WO2005099889A1 - Process for producing hollow nanoparticle with use of liposome as template - Google Patents

Abstract

Liposome having its surface laminated with two or more layers of a compound containing at least one polymer; a process for producing the same; and a method of use thereof. There is provided a liposome characterized in that a positively or negatively charged liposome is alternately laminated with a compound containing at least one negatively or positively charged polymer by electrostatic interaction. Further, there is provided a liposome characterized in that a liposome having a reactive functional group is alternately laminated with a compound containing at least one type of polymer through chemical bond.

Description

 Description Method for producing hollow nanoparticles with ribosomes as type II

 The present invention provides a method for producing nano-sized hollow particles by laminating a compound containing at least one polymer on the surface of a lipid membrane such as a ribosome, and a compound containing at least one polymer produced by the method. It relates to nano-sized hollow particles composed of liposomes stacked on the surface. Background art

 In recent years, nanotechnology has been attracting attention as a driving force for a new era of science and technology.

Nanotechnology is the creation of new materials, devices, and systems that use ultra-fine, high-performance, and low-energy consumption by controlling the structure of atoms and molecules in ultra-fine fields on the order of nanometers. Generally, there are only about 100 types of atoms in the region below the nanometer order, and it can be said that this is a world with poor individuality. On the other hand, in the nanometer-order "" region, atoms are arranged to form molecules and have various properties. Examples include various substances consisting of carbon. Diamonds, graphite, carbon nanotubes, and fullerenes all differ greatly in their properties, even though carbon is a constituent. It can be seen that characteristics that are not carbon atoms alone show various characteristics in the nanometer range depending on the spatial combination of carbon atoms. In addition, DNA is mentioned as an example in vivo. Despite the fact that DNA is a sequence of several atoms, it plays a major role in controlling genetic information in vivo. Even a slight difference in the base sequence of DNA greatly changes the individuality of living organisms. In other words, a small change in DNA on the order of nanometers will cause a large change in a macroscopic region. In this way, it can be said that the fundamental information for forming various individuals is accumulated in the nanometer range. What is nanotechnology? —It is a technology widely used to try to control molecules in the torr region, elucidate phenomena in various individuals, and develop new substances.

 At present, the concept of nanotechnology is widely used, and one of them is nanotechnology in life science. Living organisms are often created using a method called “bottom-up” in which a very small one is assembled into a large one. For example, the fact that proteins are controlled in vivo by the DNA base sequence to form an individual can be said to be exactly a bottom-up construction. At present, research is being conducted on elucidation of self-organizing behavior in vivo and development of new biomaterials by organizing nano-sized fine particles using the concept of nanotechnology in nanotechnology. As a material for such research, the development of particles in which nano-sized particles are coated with an appropriate polymer material has been attempted.

 For example, it has been reported that an uncharged hollow polymer capsule is coated with a charged surfactant, and a charged polymer substance is laminated thereon (see Patent Document 1). In addition, a technique has been reported in which a polymer is laminated on metal particles and only the laminated polymer is produced (see Non-Patent Document 1). In addition, there is a report that an appropriate surfactant or polymer is bonded to the surface of charged silica particles or the like (see Non-Patent Document 2).

 However, in these conventional studies, the nano-sized particles are hard particles such as metal particles and silica particles, and the polymer to be bonded to the particles has only one layer. And was not suitable for many uses.

 Ribosomes are used as nano-sized particles and as drug transport and release carriers. However, when the ribosome is used for such a purpose, when the drug is encapsulated in the ribosome, there is a problem that the ribosome itself is ruptured due to its low strength.

There is an example in which only one polymer is adsorbed by electrostatic interaction (see Non-Patent Document 3). This was done to improve the dispersion stability by partially adsorbing the polymer to the ribosome, and was intended to produce hollow particles. It's not something I've done. Furthermore, a single layer of polymer cannot form a stable hollow layer in the first place. -Patent Document 1 JP 2003-519565 Gazette

 Patent Document 1 Ant ipov.A.A. et al., Colloids and Surfaces A: Phys icochem.Eng.Aspects 224 (2003) 175-183

 Non-Patent Document 2 Caruso F. / Adv. Mater, 2001, 13, No. 1, January 5 Non-Patent Document 3 Ge L. et al., Col loids and Surfaces A, Phys icochem. Eng. Aspects 221 (2003) 49 -53 Disclosure of the Invention

 An object of the present invention is to provide a ribosome having at least two layers of a compound containing at least one polymer on the surface, a method for producing the ribosome, and use thereof.

