KR20170085784A - Nanoparticle sensitive to bioenvironment comprising poly(amino acid) for treatment of virus infection - Google Patents

Abstract

The present invention relates to a polyamino acid-based bio-environment sensitive nanoparticle for the treatment of a viral infection, and more particularly, to a bio-environment sensitive nanoparticle which recognizes a virus-infected cell by using a polyamino acid- And maintains the homeostasis of cells by regulating active oxygen, thereby providing an effect of treating a viral infection.

Description

[0001] The present invention relates to a polyamino acid-based bio-environment-responsive nanoparticle for the treatment of viral infection,

The present invention relates to a bio-environment-responsive nanoparticle based on polyamino acid for the treatment of a viral infection.

Reactive oxygen species (ROS), the superoxide ion (O ₂ ^{· -)} and the like, hydroxy radical (OH ^·). ROS occurs naturally in living organisms, and it is essential for the biological system by destroying bacteria, participating in enzyme activation, drug detoxification, and the like. However, more than necessary ROS causes inflammation and changes the functions of cells by modifying proteins and DNA.

Cells have genes that produce ROS to regulate homeostasis of ROS, as well as genes that constantly remove them, and they have sophisticated mechanisms to control them. However, virus-infected cells do not maintain homeostasis of ROS, resulting in disproportionate ROS damaging intracellular DNA, causing abnormalities in gene expression, altering intracellular proteins and causing cell proliferation abnormalities. In particular, high concentrations of H ₂ O ₂ generate OH ^{· ·} ROS, which is catalyzed by interfacial Fe ^{2 +} , which activates genes that promote oxidation-reduction-sensitive transcription factor activity and leads to DNA damage.

 Mohammad Latif Reshi et al., International Journal of Cell Biology, vol.2014 (Apr. 20, 2014)

It is an object of the present invention to provide a pharmaceutical use of polyamino acid-based nanoparticles for imaging that can reduce in vivo toxicity and respond to biological environment to treat viral infection.

In order to achieve the above object,

A core, a plurality of conductive polymer layers surrounding the core, and a shell surrounding the conductive polymer layer, the shell comprising a copolymer of a hydrophilic polymer and a polyamino acid; And

A composition for treating a viral infection, comprising a pharmaceutically acceptable carrier.

The nanoparticles

Preparing a nanoparticle in which two or more conductive monomers are supported by mixing a copolymer of a hydrophilic polymer and a polyamino acid and two or more different conductive monomers; And

And inducing polymerization of the two or more different conductive monomers to form a plurality of conductive polymer layers in the nanoparticles.

The present invention provides an effect of treating virus infection by recognizing virus-infected cells and controlling active oxygen using polyamino acid-based nanoparticles that specifically respond to a living environment, thereby maintaining homeostasis of cells.

1 shows the structure of the nanoparticles of the present invention and a method for producing the same.
2 shows the NMR, FT-IR and TEM photographs of the nanoparticles of the present invention.
Figure 3 is a TEM and SEM photograph showing the morphology of the nanoparticles (PASome), the polyaniline nanoparticles (Pani) and the polypyrrole nanoparticles (Ppy) of the present invention, Figures (a) and (d) e) Pani, (c) and (f) are images of PASome.
FIG. 4 shows the results of measuring changes in ROS levels in virus-infected cells.
FIG. 5 shows the ROS inhibitory effect of nanoparticles in virus-infected cells, and the lower graph shows the ROS inhibitory effect obtained by treating diluted nanoparticles.
FIG. 6 shows the effect of inhibiting the cell death of nanoparticles in virus-infected cells, and the lower graph shows the cell death inhibition effect obtained by treating the diluted nanoparticles.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention

A core, a plurality of conductive polymer layers surrounding the core, and a shell surrounding the conductive polymer layer, the shell comprising a copolymer of a hydrophilic polymer and a polyamino acid; And

To a composition for treating a viral infection, comprising a pharmaceutically acceptable carrier.

The nanoparticle of the present invention is a polyamino acid-based nano platform that specifically responds to a living environment. The nanoparticle of the present invention recognizes a biological environment that is specifically generated in a virus-infected cell, regulates active oxygen species, Thereby treating a viral infection. More specifically, the nanoparticles of the present invention have a structure including a conductive polymer layer inside a self-assembly of an amphipathic polymer. The conductive polymer forming the conductive polymer layer has a function of transferring and transferring electrons from inside and has a function of absorbing and releasing electrons. The nanoparticles of the present invention can convert electrons to reactive oxygen species by oxidation-reduction reaction, thereby converting them into nontoxic substances. It is known that ROS is involved in the process of viral proliferation. Thus, nanoparticles can control viral infection by blocking the viral growth phase by controlling the level of ROS.

