JP2007106752A - Substance conveying body using hollow nanoparticle of hepatitis b viral protein and liposome and method for transferring substance to cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composite particle for specifically, efficiently and safely transporting and transferring a transfer substance to target cells or target tissue and to provide a method for producing the same. <P>SOLUTION: The method for producing a composite particle of a transfer substance-containing nanoparticle and liposome comprises fusing a hollow nanoparticle containing hepatitis B viral protein or its variant with liposome with an encapsulated a transfer substance. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method for encapsulating an introduced substance in hollow nanoparticles, which are carriers capable of introducing the substance into specific cells and tissues, and a composite particle encapsulating the introduced substance.

In recent years, a method called a drug delivery system (DDS) has attracted attention in order to reduce the side effects of drugs. DDS is a method in which an active ingredient such as a drug can be specifically transported to a target cell or target tissue such as an affected part by a carrier, and the active ingredient can act on the target site.
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 (WO01 / 64930, WO03 / 082330, WO03 / 082344). In the past, electroporation was used to encapsulate drugs, genes, proteins and other introduced substances in the particles. Since the electroporation method requires specialized knowledge about the electroporation dedicated device and the operation of the device, certain restrictions are imposed on the method of encapsulating the introduced substance in the hollow nanoparticles by this method. was there.
As shown in WO97 / 17844, Sendai virus particles containing lipids with a lipid content of about 70% as a main component can easily form complex particles with liposomes that are the same lipid, and encapsulate the introduced substance in the virus particles. Although known, the interaction between HBsAg protein particles and liposomes was not known.

  It is an object of the present invention to provide composite particles for transporting and introducing a substance to be introduced into a target cell or target tissue specifically, efficiently and safely, and a method for producing the same.

  As a result of repeated studies in view of the above problems, the present inventor has formed a composite particle by mixing with liposomes even if it is a hollow nanoparticle mainly composed of protein such as HBsAg particles. The present inventors have found that the hollow nanoparticle can contain the introduced substance.

The present invention relates to the following composite particles and methods for producing the same.
1. A method for producing a composite particle of nanoparticles and liposomes encapsulating an introduced substance, comprising fusing hollow nanoparticles containing a hepatitis B virus protein or a variant thereof with a liposome encapsulating the introduced substance.
2. Item 2. The method according to Item 1, wherein the hollow nanoparticles have a particle size of 80 to 130 nm.
3. Item 2. The method according to Item 1, wherein the composite particles have a particle size of 130 to 500 nm.
4). Item 2. The method according to Item 1, wherein the composite particles have a particle size of 150 to 400 nm.
5. Hollow nanoparticles containing hepatitis B virus protein or variant thereof are composed of 70 to 90 parts by weight of hepatitis B virus protein or variant thereof, 5 to 15 parts by weight lipid, and 5 to 15 parts by weight sugar chain Item 2. The method according to Item 1.
6). Item 6. The method according to any one of Items 1 to 5, wherein the hollow nanoparticles are previously freeze-dried or spray-dried. A composite particle comprising a nanoparticle part composed of hepatitis B virus protein or a variant thereof, phospholipid, lipid and sugar chain, and an exogenous introduction substance encapsulated in the nanoparticle part.
8). Item 8. The composite particle according to Item 7, wherein the composite particle has a particle size of 150 to 400 nm.
9. Item 8. The nanoparticle part is composed of 70 to 90 parts by weight of hepatitis B virus protein or a variant thereof, 6 to 75 parts by weight of lipid, and 5 to 15 parts by weight of a sugar chain. Composite particles.
10. Item 7. A composite particle of nanoparticles and liposomes encapsulating an introduced substance, which can be obtained by the method according to any one of Items 1 to 6.
11. Item 10. A method for introducing an introduction substance into target cells, which comprises causing the composite particles according to any one of Items 7 to 9 or the composite particles according to Item 10 to act on target cells.

  According to the present invention, the introduced substance can be easily encapsulated in hollow nanoparticles having high protein content such as HBsAg particles and being rigid. The introduced substance may be a substance comparable to or much larger than the hollow nanoparticles before inclusion of the introduced substance, such as 100 nm to 500 nm.

  In addition, the composite particles of the present invention in which the introduction substance is encapsulated using liposomes have a higher introduction efficiency of the introduction substance than the particles in which the introduction substance is encapsulated in the hollow nanoparticles by the electroporation method.

  Furthermore, DNA and RNA such as plasmids can be made into composite particles having a size smaller than that of DNA and RNA by fusing with hollow nanoparticles after encapsulating in liposomes.

  By using the composite particles of the present invention, a substance can be specifically introduced into a specific cell or tissue in vitro or in vivo.

  In this specification, HBsAg protein particle etc. are illustrated as a hollow nanoparticle containing the introduction | transduction substance and containing the hepatitis B virus protein or its modification. The HBsAg protein may be combined with a hepatitis B virus internal core antigen protein to form particles.

  In the present specification, the size of the composite particles, hollow nanoparticles, introduced substances (nucleic acid, protein, drug, etc.) may be measured by an electron microscope, and optically measured by a zeta sizer nano-ZS (Malvern Instruments) or the like. You may measure.

  The hollow nanoparticle for encapsulating the introduced substance in the present invention includes a hepatitis B virus protein as a main component, and the protein may have a sugar chain. The hollow particles may contain a lipid component.