 The present inventors have attempted to develop a novel method for producing fine particles by a bottom-up method and to construct a novel functional material by assembling a structure from the fine particles. First, we focused on nano-sized fine particles and prepared hollow nanoparticles consisting of biologically-derived components with uniform size using ribosomes as templates. Since ribosomes can easily control lipid composition and nanometer size, and can be modified on the surface, they can be used as a biological membrane model to study lipid properties and permeability.Material structure and function research and transport. Materials for studying the structure and function of systemic proteins and proteins that transmit information through membranes. ・ They have been used to elucidate the mechanism of action of toxins and drugs that exert their actions by binding to glycolipids and cholesterol. In recent years, it has been clarified that substances that can be encapsulated in ribosomes can be stably retained in lipid bilayers, including macromolecules such as enzymes and plasmids, and hydrophobic and amphiphilic substances. For this reason, the effectiveness of liposomes as carriers has attracted attention. Utilizing these characteristics, ribosomes are being studied in the medical field, such as basic medicine, diagnosis, treatment, and prevention. The present inventors have proposed that Dimyris toyl phosphat idylchol ine

Ribosomes were prepared using (DMPC) and Di lauroyl phosphat idylac id (DLPA) as constituent lipids. DMPC has one negative charge and one positive charge at the same time under neutral conditions, and is neutral as a whole. On the other hand, DLPA shows a negative charge under neutral conditions because it has no positive charge. This The ribosome composed of these two lipids has a negative charge as a whole. Applying a charge to the ribosome not only prevents liposomes from aggregating due to electrostatic repulsion, but also creates an adsorption site on the ribosome surface that can be used for adsorption using electrostatic interaction. Make it possible.

 Further, the present inventors have attempted to adsorb a substance having a different charge to such a negatively charged ribosome surface in a Layer-by-Layer manner. Layer-by-Layer (alternative adsorption method) focuses on the many charges of a water-soluble polymer chain and uses the electrostatic interaction with a polymer having the opposite charge. In this technology, layers are sequentially stacked. When a charged template and a polymer electrolyte are present in the system, the polymer electrolytes cooperatively adsorb to the template when their respective critical concentrations exceed their respective critical concentrations. At this time, the surface charge is reversed because the polymer electrolyte is excessively adsorbed in excess of the charge amount of the template.

 Thus, by adsorbing the substance on the ribosome surface, the strength of the liposome itself could be increased.

 Another feature is that it can be applied not only to synthetic polymers but also to biological polymers such as proteins and nucleic acids as adsorbates. The use of biopolymers makes it possible to produce biocompatible thin films and particles. In particular, by imparting stimulus responsiveness to the laminated film on the liposome surface, it can be used for various applications such as controlled release. In order to produce a biocompatible material, the present inventors selected polypeptide as an adsorbate and produced nanoparticles composed of ribosome and polypeptide.

Since the template of the present invention is a ribosome, its size can be easily controlled at the stage of preparation. In addition, since it is possible to enclose various substances, it can be expected to be applied as a carrier. Interactions between materials that form the surface layer can be established by other intermolecular forces, bioaffinities, covalent bonds, etc., in addition to electrostatic interactions. As the polymer of the adsorbate, not only a polypeptide but also a polysaccharide or DNA can be used. By producing hollow nanoparticles composed of naturally occurring components and organizing them to construct a structure as in this study, it will lead to the creation of biomaterials with excellent biocompatibility. That is, the present invention is as follows.

 [I] a liposome in which at least two compounds containing at least one polymer are laminated on the surface,

 [2] The ribosome of [1], wherein the compound is a biocompatible polymer,

 [3] The ribosome of [1], wherein the compound is a biodegradable polymer,

 [4] the ribosome according to any of [1] to [3], wherein the compound is at least one selected from the group consisting of proteins, polyamino acids, polysaccharides, and nucleic acids;

 [5] The liposome according to any one of [1] to [4], wherein a negatively or positively charged compound is alternately stacked on a positively or negatively charged ribosome by electrostatic interaction.

 [6] A ribosome in which the ribosome is negatively charged, and a lipid selected from the group consisting of phosphatidic acid, phosphatidylglycerols, phosphatidylserine, and phosphatidylinositol as a constituent lipid of the ribosome. The ribosome of [5], including at least one of

 [7] The ribosome according to [6], wherein the constituent lipid of the ribosome is dilauroylphosphatidic acid (DLPE),

 [8] The negatively charged compound is selected from the group consisting of poly-L-lysine, hyaluronic acid and nucleic acid, and the positively charged compound is selected from the group consisting of poly-L-arginic acid and chitosan [5 ] To any of the ribosomes of [7],

[9] The ribosome according to any of [1] to [4], wherein a compound containing at least one polymer is alternately stacked on the ribosome having a reactive functional group via a chemical bond. ,

 [10] The ribosome according to any one of [1] to [4], wherein the compounds are alternately stacked by utilizing intermolecular force acting between the ribosome and the compound or between the compound and the compound.