Therefore, the biological environment may include intracellular environmental factors related to apoptosis such as ROS.

The nanoparticles of the present invention have a structure in which a self-assembly of an amphipathic polymer, which is a biocompatible hydrophilic polymer and a polyamino acid copolymer, contains a conductive polymer layer therein.

The nanoparticles

Preparing a nanoparticle in which two or more conductive monomers are supported by mixing a copolymer of a hydrophilic polymer and a polyamino acid and two or more different conductive monomers; And

And inducing polymerization of the two or more different conductive monomers to form a plurality of conductive polymer layers in the nanoparticles.

The method for producing nanoparticles of the present invention will be described in detail with reference to Fig.

First, a biocompatible amphipathic block copolymer that forms a shell of nanoparticles can be prepared by chemical bonding of a hydrophilic polymer and a polyamino acid.

Wherein the hydrophilic polymer is selected from the group consisting of polyalkylene glycols, polyethylene oxides, polyoxazolines, poly (N-vinylpyrrolidone), polyvinyl alcohol, polyhydroxyethyl methacrylate, dextran, polyserin, polythreonine, , Polylysine, polyarginine, polyhistidine, polyaspartic acid or polyglutamic acid, etc. may be used alone or in combination of two or more. Preferably, a polyalkylene glycol having a molecular weight of 1000 to 5000 or a derivative thereof can be used. More preferably, methoxyaminopolyethylene glycol having a molecular weight of 1000 to 5000 can be used.

The hydrophilic polymer may be suitably modified for bonding with polyamino acid using a known technique. According to one embodiment, after the deformation process of the mPEG-OH → mPEG-TsCl → mPEG-N 3 → mPEG-NH 2 may be used in the form of mPEG-NH _2.

The polyamino acid may be a homopolyamino acid represented by the following formula (1): < EMI ID =

[Chemical Formula 1]

(poly-M) n

here,

M is leucine, isoleucine, valine, phenylalanine, proline, glycine or methionine,

and n represents 10 to 100.

According to an embodiment of the present invention, an amphiphilic block copolymer (A), mPEG-b-poly phenylalanine (mPEG-b-pPhe) can be synthesized through peptide bonding between the modified hydrophilic polymer and hydrophobic polyphenylalanine .

Next, in the nanoparticle of the present invention, a plurality of conductive polymer layers form an intermediate layer in a shell, and the intermediate layer surrounds a hollow core.

The conductive polymer layer may be formed of a material selected from the group consisting of polyacetylene, polyaniline, polypyrrole, polythiophene, poly (1,4-phenylenevinylene) At least one conductive polymer selected from the group consisting of poly (1,4-phenylene sulfide), poly (fluorenyleneethynylene), poly (fluorenyleneethynylene) And the like. More preferably, it may be a double layer of a polypyrrole layer and a polyaniline layer.

When the hydrophilic polymer and the polyamino acid copolymer, the pyrrole monomer, and the aniline monomer are mixed and stirred, the nanoparticles of the present invention undergo self-assembly of the copolymer, and the above-mentioned self assembly of the nanoparticles of the present invention is a state in which the pyrrole monomer and the aniline monomer are supported .

When an oxidizing agent is added to the above magnetic assembly, the polymerization of the pyrrole monomer and the aniline monomer is induced to form a polypyrrole layer and a polyaniline layer.

Therefore, the copolymer of the hydrophilic polymer and the polyamino acid forms a shell, and a polyaniline layer and a polypyrrole layer are sequentially formed in the shell, and the core inside the polypyrrole layer has a hollow structure.

The pyrrole monomer may be contained in an amount of 282 to 330 parts by weight based on 1 part by weight of the copolymer of the hydrophilic polymer and the polyamino acid.

The aniline monomer may be contained in an amount of 282 to 330 parts by weight based on 1 part by weight of the copolymer of the hydrophilic polymer and the polyamino acid.

The mixing ratio of the pyrrole monomer and the aniline monomer may be 1: 1 molar ratio.

