JP2007089440A - Introduction of substance into cell with nanoparticle presenting cell membrane-permeable peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To further enhance the efficiency of the introduction of an objective substance into a target cell or a target tissue. <P>SOLUTION: A nanoparticle having at least one selected from the group consisting of proteins, lipids and organic polymers as a constituent and capable of encapsulating an introduced substance, wherein a cell-permeable peptide is covalently bound to at least one of the constituents, and the cell-permeable peptide is presented on the surface of the nanoparticle. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to nanoparticles presenting cell membrane penetrating peptides.

  In the past, the present inventors have introduced a biorecognition molecule into particles (HBsAg particles) composed of hepatitis B virus surface antigen protein, and transport the introduced substance specifically and safely to the target location. And found to be effective as a DDS carrier for introduction (Patent Documents 1 to 3).

  Liposomes are widely known as another means for introducing substances into cells.

On the other hand, particles composed of proteins or lipids, such as HBsAg particles and liposomes, are foreign to the living body and have problems such as antigenicity, and their dosage should be as small as possible. There is a need to further increase the efficiency of introduction into the market.
WO01 / 64930 WO03 / 082330 WO03 / 082344

  An object of the present invention is to further increase the efficiency of introduction of a target substance into a target cell or target tissue.

As a result of repeated studies in view of the above problems, the present inventor introduced a specific cell-introducing peptide into nanoparticles such as virus nanoparticles and liposomes and presented them on the particle surface, thereby introducing substances into cells or tissues. It has been found that the efficiency can be significantly increased.
The present invention relates to the following hepatitis B virus nanoparticles.
1. A nanoparticle capable of encapsulating a substance to be introduced, comprising at least one selected from the group consisting of proteins, lipids and organic polymers as a constituent, wherein a cell-permeable peptide is covalently bound to at least one of the constituents; A nanoparticle obtained by presenting a cell-permeable peptide on the surface of the nanoparticle.
2. Item 2. The nanoparticle according to Item 1, wherein the nanoparticle is a virus particle containing a virus protein or a liposome containing a phospholipid.
3. Item 2. The nanoparticle according to Item 1, wherein the component comprises a hepatitis B virus protein.
4). Item 4. The nanoparticle according to any one of Items 1 to 3, wherein the cell-penetrating peptide has an amino acid sequence of PLSSIFSRIGDP.
5. Item 5. The nanoparticle according to any one of Items 1 to 4, which has an introduction substance in the particle.
6). A variant of hepatitis B virus surface antigen protein, which is a hepatitis B virus surface antigen protein in which part or all of the hepatocyte binding region is deleted and has a cell-penetrating peptide.
7). Item 7. The variant according to Item 6, which has the sequence represented by SEQ ID NO: 4.

  According to the present invention, the efficiency of introducing a substance into cells / tissues can be greatly increased, the dosage of nanoparticles for substance introduction can be reduced, and macromolecules such as proteins and genes can be easily Can be introduced into the organization.

  In this specification, as the nanoparticles encapsulating the introduction substance, virus particles containing virus-derived proteins such as virus protein particles, liposomes mainly composed of lipids such as phospholipids, or organic polymers are used. Nano carrier as element (Kakizawa Y, Kataoka K. Block copolymer micelles for delivery of gene and related compounds. Adv Drug Deliv Rev. 2002 Feb 21; 54 (2): 203-22. Review; Otsuka H, Nagasaki Y, Kataoka K. PEGylated nanoparticles for biological and pharmaceutical applications. Adv Drug Deliv Rev. 2003 Feb 24; 55 (3): 403-19. Review; Nishiyama N, Kataoka K. Polymeric micelle drug carrier systems: PEG-PAsp (Dox) and second generation of micellar drugs. Adv Exp Med Biol. 2003; 519: 155-77. Review.).

  The virus particles are not particularly limited as long as they are nano-sized particles that can encapsulate the introduced substance. For example, nanoparticles containing virus proteins such as hepatitis B virus, Sendai virus, etc. Nanoparticles containing the protein of are particularly preferred. Examples of hepatitis B virus protein include surface antigen protein (HBsAg) and core protein, and HBsAg is preferably exemplified. HBsAg may be a natural protein (SEQ ID NO: 5; serotype y type)), and the N-terminal preS1 region (108 amino acids) and part of the preS2 region (55 amino acids) are deleted. Also good. In the serotype y type, a region spanning the preS1 region and the preS2 region, for example, 21-153, 33-153, 50-153 can be deleted. The average particle size of the virus particles is about 50 to 600 nm, preferably about 100 to 500 nm, more preferably about 100 to 400 nm.

