes a poly (N-formylethyleneimine) block or poly (methyloxazoline) block to be hydrolyzed. The amount may be at least equivalent to twice the number of moles of monomer units, and is preferably within 50 times. If it exceeds 50 times, the poly (ethyloxazoline) block is susceptible to hydrolysis. The most preferable range is 2 to 5 times.

  The hydrolysis reaction temperature is preferably 100 ° C. or lower, and may be set according to the concentration of the acid or alkali used. When the acid or alkali concentration is high, the temperature is set low, for example, about room temperature, and when the acid or alkali concentration is low, the reaction temperature is set high.

  In the hydrolysis reaction system of an O / W emulsion using acid, the O / W emulsion in the reaction system is converted into micelles in water as the reaction proceeds. The end point can be determined. For example, at the beginning of the reaction, a poly (N-formylethyleneimine) block or poly (methyloxazoline) block dissolves in an aqueous phase to form an O / W emulsion, whereas a poly (N-formylethyleneimine) block Or the hydrochloride of the poly (ethyleneimine) block produced by hydrolysis of the poly (methyloxazoline) block in the presence of hydrochloric acid becomes polymer crystals in the acidic aqueous phase. Since the polymer crystal is insoluble in the acidic aqueous phase, the emulsion is broken, and as a result, the polymer crystal becomes a core, and core-corona type micelles surrounded by a water-soluble poly (ethyloxazoline) block are formed. It is formed. The aqueous phase becomes an opaque micelle dispersion, and the organic solvent is separated from the aqueous phase as an oil phase, not as oil droplets. This phenomenon can be clearly observed visually and can be used as a measure of the end point of the reaction.

  In general, the hydrolysis reaction is preferably 2 to 48 hours, but may be appropriately selected depending on conditions such as the acid concentration and reaction temperature. By the hydrolysis reaction, the poly (N-formylethyleneimine) block or poly (methyloxazoline) block of the precursor copolymer is preferentially hydrolyzed to become a poly (ethyleneimine) block, and the target block copolymer is obtained. At this time, the poly (ethyloxazoline) block also undergoes some hydrolysis to ethyleneimine units.

  However, this hydrolysis reaction does not occur at random positions in the poly (ethyloxazoline) block, but a poly (N-propionylethyleneimine) block dissolved in an organic solvent incompatible with water, Occurs in the part drawn into the aqueous phase by stirring or thermal motion of the molecular chain. That is, as a result of hydrolysis of the (ethyloxazoline) unit in the portion adjacent to the poly (ethyleneimine) block, the target block is simply compared with the case where the poly (ethyloxazoline) block was not hydrolyzed. The molecular chain length of the poly (ethyleneimine) block in the polymer is somewhat longer and the molecular chain length of the poly (ethyloxazoline) block is only somewhat shorter.

  A block copolymer comprising a cationic poly (ethyleneimine) block and a poly (ethyloxazoline) block is obtained by protonating the nitrogen atom of ethyleneimine in the obtained block copolymer under acidic conditions. be able to.

  In the above synthesis method, a preferable blending example for forming an O / W emulsion is 5 to 40 ml of water and 0.5 to 6 ml of an organic solvent incompatible with water with respect to 1 g of the target block copolymer. is there.

[Anionic biological substances]
Any anionic biological substance may be used as long as it is ion-bonded with the above-described cationic poly (ethyleneimine) block and becomes water-insoluble. Examples of such a biological substance include DNA, protein, cell, or virus having an anionic group in its structure.

  As such an anionic biological substance, it is particularly preferable to use a substance related to a gene, and these are preferably substances such as nucleic acids, polynucleotides and oligonucleotides such as DNA or RNA.

  The nucleic acid may be a nucleic acid fragment or a part of a nucleic acid. The nucleic acid may be a single strand or double strand, linear strand or circular strand, DNA or RNA fragment such as natural or synthetic, but as a nucleic acid for gene therapy, genomic DNA, cDNA, mRNA, tRNA, antisense RNA, ribosomal RNA, ribozyme, or DNA encoding RNA is particularly preferred.

[Colloidal particles]
The colloidal particles of the present invention are prepared by stirring or vibrating an aqueous solution in which the block copolymer and the biological material are dissolved in water, or stirring or vibrating the aqueous solution of the block copolymer and the aqueous solution of the biological material. It can be easily obtained by mixing under vibration. In an aqueous solution, the cationic poly (ethyleneimine) block in the copolymer and the anionic group in the biological substance are ionically bonded, and the ion-bonded portion becomes water-insoluble. As the copolymer or biological material to be used, only a single type or a plurality of types having different structures may be used.