  In one preferred embodiment, the hollow nanoparticles are composed of 70-90 parts by weight of hepatitis B virus protein or a variant thereof, 5-15 parts by weight lipid, 5-15 parts by weight sugar chain. The hollow nanoparticles used in the examples of the present application consist of 80 parts by weight of hepatitis B virus protein, 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.). Sendai virus, which is known for fusion with liposomes, has a structure in which lipid is the main component and a small amount of protein floats in the liposome (Methods Enzymol. 1993; 221: 18-41. Okada Y. Sendai virus -induced cell fusion.). Sendai virus is thought to fuse by the membrane fusion action of the F protein stuck in the membrane. As for the hollow nanoparticles of the present invention, there is no report that the hepatitis B virus protein itself pierced in the membrane has a membrane fusion activity, and the fusion with liposomes is observed for the first time when the present inventors have found the same activity. It was. Prior to the filing of this application, no one could have predicted that virus particles with high protein content had membrane fusion activity.

  In a preferred embodiment of the present invention, the nanoparticle part encapsulating the complex after fusing with the liposome (the structural part other than the encapsulated substance such as the complex) is 70 to 90 parts by weight of hepatitis B virus protein. Or a variant thereof, 6 to 75 parts by weight of lipid (including 5 to 15 parts by weight of eukaryotic membrane constituent lipids and 1 to 60 parts by weight of liposome constituent lipids), 5 to 20 It may be composed of parts by weight of sugar chains.

  The composite particle of the present invention has a structure in which at least one liposome and a lipid part of at least one hollow nanoparticle are partially or completely fused, and this lipid membrane is stuck with a hepatitis B virus protein or a variant thereof. To obtain (FIG. 1B).

  In a preferred embodiment of the present invention, the nanoparticle part (structure part other than the encapsulated substance such as the introduced substance) encapsulating the introduction substance of the composite particle after being fused with the liposome is 70 to 85 parts by weight of hepatitis B It is composed of 5-15 parts by weight of lipid (component of eukaryotic membrane), 2-30 parts by weight of lipid (component of liposome), 5-15 parts by weight of sugar chain. obtain.

  As the liposome, a 100% phospholipid liposome can also be used, but this liposome tends to be less flexible. As the liposome used in the present invention, those containing phospholipid as a main component and containing a small amount of other lipid components such as cholesterol are preferable because they increase the fluidity of the membrane and give flexibility. The phospholipid component in the liposome is preferably about 80% or more with respect to the total weight of the liposome, and other lipid components such as cholesterol are preferably about 20% or less with respect to the total weight of the liposome. Lipid lipids fused with hollow nanoparticles constitute a lipid membrane of composite particles. The hollow nanoparticles and the liposomes may be fused in a 1: 1 manner, and a plurality of liposomes may be fused to one hollow nanoparticle, and further, one liposome and a plurality of hollow nanoparticles are fused. It is also possible, and a plurality of hollow nanoparticles and a plurality of liposomes can be fused.

  The endoplasmic reticulum membrane of eukaryotic cells that is taken up by hollow nanoparticles (and thus a component of hollow nanoparticles) can be composed mainly of phospholipids. Phospholipids include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), etc. For example, phosphatidylcholine (PC) is the main component in yeast. The composition of the endoplasmic reticulum membrane varies depending on the type of eukaryotic cell. Examples of eukaryotic cells used to produce hollow nanoparticles include mammalian cells (CHO cells, HEK293 cells, COS cells, etc.), insect cells (Sf9, Sf21, HighFive strains, etc.), yeast, etc. The endoplasmic reticulum membrane of eukaryotic cells is a component of the lipids of the hollow nanoparticles.

  As shown in FIG. 3, the composite particles of the present invention are much more excellent in substance introduction efficiency than electroporation.

  In this specification, “hollow nanoparticle” means a particle before encapsulating the introduction substance, and “nanoparticle” means a particle after encapsulating the introduction substance. “Composite particle” refers to a particle in which hollow nanoparticles and liposomes encapsulating an introduction substance are fused, and the introduction substance is encapsulated in the nanoparticles.

  S protein (226 amino acids), which is included in HBsAg and is a component of S particles, has the ability to form particles. Pre-S2 consisting of 55 amino acids added to S particles is M protein (M particle constituent protein), and Pre-S1 consisting of 108 amino acids (subtype y) or 119 amino acids (subtype d) in M protein L protein (protein constituting L particle) is added.

  In the present specification, unless otherwise specified, the numbering of amino acid positions in the Pre-S1 region is performed based on subtype y of 108 amino acids.

  L protein and M protein have the ability to form particles, as does S protein. Therefore, the two regions of PreS1 and PreS2 may be arbitrarily substituted, added, deleted, or inserted. For example, by using a modified protein that lacks the hepatocyte recognition site contained in position 3-77 (subtype y) of the Pre-S1 region, hollow particles that have lost the ability to recognize hepatocytes can be obtained. In addition, since the PreS2 region includes a site that recognizes hepatocytes via albumin, this albumin recognition site can also be deleted. On the other hand, since the S region (226 amino acids) is responsible for particle forming ability, modification of the S region must be performed so as not to impair the particle forming ability. For example, even if the S region 107-148 is deleted, the particle forming ability is retained (J. Virol. 2002 76 (19), 10060-10063), so substitution, addition, deletion, insertion, etc. may be performed. Similarly, the hydrophobic 154-226 residues at the C-terminal part can retain the particle forming ability even when substitution, addition, deletion, insertion, and the like are performed. On the other hand, 8-26 residues (TM1) and 80-98 residues (TM2) of S region are transmembrane helix (transmembrane sequence), and this region is not mutated, deleted, added or replaced. Etc. are preferably performed leaving a hydrophobic residue so as to maintain transmembrane properties.