Various serotypes of HBV are known, but since they have high amino acid homology, the corresponding site of HBV of each serotype can be easily identified. SEQ ID NO: 5 in the present specification shows the entire amino acid sequence of HBsAg from which 11 amino acids on the N-terminal side of the adr type are deleted, and corresponds to the ayw type HBsAg. In this specification, it describes using the amino acid number of HBsAg (corresponding to ayw type) corresponding to SEQ ID NO: 5, but in the case of HBsAg of other serotypes or subspecies, the corresponding amino acid position corresponds. The position can be easily recognized by those skilled in the art.
The liposome may be either a multilamellar liposome or a single membrane liposome. The average particle size of the liposome is about 50 to 500 nm, preferably about 80 to 400 nm, and more preferably about 100 to 200 nm. Liposomes can be produced by ultrasonic treatment, reverse phase evaporation, freeze-thaw, lipid dissolution, spray drying, and the like. Examples of liposome components include phospholipids, cholesterols, fatty acids, and the like. Specifically, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean In addition to natural phospholipids such as lecithin and lysolecithin, or those hydrogenated by conventional methods, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, eleostearoyl phosphatidylcholine, eleostearoyl phosphatidylethanol Amine, eleostearoyl phosphatidylserine Like synthetic phospholipids of.

  Examples of the nanoparticles having an organic polymer as a constituent element include the aforementioned nanocarriers.

  Examples of nanocarriers include polyethylene glycol and lysine, amino acids such as aspartic acid and glutamic acid, and block copolymers of polymers such as lactic acid, glycolic acid, and ε-caprolactone. For example, a block copolymer of polyethylene glycol and poly-L-lysine. Polymer {PEG-PLL}, alpha-acetal-poly- (ethylene glycol) -block-poly (D, L-lactide) {PEG-PLA}, block copolymer of polyethylene glycol and poly-L-glutamic acid, polyethylene Examples thereof include a block copolymer of glycol and poly-L-aspartic acid.

  Introduction of the cell penetrating peptide presented on the nanoparticle surface can be performed as follows.

  When the nanoparticle includes a protein as a constituent element, a cell-penetrating peptide may be linked to the inside of the protein or to the N-terminus or C-terminus. The cell penetrating peptide can preferably bind to the main chain, but can also be linked to the side chain of an amino acid. In addition, since the cell-penetrating peptide needs to be presented on the surface of the nanoparticle, it is connected to the outer surface of the nanoparticle rather than the inner surface of the particle or the inner surface of the hollow nanoparticle. Is preferred. For example, in the case of HBsAg, the a antigen determination site (a epitope) in the middle of Pre-S1 (108 amino acid residues), Pre-S2 (55 amino acid residues), and S region (226 amino acid residues) is outside the particle. It is possible to insert a cell-penetrating peptide into the exposed area and present it outside the nanoparticles. Specific examples of the site exposed to the outside of the S region include the 105th to 155th amino acid residues of the S region.

  When the nanoparticles are composed of lipids (particularly phospholipids), for example, phospholipids such as phosphatidylserine and phosphatidylethanolamine may be linked to cell-permeable peptides, and cell-permeable to other lipids such as cholesterol. Peptides may be linked to present the cell permeable peptide on the liposome surface.

When the nanoparticle is composed of an organic polymer (for example, a polymer micelle such as a nanocarrier), the cell-permeable peptide is an amino acid (lysine, glutamic acid, aspartic acid, etc.) or hydroxycarboxylic acid (lactic acid) present in the organic polymer. , Glycolic acid, and the like) and functional groups such as COOH, NH ₂ and OH can be linked via an amide bond or an ester bond.

The cell penetrating peptide (TLM) is not particularly limited as long as it is presented on the surface without destroying the structure of the nanoparticles, and examples thereof include the following:
1) Cell penetrating peptides (TLM) that can be designed as HBV-derived or HBV variants:
PLSSIFSRIGDP;
PISSIFSRTGDP;
AISSILSKTGDP;
PILSIFSKIGDL;
PLSSIFSKIGDP;
PLSSIFSHIGDP; and
PLSSIFSSIGDP.
2) Cell penetrating peptides derived from proteins:
RKKRRQRRR (Tat (49-57));
RQIKIWFQNRRMKWKK (Penetrarin (43-58));
DAATATRGRSAASRPTERPRAPARSASRPRRPVD (VP22);
AAVALLPAVLLALLAP, AAVLLPVLLAAP (Kaposi FGF signal sequences);
VTVLALGALAGVGVG (Human beta3 integrin signal sequence);
GALFLGWLGAAGSTMGA (gp41 fusion sequence);
MGLGLHLLVLAAALQGA (Caiman crocodylus Ig (v) light chain); and
LGTYTQDFNKFHTFPQTAIGVGAP (hCT derived peptide).
3) Synthetic / chimeric cell penetrating peptide
GWTLNSAGYLLKINLKALAALAKKIL (Transportan);
(TPPKKKRKVEDPKKKKK) 8- (Loligomer);
RRRRRRR (Arginine peptide); and
KLALKLALKALKAALKLA (Amphiphilic model peptide).