The self-assembly of a copolymer of a hydrophilic polymer and a polyamino acid may be performed by dispersing or dissolving a mixture of a hydrophilic polymer and a polyamino acid copolymer, a pyrrole monomer and an aniline monomer in a solvent, applying ultrasonic waves, or dispersing or dissolving the mixture in an organic solvent, The organic solvent may be prepared by extraction or evaporation, or dispersion or dissolution in an organic solvent followed by dialysis with excess water.

The organic solvent may be chloroform, hexane, heptane, methylene chloride, benzene, toluene, tetrahydrofuran, acetone or a mixture thereof, but is not particularly limited thereto.

The polymerization of the pyrrole monomer and the aniline monomer carried in the self-assembly of the copolymer of the hydrophilic polymer and the polyamino acid can be induced through oxidation-reduction of the pyrrole monomer and the aniline monomer through the introduction of an oxidizing agent.

As the oxidizing agent, persulfates such as ammonium persulfate, hydroperoxide and iron (divalent) ions may be used, but there is no particular limitation thereto.

The nanoparticles prepared through the above process may have an average particle diameter of 200 nm or less. Preferably 50 to 200 nm.

In addition, the nanoparticles of the present invention may further comprise a pharmaceutically active ingredient for the treatment of a viral infection. The pharmaceutically active ingredient may be carried on the core or physicochemically encapsulated or bound to the intermediate layer or shell.

The pharmaceutically active ingredient may be selected from the group consisting of anticancer agents, antibiotics, hormones, hormone antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, Alkylphosphocholine, radioisotope labeled compounds, cardiovascular drugs, gastrointestinal drugs, or nervous system drugs may be used alone or in combination of two or more.

Examples of viruses that can be treated with the nanoparticles of the present invention include, but are not limited to, influenza A virus H1N1, canine influenza virus H3N2, avian influenza virus H9N2, and the like.

Examples of diseases caused by the virus include H1N1 virus, avian flu, influenza virus, and the like.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline Can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the composition of the present invention may be in the form of a sterile injectable preparation of a sterile injectable aqueous or oily suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (for example, a solution in 1,3-butanediol). Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil including synthetic mono or diglycerides and less irritant may be used.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 > Preparation of amphiphilic nanoparticles

FIG. 1 shows the structure of a nanoparticle and its production process. In order to prepare a block copolymer of a hydrophilic polymer and a polyamino acid, methoxyaminopolyethylene glycol (mPEG-NH ₂ , 0.2 mmol) and phenylalanine-NCA were used. For this, phenylalanine-NCA was formed using phenylalanine and triphosgene.

The amphiphilic block copolymer prepared above was mixed with an equivalent molar ratio of pyrrole monomer and aniline monomer and stirred to prepare a self assembly of the copolymer carrying the pyrrole monomer and the aniline monomer. Addition of an oxidizing agent (ammonium persulfate) thereto induced polymerization of pyrrole monomer and aniline monomer to produce nanoparticles containing a polypyrrole layer and a polyaniline layer.

( ¹ H-NMR)

The ¹ H-NMR of the polymer prepared in Example 1 was measured using CDCl ₃ .

(FT-IR)

The FT-IR spectrum of the polymer prepared in Example 1 was measured using a PerkinElmer infrared spectrometer.

FIG. 2 shows the ¹ H-NMR and FT-IR results of the nanoparticles prepared in Example 1. The mPEG fraction of the amphiphilic block copolymer was confirmed by ¹ H-NMR. In addition, presence and copolymerization of methoxypolyethylene glycol and phenylalanine in the copolymer were confirmed through FT-IR.

Comparative Example 1 Production of polyaniline nanoparticles and polypyrrole nanoparticles

Except that the polyaniline nanoparticles or the polypyrrole nanoparticles in which the conductive polymer layer was formed into a single layer by using the amphiphilic block copolymer and the aniline monomer or the pyrrole monomer of Example 1 were prepared, Were used.

3 is a TEM and SEM photograph of the nanoparticles (PASome), polyaniline nanoparticles (Pani) and polypyrrole nanoparticles (Ppy) prepared in Example 1, PASome (c, f) and Ppy Pani (b, e) is a cylindrical shape with a diameter of 1 μm and a thin thickness. PASome is separated by one compared with Ppy and has good dispersion to water.

<Example 2> Measurement of ROS level change in virus-infected cells

(ROS activator, L-Buthionine sulphoximine) after infecting the kidney epithelial cells, MDCK (Madin-Darby Canine Kidney Epithelial) cells, uninfected cells, and viruses infected with H1N1, H3N2 and H9N2 Changes in ROS levels of cells treated with NAC (ROS inhibitor, N-acetyl cysteine) were measured over time.