  In one preferred embodiment, various variants of hepatitis B virus protein are widely encompassed as long as they have the ability to form hollow nanoparticles. Taking HBsAg as an example, the PreS1 and PreS2 regions are optional. Of the number of substitutions, deletions, additions and insertions, and with respect to the S region, one or several or plural, for example, 1 to 120, preferably 1 to 50, more preferably 1 to 20, Preferably 1 to 10, especially 1 to 5 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.

  In the case of hollow bio-nanoparticles composed of proteins capable of recognizing hepatocytes such as L protein and M protein as hepatitis B virus protein or a variant thereof, it is not necessary to introduce a cell recognition site. On the other hand, a hollow bio-nanoparticle composed of a modified protein in which the hepatocyte recognition site contained in amino acids 3 to 77 of the Pre-S1 region has been deleted, or a protein in which both the PreS1 and PreS2 regions have been deleted. In this case, since cell recognition cannot be performed as it is, a cell recognition site can be introduced to recognize any cell other than hepatocytes, and nucleic acids can be introduced into various target cells. Such cell recognition sites for recognizing specific cells include, for example, cell function regulatory molecules consisting of polypeptides such as growth factors and cytokines, cell surface antigens, tissue-specific antigens, receptors and other cells and tissues. Polypeptide molecules derived from these, polypeptide molecules derived from viruses and microorganisms, antibodies, sugar chains 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.

The ZZ tag is defined as an amino acid sequence having the ability to bind to the Fc region of immunoglobulin G, and consists of the following twice-repeated sequence (ZZ tag sequence (SEQ ID NO: 1):
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK).

  When the cell recognition site is a polypeptide, the DNA encoding the hepatitis B virus protein or a variant thereof and the DNA encoding the cell recognition site are in-frame via the DNA encoding the spacer peptide as necessary. Hollow bio-nanoparticles that recognize any target cell can be obtained by ligation, incorporation into a vector or the like, and expression in eukaryotic cells.

  When the cell recognition site is an antibody, a DNA encoding a hepatitis B virus protein or a variant thereof and a DNA encoding a ZZ tag are linked in-frame via a DNA encoding a spacer peptide, if necessary. Is incorporated into a vector or the like, expressed in a eukaryotic cell, and the resulting hollow bionanoparticle and an antibody capable of recognizing the target cell are mixed to obtain the desired hollow bionanoparticle.

  When the cell recognition site is a sugar chain, biotin is added to the hollow nanoparticle by chemical modification, and a biotin-labeled sugar chain or the like is presented via streptavidin or the like.

  The variant of HBsAg protein may be a variant with altered antigenicity (a variant in which a site involved in antigenicity such as an epitope is deleted / replaced), particle structure stability, cell selectivity, etc. .

  The size of the hollow nanoparticles is about 50 to 500 nm, preferably about 80 to 130 nm. The hollow nanoparticles are desirably larger than a certain size, particularly when the introduced substance to be encapsulated is large. To increase the size of the hollow nanoparticles, for example, increase the size of the Pre-S region added to the N-terminus of the S region (from 163 residues (subtype y) plus Pre-S1 + Pre-S2) It has been confirmed by the inventor that the size of the particles increases.

  What is necessary is just to use a longer thing as the hepatitis B virus protein which comprises this particle | grain, or its modification. For example, when targeting hepatocytes, L particles or hollow bio-nanoparticles of the same size are preferably used, and hepatocytes contained in S protein / M protein or 3 to 77 amino acid residues in the Pre-S1 region By introducing a cell recognition site into a protein lacking the recognition site, hollow particles targeting cells other than hepatocytes can be obtained.

  When the HBsAg protein 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.

  In the production method of the present invention, it is particularly preferable that the hollow nanoparticles containing HBsAg protein as a constituent component are freeze-dried and then fused and complexed with liposomes. Spray-dried ones can be used instead of freeze-drying. By using freeze-dried or spray-dried hollow nanoparticles, the fusion efficiency is significantly improved.

  In one embodiment of the present invention, nanoparticles containing HBsAg protein as a constituent component are once freeze-dried or spray-dried and then mixed with liposomes to form composite particles, which are introduced into liposomes enclosed in hollow nanoparticles. Substances can be taken up. In the conventional method, the introduction substance is taken into the hollow nanoparticle by electroporation, but the introduction substance can be more easily taken into the hollow nanoparticle by the method of the present invention, and the introduction into the cell is possible. Efficiency can also be improved.

  It is also possible to perform freezing / rapid thawing and heat treatment instead of freeze drying or spray drying.

  Mixing of lyophilized hollow nanoparticles and liposomes as necessary is performed at a temperature of about 37 ° C., for example, using about 0.1 to 10 mg, preferably about 0.5 to 2 mg of liposomes per 1 mg of hollow nanoparticles (lyophilized product). For about 10 minutes. If the amount of liposome used for the hollow nanoparticles is too large, the efficiency of introducing the introduced substance into the cells will be reduced. On the other hand, if the amount of liposomes is too small, the efficiency of encapsulating the introduced substance in the hollow nanoparticles decreases.

  In a preferred embodiment of the present invention, the particle size of the hollow nanoparticles before complexing with the liposome is about 80 to 130 nm, and complexing after complexing with the liposome and incorporating the introduced substance into the interior of the nanoparticle The particle size of the nanoparticles (composite particles) is about 130 to 500 nm, for example, 150 to 400 nm, more preferably about 200 to 400 nm.