  Examples of virus hollow nanoparticles containing hepatitis B virus protein or a modified form thereof include HBsAg protein particles. HBsAg protein may be combined with hepatitis B virus internal core antigen protein to form particles.

  The virus nanoparticles for encapsulating the introduced substance in the present invention include a hepatitis B virus protein or a variant thereof as a main component, and the protein may have a sugar chain. The nanoparticles may contain a lipid component.

  In one preferred embodiment of the present invention, the virus nanoparticles are composed of 75 to 85 parts by weight of hepatitis B virus protein variant, 5 to 15 parts by weight lipid, and 5 to 15 parts by weight sugar chain. The virus hollow nanoparticles used in the examples of the present application consist of 80 parts by weight of hepatitis B virus protein variant, 10 parts by weight of sugar chain, and 10 parts by weight of lipid (J Biotechnol. 1992 Nov; 26 (2- 3): 155-62. Characterization of two differently glycosylated molecular species of yeast-derived hepatitis B vaccine carrying the pre-S2 region. Kobayashi M, Asano T, Ohfune K, Kato K.).

  The variant of the present invention may be further introduced with various mutations as long as it has the ability to form virus nanoparticles in addition to the deletion of the preS region and the introduction of a cell-penetrating peptide. For example, one or several or a plurality of hepatitis B virus proteins, for example 1 to 50, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly 1 to 3 The amino acids may be substituted, added, deleted or inserted. As a method for introducing mutations such as substitution, addition, deletion, insertion and the like, in the DNA encoding the protein, for example, cytospecific mutagenesis (Methods in Enzymology, 154, 350, 367-382 (1987); , 468 (1983); Nucleic Acids Res., 12, 9441 (1984)), chemical synthesis means such as the phosphate triester method and phosphate amidite method (for example, using a DNA synthesizer) ( J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981)). Codon selection can be determined by taking into account the codon usage of the host.

  Encapsulation of the substance in the virus hollow particle may be performed by an electroporation method. By encapsulating the introduced substance in the liposome and inducing the liposome and hepatitis B virus nanoparticle, The introduction substance may be included. Fusion of virus nanoparticles and liposomes can be easily performed by mixing liposomes and virus particles in water or an aqueous medium, and stirring or shaking as necessary.

  Hereinafter, as a nanoparticle of the present invention, an HBsAg variant (a variant containing a cell-penetrating peptide and lacking part or all of the preS1 site involved in hepatocyte specificity as described in SEQ ID NO: 4) is used. The description will be made mainly using virus particles as the main component, but other virus particles, liposomes, nanocarriers, and the like can be similarly applied.

  In addition to improving the efficiency of substance introduction, the substance can be introduced specifically into target cells or tissues by introducing molecules that recognize specific cells into the components of particles such as HBsAg variants. You can also. Examples of such molecules that recognize specific cells include cell function regulatory molecules such as growth factors and cytokines, cell surface antigens, tissue-specific antigens, molecules such as receptors for identifying cells and tissues, viruses and microorganisms. Molecules derived from, antibodies, sugar chains, lipids and the like are preferably used. Specifically, antibodies against EGF receptor and IL-2 receptor that appear specifically in cancer cells, EGF, and receptors presented by HBV are also included. Alternatively, a protein capable of binding an antibody Fc domain (for example, a ZZ tag), a strept tag showing biotin-like activity to present a biorecognition molecule labeled with biotin via streptavidin, and the like can also be used. These are appropriately selected according to the target cell or tissue. A cell recognition molecule can be introduced into a component of a nanoparticle such as a modified HBsAg according to a known method.

  When the HBsAg variant is expressed in eukaryotic cells, the protein is preferably expressed and accumulated as a membrane protein on the endoplasmic reticulum membrane and released as nanoparticles. As eukaryotic cells, animal cells such as mammals, yeast and the like can be applied. Since such particles do not contain any HBV genome, they are extremely safe for the human body. Moreover, the cell selectivity of the nanoparticles of the present invention for hepatocytes or other cells can be enhanced by introducing a cell recognition molecule into at least a part of the protein constituting the particles as necessary.