  The ratio of the block copolymer to the biological substance is such that the ratio of the total number of cationic groups in the copolymer to the total number of moles of the coloring compound anionic group is 10/1 to 1/4. If it exists, the colloidal particle containing the biological material of this invention can be adjusted suitably, and if it is the range of 2/1-1/2, it is more preferable.

  The resulting colloidal particles are such that the anion binding portion between the cationic poly (ethyleneimine) block in the copolymer and the biological material, for example, the phosphate anion binding portion when the biological material is a nucleic acid, becomes water-insoluble. Further, the portions bonded to the biological material grow into polyion aggregates of a certain size by hydrophobic bonds. The periphery of the polyion aggregate is covered with a hydrophilic portion composed of a nonionic poly (ethyloxazoline) block, and colloidal particles that are ion complex micelles can be easily formed.

  When the copolymer is a star-shaped block copolymer, a structure in which the star-shaped central portion is bonded to the end of a cationic poly (ethyleneimine) block, or the star-shaped central portion is nonionic. There can be a structure bonded to the poly (ethyloxazoline) block, but in any structure, the water-insoluble part in which the cationic (polyethyleneimine) block and the biological substance are ion-bonded is hydrophobically associated and grows into colloidal particles. can do.

  The particle size of the resulting colloidal particles can be easily controlled by adjusting the degree of polymerization of the copolymer used and the composition of the cationic poly (ethyleneimine) block in the copolymer. The particle size tends to increase as the value of the ionic hydrophilic polymer unit (a) increases. The degree of polymerization and the composition of the cationic poly (ethyleneimine) block need to be appropriately adjusted depending on the type of copolymer used, but the average particle size of the colloidal particles should be controlled in the range of about 20 nm to 5 μm. Can do.

  In the colloidal particles of the present invention, an outer layer made of a poly (ethyloxazoline) block is formed around an inner layer formed by ionic bonding between a cationic poly (ethyleneimine) block and an anionic biological substance. Poly (ethyloxazoline) layer even when injected into a living body because it has high physical stability and can prevent contact with external materials such as DNA incorporated inside. Protects biological substances such as DNA from degrading enzymes in the body and can safely transport genes to target cells.

Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples.
Unless otherwise specified, “%” represents “mass%”.

<Molecular weight measurement method>
It was measured using a high performance liquid chromatography “HLC-8000” (RI detector, TSKge12000 × 1 + 3000Hxl + 5000Hxl + guardcolumnHx1-H, solvent dimethylformamide, flow rate 1.0 ml / min, temperature 40 ° C.) manufactured by Tosoh Corporation.

<Measuring method of particle size and particle size distribution>
Using a UPA particle size analyzer “Microtrac 9203” manufactured by Nikkiso Co., Ltd. (laser beam wavelength 780 nm, reflection angle 180 ^° , temperature 25 ° C.), the particle size and particle size distribution by dynamic light scattering (DLS) method were measured. .

(Synthesis Example 1)
<Synthesis of Precursor of Star Block Copolymer (1-1)>
0.022 g of tetra (4-chloromethylphenyl) porphyrin, which is a tetrafunctional cationic ring-opening living polymerization initiator and serves as a star-shaped block precursor nucleus (1), in a reaction vessel substituted with argon gas inside. (0.027 mmol), 0.12 g of sodium iodide, and 6 ml of N, N-dimethylacetamide were added, and the mixture was stirred at room temperature for 3 hours. To this solution, 0.99 g (10 mmol) of 2-ethyl-2-oxazoline was added, heated to 100 ° C. and stirred for 24 hours.

After the reaction solution temperature was lowered to 60 ° C., 1.53 g (18 mmol) of 2-methyl-2-oxazoline was added, the temperature was raised to 100 ° C., and the mixture was stirred for 24 hours. The temperature of the reaction mixture was lowered to room temperature, 10 ml of methanol was added, and the reaction mixture was concentrated under reduced pressure. This concentrated solution was poured into 100 ml of diethyl ether to precipitate a polymer.
The methanol solution of the obtained polymer was poured into diethyl ether for reprecipitation, and after suction filtration, the filtrate was vacuum-dried to obtain 2.4 g of a star-shaped block precursor (1-1). The yield was 95%.