  The liposome may be either a multilamellar liposome or a monolayer liposome. The size of the liposome is about 50 to 300 nm (for example, 100 to 300 nm), preferably about 80 to 250 nm, more preferably about 100 to 200 nm, and particularly preferably about 100 to 150 nm. The size of the liposome is preferably about 0.5 to 2 times that of the hollow nanoparticle.

  If the liposome is too large or too small relative to the nanoparticle, formation of a smooth composite particle is prevented.

  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. It is desirable to use phospholipids in combination with lipids having various degrees of saturation. In addition, cholesterols include cholesterol, phytosterol, and the like, and examples of fatty acids include oleic acid, palmitooleic acid, linoleic acid, and fatty acid mixtures containing these unsaturated fatty acids. Liposomes containing unsaturated fatty acids with small side chains are effective in producing small liposomes because of curvature.

  An example of a method for producing liposomes will be described in detail. For example, the above-described phospholipids, cholesterol and the like are dissolved in an appropriate organic solvent, put in an appropriate container, the solvent is distilled off under reduced pressure, and the inner surface of the container is removed. A phospholipid membrane is formed, and an aqueous solution containing an introduction substance, preferably a buffer solution, is added thereto and stirred to obtain a liposome encapsulating the introduction substance. The liposome is directly or once lyophilized, and then mixed with the freeze-dried nanoparticles of the present invention, whereby composite particles of liposomes and nanoparticles can be obtained.

  The composite particles of the present invention are useful as those for specifically introducing an introduced substance into specific cells. For example, if the composite particle is administered into the body by intravenous injection or the like, the particle circulates in the body and is guided to the target cell by a substance selective / specific to hepatocytes or other cells presented on the particle surface, An introduced substance is introduced into the target cell.

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

  The substance to be introduced into the cell is not particularly limited. For example, various drugs that are introduced into the cell to cause physiological action, for example, physiologically active proteins such as hormones, lymphokines, enzymes, etc .; antigenic proteins that act as vaccines; Genes expressed in, polynucleotides such as plasmids, or polynucleotides involved in specific gene expression that induces or induces expression; various genes introduced for gene therapy and antisense DNA / RNA, etc. it can. The “gene” to be introduced includes not only DNA but also RNA. The introduced substance is preferably 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).

  In the following, “BNC” is an abbreviation for bio-nanocapsule, and is used to mean both HBsAg particles and composite particles in which HBsAg particles and liposomes are fused (particles that contain an introduced substance such as a gene in HBs particles).

Experimental method <br/> Materials
BNC was prepared from yeast according to a method described in a known paper (Kuroda, S., Otaka, S., Miyazaki, T., Nkao, M. and Fujisawa, Y. (1992) J. Biol. Chem. 267 ( 3), 1953-1961) and AKTA (Amershambiosciences co., Japan). Liposomes (Coatsome EL-01-A, Coatsome EL-01-D) were purchased from NOF corporation (Tokyo, Japan). Fluorophore carboxylate-modified microspheres (100-nm polystyrene beads) were purchased from Molecular probes Inc. (Eugene, OR). Plasmid pEGFP-C1 was purchased from Clonetech laboratories Inc. (Takara bio Inc., Japan), and plasmids pcDNA6.2 / C-EmGFP and pAD / CMV-GFP were purchased from Invitrogen co. (Carlsbad, CA).

Fusion of BNC and liposome
To encapsulate 100-nm polystyrene beads in liposomes, lyophilized liposomes (Coatsome-El-01-A) were dissolved in an aqueous solution of 100-nm polystyrene beads (0.2% w / v) and then gently shaken at room temperature. did. For encapsulation of DNA in liposomes, lyophilized liposomes (Coatsome-EL-01-A) were dissolved in an aqueous DNA solution (250 μg / mL) at room temperature for 15 minutes. Liposomes containing the transduct (100-nm polystyrene beads or genes) were added to lyophilized BNC and treated for 15 minutes at room temperature.

Transmission electron microscope (TEM)
To visually examine liposome-fused BNC, the literature (Kuroda, S., Otaka, S., Miyazaki, T., Nkao, M. and Fujisawa, Y. (1992) J. Biol. Chem. 267 (3) , 1953-1961), and observed with TEM (JEOL, Japan).

Particle size The particle size was measured at 25 ° C. using a Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). This measurement was based on dynamic light scattering; the Z-average particle size was estimated based on the Einstein-Stokes equation.

cell
HepG2 (human hepatocellular carcinoma) cells and A431 (human epidermoid carcinoma) cells were maintained in Dulbecco's modified MEM medium supplemented with 10% fetal bovine serum (FBS). WiDr (human colon adenocarcinoma) and NuE (human hepatocellular carcinoma, obtained from T. Tadakuma, National Defense Medical College) cells were maintained in RPMI 1640 medium supplemented with 10% FBS. These cells were incubated at 37 ° C. in 5% CO ₂ .

Mouse xenograft model
5-week old male BALB / c nude (nu / nu) mice were purchased from CLEA Japan, Inc. (Osaka, Japan). Animals were treated according to the Ministry of Education, Culture, Sports, Science and Technology guidelines. Approximately 5 × 10 ⁶ carcinoma cells (NuE and A431) were injected subcutaneously into the back of the mice. Two weeks later, mice were injected intravenously with BNC or GFP plasmid containing 100-nm polystyrene beads.

Histological analysis The mice were anesthetized with pentobarbital (Dainippon sumitomo pharma Co., Japan), and the tumor, liver and kidney were removed. These tissues were fixed with 4% (wt / vol) paraformaldehyde and embedded in synthetic resin with Technovit 8100 (Kluzer, Germany). Blocks were sectioned at a width of 5 μm and then viewed with an LSM5 PASCAL laser scanning confocal microscope (Carl ziess, Germany).