  The nanoparticle of the present invention encapsulating an introduction substance can be preferably used to introduce a substance that expresses a function in a cell / tissue but is difficult to move into the cell / tissue. For example, when the nanoparticles of the present invention are administered into the body by intravenous injection or the like, the particles circulate in the body and can efficiently introduce substances into various cells. Moreover, even if the substance has a short half-life in vivo, the substance is protected in the nanoparticle of the present invention until it is introduced into the cell / tissue, and therefore can act effectively.

  The nanoparticles of the present invention can also be preferably used as a cell introduction reagent by mixing with target cells in vitro.

  There are no particular limitations on the substance to be introduced, for example, various drugs that are introduced into cells to produce physiological effects, such as physiologically active proteins such as hormones, lymphokines, and enzymes; antigenic proteins that act as vaccines; genes that are expressed in cells And genes such as plasmids, or genes involved in specific gene expression that induces or induces expression; various genes and antisense introduced for gene therapy. The “gene” to be introduced includes not only DNA but also RNA. The introduced substance is preferably exemplified by a high molecular weight physiologically active substance such as a protein or a gene, but preferable results can be obtained even when applied to various low molecular weight drugs. In addition, genes and proteins may be natural or synthesized, and may be modified genes and proteins.

  Hereinafter, embodiments will be described with reference to the accompanying drawings, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.

  In the following examples, HBsAg refers to hepatitis B virus surface antigen. When expressed in eukaryotic cells, HBsAg is expressed and accumulated as a membrane protein on the endoplasmic reticulum membrane. After that, aggregation occurs between molecules, and it is released as HBsAg particles to the lumen side in a budding manner while taking up the endoplasmic reticulum membrane.

The HBsAg particles are obtained after expression using eukaryotic cells such as yeast cells, insect cells, and mammalian cells and purification thereof (Patent Documents 1 to 3).
Example 1
Plasmid construction
TLM was inserted into three types of deletion mutagenesis proteins (21-153, Δ33-153, Δ50-153) constructed by genetic engineering techniques based on the hepatitis B virus L protein (SEQ ID NO: 5). Plasmids for protein expression were constructed.
1. An oligo DNA encoding TLM was synthesized.

PreS2-TLM (F) (SEQ ID NO: 1)
5'- ggg ggc ggc cgc ccc tta tcg tca atc ttc tcg agg att ggg gac cct ggc ggc cgc ggg -3 '
PreS2-TLM (R) (SEQ ID NO: 2)
5'- ccc gcg gcc gcc agg gtc ccc aat cct cga gaa gat tga cga taa ggg gcg gcc gcc ccc -3 '

2. Two types of oligo DNA (100 μM) were annealed at 10 μl each under the following conditions.

3. The vector pGLDLII-P39-RcT (Δ21-153, Δ33-153, Δ50-153) and the insert were digested with NotI and ligated.

Production by yeast The gene constructed was transformed into the yeast Saccharomyces cerevisiae AH22R ⁻ strain by the spheroplast method, and a high-producing strain was screened. The obtained high-producing strain was cultured in large quantities with an industrial medium, and the cells were collected. The yeast was crushed with glass beads, the cell extract was collected, and the particles were purified by performing cesium chloride density gradient ultracentrifugation twice and sucrose density gradient ultracentrifugation once.

It was treated by PNGaseF to examine the analysis <br/> sugar processing glycosylation particles.

We confirmed the presence of TLM on the particle surface by digesting the domain existing on the surface of trypsin-treated particles with trypsin and examining changes in the molecular weight of the protein.
Example 2
Calcein introduction experiment The purified particles were used for particle analysis and introduction experiment into various cells. In the introduction experiment, the fluorescent substance calcein was used as a drug model. First, TLM-presenting particles (about 30 μg), calcein (final concentration 0.5 mM), and Buffer B (200 μl) were mixed and made up to 500 μl with DW. This was added to a 4 mm cuvette, and calcein was encapsulated in the particles by the electroporation method (160 V, 950 μF). After adding to various cells, about 8 hours later, observation with a fluorescence microscope and analysis by FACS were performed.
Plasmid construction
Three types of plasmids for expression of proteins in which TLM was inserted into deletion mutants obtained by genetically modifying the hepatitis B virus L protein were constructed (FIG. 2).