As shown in Fig. 4, virus-infected cells had increased intracellular ROS. When BSO was treated, the ROS level increased with increasing BSO concentration, and when NAC was treated, the ROS level decreased with increasing NAC concentration. Cells not infected with the virus did not change at the ROS level during the treatment period.

<Example 3> ROS inhibitory effect of nanoparticles in virus-infected cells

(PASome), polyaniline nanoparticles (Pani) and polypyrrole nanoparticles (Ppy) prepared in Example 1 were tested in order to examine the effect of ROS inhibition of nanoparticles in cells infected with a low-pathogenic influenza virus, H1N1, H3N2 and H9N2 The virus-infected cells were treated with 1 mmol / mL and the ROS level was measured with a ROS kit (Thermo Fisher Scientific) over time. Next, the particles were diluted to 1/2 and treated with virus-infected cells to measure ROS levels after 72 hours.

As shown in FIG. 5, when the cells infected with the three viruses were treated with three particles, the ROS level was lowered, and it was confirmed that ROS was inhibited in all three viruses (upper graph in FIG. 5).

When the particles were diluted and treated to infected cells, PASome showed the lowest ROS inhibitory effect among the three particles (lower graph in FIG. 5).

<Example 4> Effect of inhibiting the cell death of nanoparticles in virus-infected cells

The cell death inhibition effect of 1 mmol / mL of nanoparticles in cells infected with H1N1, H3N2 and H9N2 was confirmed by MTT assay.

The negative control group is a virus-free cell, and the positive control group is a virus-infected cell.

In addition, the survival rate of the cells was measured by treating the infected cells by diluting the particle concentration to 1/2.

As shown in FIG. 6, when the cells were treated with the H9N2, H3N2, and H1N1 virus-infected cells, the survival rate was high (top image in FIG. 6) (Lower graph of Fig. 6).

Claims (11)

A core, a plurality of conductive polymer layers surrounding the core, and a shell surrounding the conductive polymer layer, the shell comprising a copolymer of a hydrophilic polymer and a polyamino acid; And
A composition for treating a viral infection, comprising a pharmaceutically acceptable carrier.
The method according to claim 1,
The conductive polymer layer may be formed of at least one selected from the group consisting of polyacetylene, polyaniline, polypyrrole, polythiophene, poly (1,4-phenylenevinylene) At least one conductive polymer selected from the group consisting of poly (1,4-phenylene sulfide), poly (fluorenyleneethynylene), and derivatives thereof, Wherein the composition is a plurality of layers.
The method according to claim 1,
Wherein the conductive polymer layer comprises a polypyrrole layer and a polyaniline layer.
The method according to claim 1,
The hydrophilic polymer may be selected from the group consisting of polyalkylene glycols, polyethylene oxides, polyoxazolines, poly (N-vinylpyrrolidone), polyvinyl alcohol, polyhydroxyethyl methacrylate, dextran, polyserine, polythreonine, Wherein the composition is at least one selected from the group consisting of polylysine, polyarginine, polyhistidine, polyaspartic acid, and polyglutamic acid.
The method according to claim 1,
Wherein the hydrophilic polymer comprises methoxyaminopolyethylene glycol.
The method according to claim 1,
Wherein the polyamino acid is represented by the following formula (1):
[Chemical Formula 1]
(poly-M) n
here,
M is leucine, isoleucine, valine, phenylalanine, proline, glycine or methionine,
and n represents 10 to 100.
The method of claim 1, wherein the nanoparticles comprise
Preparing a nanoparticle in which two or more conductive monomers are supported by mixing a copolymer of a hydrophilic polymer and a polyamino acid and two or more different conductive monomers; And
Wherein the nanoparticles are prepared by inducing polymerization of two or more different conductive monomers to form a plurality of conductive polymer layers inside the nanoparticles.
The method according to claim 1,
Wherein the nanoparticles have an average particle size of 50 to 200 nm.
The method according to claim 1,
Wherein the nanoparticles are reactive oxygen species sensitive.
The method according to claim 1,
Wherein the virus is any one of swine influenza A type H1N1, canine influenza virus H3N2 or avian influenza virus H9N2.
The method according to claim 1,
Wherein the nanoparticles further comprise a pharmaceutically active ingredient.

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