Example 1 Encapsulation of substance in HBsAg particles via liposome
FITC-labeled polystyrene beads (Fluospheres ^R (diameter 100 nm), manufactured by Molecular Probe) prepared in sterile water at 10 mg / ml, 1 ml of FITC-labeled polystyrene beads solution freeze-dried with 8.5% sucrose ( COATSOME EL-01-A (manufactured by NOF Corporation) and mixed uniformly to prepare a FITC-labeled polystyrene bead-encapsulated liposome solution. The prepared FITC-labeled polystyrene bead-encapsulated liposome solution in a Beckman ultracentrifugal swing rotor SW41 equivalent product was used with a 6% sucrose solution, 3.5 ml, a 10% sucrose solution, 3.5 ml, and a 30% sucrose solution, 3.5 ml. The FITC-labeled polystyrene beads that were not included in the liposomes were removed by ultracentrifugation for 1 hour at 24,000 rpm and 4 ° C, and placed on the upper layer of the separation solution. Was recovered. Freeze-dried HBsAg particles (Vaccine. 2001 Apr 30; obtained by lyophilizing overnight a solution prepared by mixing sucrose solution with HBsAg particles to a final concentration of 5% in PBS (phosphate buffer saline); 19 (23-24): 3154-63. Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein.Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T, Fukuda H, Kondo A, Seno M, Tanizawa K, Kuroda S.) and FITC-labeled polystyrene bead-encapsulated liposomes are mixed uniformly to fuse HBsAg particles with FITC-labeled polystyrene bead-encapsulated liposomes. As a result, HBsAg fused particles including FITC-labeled polystyrene beads were obtained. This fusion reaction further promoted the fusion of liposomes and HBsAg particles by heating at 37 ° C. for 1 hour after mixing. In addition, this sample was placed on the upper layer of the separation solution prepared with a density gradient using 3.5 ml of 10% sucrose solution, 3.5 ml of 30% sucrose solution, and 3.5 ml of 50% sucrose solution as before, 24,000 rpm, Only HBsAg fused particles could be recovered by ultracentrifugation at 4 ° C for 2 hours. FIG. 1 is a graph showing the state of separation of the fraction collected from the upper part of the centrifuge tube after ultracentrifugation. The peak in Fraction 8 corresponds to HBsAg fused particles. By collecting the peak fraction, HbsAg Composite particles (BNC) were obtained. Most of the BNC was fused with liposomes, and there was almost no free BNC. Liposome-fused BNC was observed by TEM (Fig. 1B). Electron micrographs of liposome-fused BNC showed that BNC was surrounded by liposomes encapsulating FITC-beads. The average size of BNC was about 200 nm (Table 1).

As shown in Table 1, the composite particle of the present invention can compact a nucleic acid (DNA / RNA) such as a GFP plasmid (reducing the size), and is suitable for a drug delivery system (DDS).

Example 2 Introduction of substance into human hepatoma cell HepG2 by HBsAg particles encapsulating substance via liposome

Human hepatoma cell HepG2 in the exponential growth phase is seeded on a 96-well plastic bottom plate at 1 × 10 ⁴ cells / well, and MEM containing 10% fetal bovine serum in the presence of 5% CO ₂ at 37 ° C. (Modified Eagle's medium) was used for overnight culture. The next day, as in Example 1, FITC-labeled polystyrene beads (diameter 100 nm), freeze-dried liposomes, freeze-dried HBsAg particles were used to prepare FITC-labeled polystyrene bead-containing HBsAg particles, which were then added to the HepG2 culture solution. ° C., and cultured overnight at the presence of 5% CO _2.

  In order to quantify the amount of FITC-labeled polystyrene beads introduced into HepG2, it was measured with a plate reader. The state of introduction of FITC-labeled polystyrene beads into HepG2 was observed with a confocal laser fluorescence microscope.

  The quantitative results of the FITC-labeled polystyrene beads are shown in the graph of FIG. A fluorescence photograph of HepG2 is shown in FIG. FIGS. 2 and 3 also show the results of the human colon cancer cell WiDr used as a control for HepG2. From the graph of FIG. 2, both HepG2 and WiDr showed about 10 times the fluorescence intensity when introduced into HepG2 using fused particles fused with HBsAg particles, compared to the case where only FITC-labeled polystyrene bead-encapsulated liposomes were used. On the other hand, even when fused particles were used, there was almost no difference with WiDr, indicating that the specificity of HBsAg particles to hepatocytes remained maintained after fusion with liposomes. In FIG. 3, the photograph at the time of using RITC labeled polystyrene beads instead of FITC labeled polystyrene beads is shown. From the results shown in FIG. 3, introduction by HBsAg particles fused with liposomes was observed only in HepG2.

  From the above, it was shown that it is possible to introduce a substance with extremely high specificity and efficiency to human hepatocytes at the level of cultured cells using HBsAg particles encapsulated with the liposome of the present invention. .

Example 3 Substance introduction into nude mice transplanted with human liver cancer by HBsAg particles encapsulating substance via liposomes

Tumor-bearing mice were nude mice (strain: BALB / c, nu / nu, microbiological quality: SPF, sex: male 5 weeks of age) subcutaneously on the dorsal dorsal area, 2 × 10 ⁵ human hepatocarcinoma-derived cells each The cells were injected and grown for 2-3 weeks until the transplanted tumor became a solid tumor of about 2 cm in diameter.