As a result of molecular weight measurement and ¹ H-NMR measurement, the mass average molecular weight was 25800, and the molecular weight distribution was 1.72.
^{From the 1} H-NMR (δ _TMS = 0, CDCl ₃ ) measurement, the side chain methyl group signal in the methyloxazoline block (CH ₃ : 1.97 ppm) and the side chain ethyl signal in the ethyl oxazoline block (CH ₃ : 1). .14 ppm, CH ₂ : 2.41 ppm) and main chain ethylene signals of both blocks (CH ₂ CH ₂ : 3.45 ppm) were confirmed. Moreover, the proton of the pyrrole ring of the porphyrin skeleton located in the center of the star polymer appeared at 8.82 ppm. ^{From the} integration ratio by ¹ H-NMR measurement, it was found that the molar composition ratio of the methyloxazoline block and the ethyloxazoline block was 71:29.

<Synthesis of star-shaped water-soluble block copolymer (1-2)>
After 0.5 g of the block precursor (1-1) described above was dissolved in 15 mL of chloroform, 0.8 mL of a 5 mol / L hydrochloric acid aqueous solution was added thereto. This mixed solution was stirred to obtain a W / O type emulsion. The emulsion was heated to 60 ° C. and stirred for 48 hours. After adding 50 ml of acetone to the reaction solution to precipitate a polymer, the solution was suction filtered and washed with acetone. The obtained polymer was dried to obtain 0.45 g of a water-soluble block copolymer (1-2) having a molecular chain composed of an ethyleneimine block and an ethyloxazoline block.

  The molar composition ratio of the ethyleneimine block and the ethyloxazoline block in the molecular chain was 79:21. Since this star-shaped water-soluble block copolymer has a porphyrin structure in the nucleus, the absorption spectrum of the aqueous solution of the block copolymer has a strong 420 nm (free base) derived from the Soret band inherent to porphyrin. Absorption was observed.

(Synthesis Example 2)
<Synthesis of comparative star-shaped polyethyleneimine>
0.022 g of tetra (4-chloromethylphenyl) porphyrin, which is a tetrafunctional cationic ring-opening living polymerization initiator and serves as a star-shaped block precursor nucleus (1), in a reaction vessel substituted with argon gas inside. (0.027 mmol), 0.12 g of sodium iodide, and 6 ml of N, N-dimethylacetamide were added, and the mixture was stirred at room temperature for 3 hours. To this solution, 1.53 g (18 mmol) of 2-methyl-2-oxazoline was added, heated to 100 ° C. and stirred for 24 hours. The temperature of the reaction mixture was lowered to room temperature, 10 ml of methanol was added, and the reaction mixture was concentrated under reduced pressure. This concentrated solution was poured into 50 ml of diethyl ether to precipitate a polymer. The obtained polymer was dissolved again in 5 mL of methanol, and then reprecipitated in 50 mL of diethyl ether. After suction filtration, the filtrate was vacuum dried to obtain 1.47 g of star-shaped polymethyloxazoline. The yield was 95%.
As a result of molecular weight measurement and ¹ H-NMR measurement, the mass average molecular weight was 19,800, and the molecular weight distribution was 1.5.
¹ H-NMR (δ _TMS = 0, CDCl ₃ ) measurement confirmed the side chain methyl group signal (CH ₃ : 1.97 ppm) and main chain ethylene signal (CH ₂ CH ₂ : 3.45 ppm) in the ethyloxazoline block. did.

0.5 g of the star-shaped poly (methyloxazoline) obtained above was dissolved in 4 ml of 5 mol / l hydrochloric acid aqueous solution. The solution was heated to 90 ° C. and stirred for 7 hours. After the reaction solution temperature was lowered to room temperature, 20 ml of acetone was added to completely precipitate the polymer. After suction filtration, it was washed several times with acetone, and the resulting polymer powder was dried to obtain 0.42 g of star-shaped poly (ethyleneimine). It was confirmed by ¹ H-NMR that the 1.97 ppm signal derived from the methyl group of methyloxazoline completely disappeared.

(Example 1)
<Polyionic association of star-shaped water-soluble block copolymer (1-2) and DNA>
0.015 g of the star-shaped water-soluble block copolymer (2-1) obtained in Synthesis Example 1 was dissolved in 1 ml of distilled water, and this solution was dissolved in 4 ml of an aqueous DNA solution extracted from salmon sperm (concentration: 2.). 2 mg / ml) and stirred for 1 hour. With respect to the dispersion, the formation of particles of a polyion aggregate of the star-shaped water-soluble block copolymer (2-1) and DNA was confirmed by a dynamic light scattering method. State nanoparticles were observed. Furthermore, 0.2 g of sodium chloride was added to this dispersion to adjust the salt concentration to 0.69 mol / l. When the particle size of the associated particles was confirmed again, the average center particle size was 172 nm. Nanoparticles containing DNA did not aggregate at all even in such a high concentration of salt, and did not change in particle size even after being left for half a year, and maintained extremely high stability. This high stability strongly suggests that the ethyloxazoline block of the star-shaped water-soluble block copolymer (1-2) formed a water-soluble corona layer surrounding the ion association core. It can be inferred that this neutral corona layer worked to weaken the electrostatic shielding effect of the salt.