Production by yeast
As a result of transformation of the three types of plasmids into yeast, no transformant was obtained for Δ21-TLM. Δ33-TLM produced a transformant but produced very little particles. On the other hand, a large number of transformants were obtained for Δ50-TLM, and the amount of particles produced was high. A transformant of Δ50-TLM was selected to obtain a high-producing strain. In the selection method, transformants were cultured in 3 ml of industrial medium in a test tube, and 1 ml of the cells was collected, dissolved in 250 ml of Buffer A, disrupted with glass beads, and the yeast extract was 100 times with PBS. After dilution, the amount of particles was measured with an enzyme immunoassay apparatus IMx. We also performed Western blot analysis of high-producing strains. As a result, Δ50-TLM had the highest production volume, and this was used in the subsequent experiments.

Analysis of particles Silver staining / Western blot analysis A plurality of bands were observed, but purified particles were obtained (Fig. 3).

Sugar chain treatment As a result of Western blot analysis, it was confirmed that one N-type sugar chain was added (FIG. 4).

Trypsin treatment As a result of Western blot analysis, the main band was shifted to the low molecule side, confirming that TLM was presented on the particle surface (Fig. 5).

Based on the above, it can be said that the plurality of bands confirmed by silver staining are degradation products of sugar chains and proteases.

Calcein introduction experiment <br/> Adherent cells
For HepG2 and A431, Δ50-153 and Δ50-153 + TLM were compared (FIG. 6).
Next, introduction experiments into NuE (human hepatoma cells), NA (human squamous cell carcinoma cells), Cos7 (monkey kidney cells), and PC12 (rat adrenal pheochromocytoma) were performed (FIGS. 7 to 10). A clear difference in fluorescence intensity was confirmed compared to the control L particles.

Floating cells
Experiments for introduction into H69 (human lung cancer cells) and MOLT4 (human lymphoblast cells) were performed (FIGS. 11 and 12). A slight difference in fluorescence intensity was confirmed compared to the control in which calcein and TLM presentation particles were mixed. In general, introduction into suspension cells is considered difficult, and the usefulness of the nanoparticles of the present invention has been clarified.

Reference 1. Gene Ther. 2000 May; 7 (9): 750-8.
Oess S, Hildt E
Novel cell permeable motif derived from the PreS2-domain of hepatitis-B
virus surface antigens.

2. EMBO Rep. 2003 Aug; 4 (8): 767-73.
Hafner A, Brandenburg B, Hildt E
Reconstitution of gene expression from a regulatory-protein-deficient
hepatitis B virus genome by cell-permeable HBx protein.

3. J Hepatol. 2005 Sep; 43 (3): 442-50.
Hillemann A, Brandenburg B, Schmidt U, Roos M, Smirnow I, Lemken ML, Lauer
UM, Hildt E.
Protein transduction with bacterial cytosine deaminase fused to the TLM intercellular transport motif induces profound chemosensitivity to 5-
fluorocytosine in human hepatoma cells.

The structure of the nanoparticle of this invention comprised from HBsAg is shown typically. The structure of the plasmid used in the examples is shown. The result of silver dyeing and WB of purified particles is shown. The result of sugar chain treatment (PNGase F treatment) is shown. The result of trypsin treatment is shown. A comparison of Δ50-153 and Δ50-153 + TLM is shown. The result of the introduction experiment to NuE cells is shown. The result of the introduction experiment into NA cells is shown. The result of the introduction experiment to Cos7 cells is shown. The result of the introduction experiment to PC12 cells is shown. The result of the introduction experiment to H69 cells is shown. The result of the introduction experiment to MOLT4 cells is shown.

Claims (7)

A nanoparticle capable of encapsulating a substance to be introduced, comprising at least one selected from the group consisting of proteins, lipids and organic polymers as a constituent, wherein a cell-permeable peptide is covalently bound to at least one of the constituents; A nanoparticle obtained by presenting a cell-permeable peptide on the surface of the nanoparticle. The nanoparticle according to claim 1, wherein the nanoparticle is a virus particle containing a viral protein or a liposome containing a phospholipid. The nanoparticle of claim 1, wherein the component comprises hepatitis B virus protein. The nanoparticle according to any one of claims 1 to 3, wherein the cell-penetrating peptide has an amino acid sequence of PLSSIFSRIGDP. The nanoparticle according to any one of claims 1 to 4, which has an introduction substance in the particle. A variant of hepatitis B virus surface antigen protein, which is a hepatitis B virus surface antigen protein in which part or all of the hepatocyte binding region is deleted and has a cell-penetrating peptide. The variant according to claim 6, which has the sequence represented by SEQ ID NO: 4.