  100 μg of FIB-labeled polystyrene bead-containing HBsAg fusion particles obtained by the method described in Example 1 (dissolved in 100 μl of PBS) was administered into the mouse tail vein using a 26G injection needle. 16 hours after administration, the abdomen was opened under anesthesia, and after perfusion fixation was performed according to a conventional method, the tumor, liver, and kidney were removed, and the tissue was fixed and embedded using a resin embedding kit (Technovit 8100). .

  Specifically, after laparotomy, a 21G winged injection needle was inserted into the left ventricle, the right atrial appendage was opened, and blood was removed by flowing PBS. Thereafter, 4% neutralized formaldehyde that had been ice-cooled in advance was poured, and the tissue was filled with the formaldehyde solution. After tissue removal, the tissue was further fixed by immersion in 4% neutralized formaldehyde at 4 ° C for 2 hours, and then immersed in 6.8% sucrose-PBS solution at 4 ° C overnight. The next day, dehydration was carried out with 100% acetone, and then immersion was carried out with Technonov 8100 within 4 ° C. for 24 hours. After removal, the polymer was allowed to stand at 4 ° C. for polymerization reaction.

  According to a conventional method, sections were prepared, and the fluorescence of FITC-labeled polystyrene beads in the HBsAg particle-administered group and the non-administered group was compared with a confocal laser fluorescence microscope (FIG. 4).

  From FIG. 4, fluorescence derived from FITC-labeled polystyrene beads was observed in the tumor part of the tumor-bearing mouse by human liver cancer-derived NuE cells. However, no fluorescence was observed in the liver and kidney simultaneously removed from the mouse. In addition, with the FITC-labeled polystyrene beads alone or the FITC-labeled polystyrene bead-encapsulated liposomes alone, no fluorescence was observed in the tissues of tumor-bearing mice including the tumor site.

  From the above, it was found that HBsAg particles encapsulated with liposomes enable the introduction of substances with extremely high specificity and efficiency even at the experimental animal level for human hepatoma cells.

Example 4 Substance Introduction into Human Squamous Cell Carcinoma Cell A431 Using ZZ Tag Surface Displayed HBsAg Particles (ZZ-HBsAg Particles) Encapsulating Substances via Liposomes

Exponentially growing human squamous cell carcinoma cell A431 is seeded in a 96-well plastic bottom dish plate at 1 × 10 ⁴ cells / well, and 10% fetal bovine serum in the presence of 37 ° C and 5% CO ₂ Incubated overnight with MEM containing. The next day, as in Example 1, FITC-labeled polystyrene beads, freeze-dried liposomes, and freeze-dried ZZ-HBsAg particles (PCT / JP03 / 03694) were used to prepare ZZ-HBsAg-fused particles containing FITC-labeled polystyrene beads. Then, 8μg of monoclonal antibody against human epidermal growth factor receptor (hEGFR) (anti-hEGFR antibody) and 100μg of the prepared ZZ-HBsAg particles were mixed uniformly and then at 4 ℃ for 1 hour. A binding reaction was performed. Anti-hEGFR antibody-presenting ZZ-HBsAg fused particles obtained by this binding reaction were added to the A431 culture solution and cultured overnight at 37 ° C. in the presence of 5% CO ₂ .

  In order to quantify the amount of FITC-labeled polystyrene beads introduced into A431, the amount was measured with a plate reader. The state of introduction of FITC-labeled polystyrene beads into A431 was observed with a confocal laser fluorescence microscope.

  The quantitative results of the FITC-labeled polystyrene beads are shown in the graph of FIG. A fluorescent photograph of A431 is shown in FIG. FIGS. 5 and 6 also show the results of ZZ-HBsAg fused particles that did not undergo a binding reaction with an anti-hEGFR antibody as a control. From the graph of FIG. 5 and the photograph of FIG. 6, it was found that the ZZ-HBsAg fused particles can introduce FITC-labeled polystyrene beads encapsulated in A431 via an anti-hEGFR antibody. This result shows that the ZZ-HBsAg particles maintain the antibody binding ability of the ZZ tag even after fusing with the liposome, and can introduce substances through the antibody bound to the ZZ tag. It was.

  In addition, in FIG. 7, as described above, the substance is also applied to breast cancer-derived cell MCF-7 expressing RIGF labeled polystyrene beads instead of FITC labeled polystyrene beads and expressing the EGF receptor on the cell surface in the same manner as A431. The photograph at the time of introduction was shown. From the results shown in FIG. 7, even with the HBsAg particles presenting the foreign functional protein on the surface, it is possible to encapsulate substances inside the particles while maintaining the function of the particles by using the method of Example 1. It was shown that.

Example 5 Gene transfer into human hepatoma cell HepG2 using HBsAg particles encapsulating genes via liposomes

Exponentially growing human hepatoma cell HepG2 is seeded in a 96-well plastic bottom dish plate at 1 × 10 ⁴ cells / well, and MEM containing 10% fetal bovine serum in the presence of 37% at 5% CO ₂ Was used for overnight culture. On the next day, empty liposomes prepared by freeze-drying 1.5 ml of a solution prepared by making Green Fluorescence Protein (GFP) expression plasmid (pEGFP-C1; Clontech) 50 μg / ml in sterile water with 8.5% sucrose ( COATSOME EL-A-01 (manufactured by NOF Corporation) and mixed uniformly, and allowed to stand at room temperature for 5 minutes to prepare a GFP expression plasmid-encapsulated liposome solution. 150 μl of the prepared GFP-expressing plasmid-encapsulated liposome solution mixed with HBsAg particles so that the final concentration of sucrose solution becomes 5% is freeze-dried overnight, so that it becomes homogeneous with 200 μg of freeze-dried HBsAg particles. The mixture was allowed to stand at room temperature for 5 minutes to fuse the HBsAg particles with the GFP expression plasmid-encapsulated liposomes. As a result, GFP expression plasmid-containing HBsAg fusion particles were obtained. Thereafter, this was added to a HepG2 culture solution and cultured for 48 hours at 37 ° C. in the presence of 5% CO ₂ . After 48 hours, the state of GFP expressed in the cells by the introduced GFP expression plasmid was observed with a confocal laser fluorescence microscope.