< ³¹ P-NMR measurement of polyion aggregate>
The polyion aggregate of the star-shaped water-soluble block copolymer (1-2) and DNA incorporates a phosphate group of nucleic acid, and its NMR signal was measured. 19 mg of the star-shaped water-soluble block copolymer (1-2) and 9 mg of DNA extracted from salmon sperm were dissolved in 0.6 mL of heavy water to prepare a colloid solution of polyion aggregates. The signal of phosphorus atoms in this colloidal dispersion appeared at 0.35 ppm. This was significantly different from the appearance of a phosphorus atom signal at −160 ppm in the case of DNA alone. That is, it is suggested that the polyion aggregate of the water-soluble block copolymer (1-2) and DNA was formed by ionic bond between the cation of ethyleneimine and the anion of the phosphate group in DNA.

(Comparative Example 1)
<Polyion aggregate of star-shaped water-soluble poly (ethyleneimine) and DNA>
0.015 g of star-shaped poly (ethyleneimine) obtained in Synthesis Example 2 was dissolved in 1 ml of distilled water, and this solution was dropped into 4 ml of an aqueous DNA solution (concentration of 2.2 mg / ml) extracted from salmon sperm. And stirred for 1 hour. With respect to the dispersion, when the formation of particles of a polyion aggregate of star-shaped polyethyleneimine and DNA was confirmed by a dynamic light scattering method, the average particle diameter was 68 nm. When 0.2 g of sodium chloride was added to this dispersion to adjust the salt concentration to 0.69 mol / l, precipitation due to aggregation occurred in the system. This precipitate could not be dissolved by heating or sonication. This is in contrast to the result that the polyion aggregate in Example 1 maintains stability even in the presence of sodium chloride. The block copolymer composed of poly (ethyleneimine) and poly (ethyloxazoline) in the present invention is It is thought to suggest the effectiveness of DNA as a carrier.

Claims (10)

A block copolymer having a cationic poly (ethyleneimine) block, a poly (ethyloxazoline) block, and a nucleic acid , and the cationic poly (ethyleneimine) block and the nucleic acid in the block copolymer Is an ion-bonded polyion aggregate. The block copolymer is represented by the following general formula (1)
X- (AB) n or X- (BA) n (1)
(In the formula (1), X is a monovalent or higher polymerization initiation compound residue, A is a poly (ethyleneimine) block, B is a poly (ethyloxazoline) block, and n is at least 1 within the range of the valence of X. 2. The polyion aggregate according to claim 1, wherein the polyion aggregate is represented by an integer of from 1,000 to 100,000. The block copolymer is represented by the following general formula (2)
[X- (AB) n] mY, or [X- (BA) n] mY (2)
(In formula (2), X is a monovalent or higher polymerization initiation compound residue, Y is a monovalent or higher terminal compound residue, A is a poly (ethyleneimine) block, B is a poly (ethyloxazoline) block, and n is 2. An integer of at least 1 within the range of valence of X, m is an integer of at least 1 within the range of valence of Y), and the mass average molecular weight is in the range of 3000 to 100,000. The polyion aggregate according to 1. The ratio of the cationic poly (ethyleneimine) block to the poly (ethyloxazoline) block is 5/1 to 5 in terms of the molar ratio of structural units forming each polymer (ethyleneimine unit) / (ethyloxazoline unit). The polyion aggregate according to any one of claims 1 to 3, which is in a range of 1/5. The poly (ethyleneimine) block has the following formula (i)

(In the formula, p represents an integer of 10 to 50,000.)
The poly (ethylimazoline) block is a linear poly (ethyleneimine) represented by the following formula (ii):

(Wherein q represents an integer of 10 to 5000)
The polyion aggregate according to claim 1, which is a linear poly (ethyloxazoline) represented by the formula: The polyion aggregate according to any one of claims 2 to 5, wherein X is a 1 to 12-valent polymerization initiation compound residue. The polyion aggregate according to any one of claims 3 to 5, wherein Y is a 1 to 12-valent terminal compound residue. The polyion aggregate according to any one of claims 2 to 7, wherein X is a residue of a polymerization initiation compound having any one of a benzene skeleton, a porphyrin skeleton, a phthalocyanine skeleton, and a pyrene skeleton. The polyion aggregate according to any one of claims 1 to 8 , which is in a colloidal particle form. A biological material carrier comprising the polyion aggregate according to any one of claims 1 to 9 .