  A fluorescence photograph of HepG2 is shown in FIG. In FIG. 8, the result in the human colon cancer cell WiDr used as a control of HepG2 is also shown. From the results shown in FIG. 8, the introduction of HBsAg particles fused with liposomes was observed only in HepG2, and no fluorescence was observed in WiDr.

  From the above, the present invention shows that it is possible to introduce genes with extremely high specificity and efficiency to human hepatocytes at the level of cultured cells using HBsAg particles encapsulated with liposomes. It was.

Example 6
Ex vivo and in vivo delivery of 100-nm polystyrene beads using liposome-fused BNC
Since BNC can be almost completely fused with liposomes, liposome-fused BNC encapsulating rhodamine-labeled 100-nm polystyrene beads (Rho-beads) was used without separation by ultracentrifugation. Approximately 5 × 10 ⁴ HepG2 cells, WiDr cells and A431 cells were transfected using 10 μg of liposome-fused BNC encapsulating 1 μg of Rho-beads. After 6 hours, fluorescence was observed specifically in HepG2 cells, but not in WiDr cells and A431 cells (Fig. 9). In addition, these BNCs were injected into a xenograft model carrying liver NuE cells and A431 cells. 100 μg of liposome-fused BNC encapsulating 25 μg of Rho-beads per mouse was used. After 16 hours, fluorescence was observed in NuE-derived tumors, but no fluorescence was observed in A431-derived tumors. To confirm the location of Rho-beads, FITC-labeled tomato lectin was injected before sacrificing. Rho-beads present around the blood vessels showed a green color (FIG. 10).

Example 7
Ex vivo and in vivo gene delivery using liposome-fused BNC
In order to introduce DNA into BNC, the DNA was first encapsulated in cationic liposomes and then fused with BNC in the same manner as the introduction of beads. Liposome-fused BNC encapsulating GFP plasmid (pEGFP-C1) was added to HepG2 cells and WiDr cells (2 × 10 ⁵ cells / well). 2 μg of GFP plasmid was encapsulated in 10 μg of BNC. Significant GFP expression was observed in BNC-treated HepG2 cells on the second day after transfection (FIG. 11). In the xenograft model, 50 μg of BNC was injected. After 5 days, mouse liver, mouse kidney, NuE-derived tumor and A431-derived tumor were collected. This amount was reduced compared to the amount of bead-containing BNC per mouse, but GFP expression was observed in NuE-derived tumors but not in A431-derived tumors (FIG. 12). No GFP expression was observed in mouse liver and kidney (data not shown).

Example 8
Efficient ex vivo delivery of 35-kbp GFP plasmid using liposome-fused BNC
4.7-kbp of pEGFP-C1 was efficiently introduced into BNC and delivered to HepG2 cells or NuE cells ex vivo or in vivo. In addition, 6.4-kbp pcDNA6.2 / C-EmGFP was delivered to hepatocytes (data not shown). In order to determine the size limit of DNA, we used pAD / CMV-GFP (about 35 kbp) for encapsulation in BNC. HepG2 cells and A431 cells (5 × 10 ⁴ cells / well) were seeded, and liposome-fused BNC containing 2 μg of DNA was transferred to HepG2 cells and A431 cells the next day. After 48 hours, GFP expression was observed in HepG2 cells as expected, but not in A431 cells (FIG. 13). Effective transfection with liposome-fused BNC (Fusion) was approximately 10% (n = 400), but less than 1% with electroporation (EP) (FIG. 14).

A) In the Example of this invention, it is the graph which showed the isolation | separation condition after carrying out the ultracentrifugation of the HBsAg fusion | melting particle resulting from fusing the liposome which included the introduction | transduction substance, and the hollow nanoparticle. B) Electron micrographs of BNC (right) fused with liposomes (left), BNC (middle), and liposomes containing 100-nm polystyrene beads were observed using TEM and subsequent negative staining. Scale bar, 100 nm. In the examples of the present invention, HBsAg fusion particles obtained as a result of encapsulating hollow nanoparticles via FITC-labeled polystyrene bead-encapsulated liposomes were contacted with human hepatoma-derived cells HepG2 and human colon cancer-derived cells WiDr used as control cells, respectively. 3 shows the result of quantifying the amount of FITC-labeled polystyrene beads in each cell. RFU is a relative fluorescent unit. Fig. 2 shows confocal laser fluorescence micrographs of HepG2 and WiDr when HBsAg fusion particles obtained as a result of being encapsulated in hollow nanoparticles via liposomes containing RITC-labeled polystyrene beads are brought into contact with HepG2 and WiDr, respectively. It is the figure which showed the confocal laser fluorescence micrograph showing that FITC labeled polystyrene bead inclusion HBsAg fusion particle can introduce | transduce a FITC labeled polystyrene bead very specifically to human liver cancer origin cell NuE. Tumor area of tumor-bearing mice transplanted with NuE (with fluorescence), normal liver of mice (without fluorescence). In an embodiment of the present invention, a ZZ-HBsAg fusion particle formed as a result of encapsulating a hollow nanoparticle in which a biorecognition molecule has a binding ability with an antibody through a FITC-labeled polystyrene bead encapsulated liposome, and human epithelium When the anti-hEGFR antibody-presenting ZZ-HBsAg fusion particle conjugated with a monoclonal antibody against the growth factor receptor (hEGFR) (anti-hEGFR antibody) is contacted with human squamous cell carcinoma-derived cell A431, it is in A431 cells The result of quantifying the amount of FITC-labeled polystyrene beads is shown. As a control, the results of ZZ-HBsAg fusion particles without antibody were also shown. ZZ-HBsAg fusion particles that were formed by encapsulating in ZZ-HBsAg particles via liposomes encapsulated in FITC-labeled polystyrene beads and anti-hEGFR antibodies combined with anti-hEGFR antibodies were contacted with A431 cells. The confocal laser fluorescence micrograph of the A431 cell at the time is shown. As a control, the results of ZZ-HBsAg fusion particles without antibody were also shown. Anti-hEGFR antibody display ZZ-HBsAg fused particles combined with anti-hEGFR antibody and ZZ-HBsAg fused particles that can be encapsulated in ZZ-HBsAg particles via liposome encapsulated with RITC-labeled polystyrene beads, derived from A431 cells and human breast cancer 2 shows confocal laser fluorescence micrographs of A431 cells and MCF-7 cells when cells are contacted with MCF-7, respectively. In an embodiment of the present invention, human colon cancer-derived cell WiDr using HBsAg fusion particles obtained as a result of being encapsulated in hollow nanoparticles via liposomes encapsulating a green fluorescent protein (GFP) expression gene and human liver cancer-derived cells HepG2 and control cells 2 shows confocal laser fluorescence micrographs of HepG2 and WiDr when they are contacted with each other. Ex vivo delivery of rhodamine labeled 100-nm polystyrene beads (Rho-beads) by liposome-fused BNC. Rho-beads were encapsulated in liposome-fused BNC. These BNCs were applied to HepG2 cells and WiDr cells and incubated for 6 hours. A) Cells were observed under a confocal microscope. Scale bar, 50 μm. B) RFU of cells was measured with a microplate reader. In vivo delivery of Rho-beads by liposome-fused BNC. Rho-beads were encapsulated in liposome-fused BNC. These BNCs were intravenously injected into a mouse xenograft model (nude mice bearing a tumor derived from NuE cells and a tumor derived from A431 cells). After 16 hours, FITC-labeled tomato lectin was injected intravenously and then sacrificed. Fluorescence was observed in sections of NuE derived tumors. Scale bar, 100 μm. Ex vivo gene delivery by liposome-fused BNC. The GFP plasmid was encapsulated in liposome fusion BNC. These BNCs were applied to HepG2 cells and WiDr cells. After 48 hours, GFP expression was observed under a confocal microscope (A), and RFU was calculated with Imaging J software (B). Scale bar, 100 μm. In vivo gene delivery by liposome-fused BNC. The GFP plasmid was encapsulated in liposome fusion BNC. These BNCs (50 μg) were injected intravenously into mouse xenograft models (nude mice bearing tumors derived from NuE cells and tumors derived from A431 cells). After 7 days, the tumor sections were observed for fluorescence. Scale bar, 100 μm. Delivery of large plasmids ex vivo by liposome-fused BNC. The 35-kbp GFP plasmid was encapsulated in liposome-fused BNC. These BNCs were applied to HepG2 cells and A431 cells. After 48 hours, the expression of GFP was observed under a confocal microscope. Scale bar, 100 μm. The efficiency of the uptake method. 100-nm polystyrene beads (A) and pcDNA / CMV-GFP (~ 35kbp) (B) transfected into HepG2 cells by electroporation (EP, white bars) and BNC-liposome fusion (Fusion, black bars) did.

Claims (11)

A method for producing a composite particle of nanoparticles and liposomes encapsulating an introduced substance, comprising fusing hollow nanoparticles containing a hepatitis B virus protein or a variant thereof with a liposome encapsulating the introduced substance. The method according to claim 1, wherein the hollow nanoparticles have a particle size of 80 to 130 nm. The method according to claim 1, wherein the composite particles have a particle size of 130 to 500 nm. The method according to claim 1, wherein the composite particles have a particle size of 150 to 400 nm. Hollow nanoparticles containing hepatitis B virus protein or variant thereof are composed of 70 to 90 parts by weight of hepatitis B virus protein or variant thereof, 5 to 15 parts by weight of lipid, and 5 to 15 parts by weight of sugar chain The method of claim 1, wherein: The method according to any one of claims 1 to 5, wherein the hollow nanoparticles are previously freeze-dried or spray-dried. A composite particle comprising a nanoparticle part composed of hepatitis B virus protein or a variant thereof, phospholipid, lipid and sugar chain, and an exogenous introduction substance encapsulated in the nanoparticle part. The composite particle according to claim 7, wherein a particle diameter of the composite particle is 200 to 400 nm. The said nanoparticle part is comprised from 70-90 weight part hepatitis B virus protein or its modification, 6-75 weight part lipid, and 5-15 weight part sugar chain. Composite particles. A composite particle of nanoparticles and liposomes encapsulating an introduction substance, which can be obtained by the method according to any one of claims 1 to 6. The method to introduce | transduce the introducing | transducing substance including making the composite particle in any one of Claims 7-9 or the composite particle of Claim 10 act on a target cell to a target cell.