 [I I] The liposome of [10], wherein the intermolecular force is due to complementary hydrogen bonding of the nucleic acid,

[12] The ribosome according to any of [1] to [11], wherein the ribosome contains a substance selected from the group consisting of a drug, a fluorescent substance, a bioactive substance, and a dye. [13] The ribosome according to [12], wherein the physiologically active substance is a protein or DNA; [14] The semiconductor nanoparticle is contained in a compound containing at least one polymer laminated on the surface of the ribosome [1] to [ 13] any of the ribosomes,

 [15] preparing a positively or negatively charged ribosome and alternately bonding a negatively or positively charged compound to the liposome surface by electrostatic interaction, and laminating two or more layers of polymer on the surface A method for producing a ribosome,

 [16] A method for producing ribosomes in which a compound is a biocompatible polymer [15], wherein two or more layers of the compound are stacked on the surface,

 [17] The compound is at least one selected from the group consisting of proteins, polyamino acids, polysaccharides, and nucleic acids. [15] or [16] has at least two layers of compounds containing at least one polymer on the surface. A method for producing ribosomes,

 [18] The ribosome is a negatively charged ribosome, and the ribosome comprises at least one lipid selected from the group consisting of phosphatidic acid, phosphatidylglycerols, phosphatidylserines, and phosphatidylinositols A method of producing a liposome in which at least one compound containing at least one polymer 2 is laminated on at least two layers on the surface of any of [15] to [17],

 [19] The liposome is composed of dilauroylphosphatidic acid (DLPE) whose constituent lipid is dilauroylphosphatidic acid (DLPE). A ribosome in which two or more layers of a compound containing at least one polymer are laminated on one of the surfaces How to manufacture the

 [20] The negatively charged compound is selected from the group consisting of poly-L-lysine, hyaluronic acid and nucleic acids, and the positively charged compound is selected from the group consisting of poly-L-arginic acid and chitosan [15] ] To [19], a method for producing a liposome in which at least one compound containing at least one polymer is laminated on at least two layers on the surface of any of [19].

[21] A ribosome obtained by chemically cross-linking a stack of compounds using a cross-linkable substance. [22] A solid ribosome obtained by assembling the ribosomes of any of [1] to [.14] by cross-linking the compound on the surface. Cross-linked ribosomes, and

 [23] The solid crosslinked liposome according to [22], which has a sheet or tube shape.

This specification is a description of Japanese Patent Application No. 2004-1136350, which is the basis of the priority of the present application. And / or a brief description of the drawing (s), including the content described in the drawings.

 FIG. 1 is a diagram showing a method for preparing ribosomes.

 FIG. 2 is a diagram showing the fluidity of lipids. The right shows the lipid crystal phase of T> Tc, the left shows Tc Tc (gel phase), and the center shows T = Tc.

FIG. 3 is a diagram showing the motility of lipid molecules in ribosomes above _Tc . In the figure, (a) shows lateral diffusion, (b) shows anisotropic axial rotation, and (c) shows flip-flops.

 FIG. 4 is a diagram showing a calibration curve of phospholipid concentration.

 FIG. 5 is a diagram showing a ribosome phase transition temperature. (A) shows DLPA ribosome, (b) shows DMPC / DLPA (= 1/1 by mol) ribosome, and (c) shows DMPC ribosome. FIG. 6 is a photograph showing an FE-TEM observation image of the liposome.

 FIG. 7 is a diagram showing the state of adsorption of polypeptide to liposomes.

 FIG. 8 is a diagram showing a reaction mode between CBQAC and amine.

 FIG. 9 is a diagram showing separation of a complex of ribosome and polypeptide and polypeptide by ultracentrifugation.

 FIG. 10 is a diagram illustrating the principle of the circular two-color measurement.

Figure 1 1 is a diagram showing a calibration curve of P _lys by CBQAC protein Atsusi.

 FIG. 12 is a diagram showing the structures of DLPA (top) and DMPC (bottom).

FIG. 13 is a diagram showing an adsorption isotherm of _Polys on the ribosome.

FIG. 14 is a photograph showing an SEM of the ribosome and poly-L-lysine complex after centrifugation. FIG. 15 is a diagram showing the interaction between the ribosome and the poly-L-lysine complex when the amount of poly-L-lysine is different. (A) is 5~ ² 0ppm, (b) is 50~ 100ppm, (c) is

200ρρπ! ~ 2000 ppm.

 FIG. 16 is a photograph showing the ribosome and poly-L-lysine complex after centrifugation. (A) is the case where DLPA / DMPO 0.5 / 0.5 liposome containing HPTS is used, and (b) is

DLPA / DMPC = 0.5 / 0.5 This is the case where ribosomes are used.

FIG. 17 is a diagram showing the structure of poly-L-lysine at each temperature and pH. FIG. 18 is a diagram showing a CD spectrum of poly-L-lysine. ① is a random coil (pH 7.4, 20 ° C), ② is a << helix (pH 11; 4, 20 ° C), ③ is a / 3-structure (pH 11 4, 65 ° C → 20).

 FIG. 19 is a diagram showing a CD spectrum of poly-L-lysine in the presence of ribosome (poly-L-lysine concentration is 50 ppm).

 FIG. 20 is a diagram showing the average molar ellipticity in the range of 195 to 200ηπι.

 FIG. 21 shows a CD spectrum of poly-L-lysine in the presence of ribosome (poly-L-lysine concentration: 400 ppm).

 FIG. 22 is a diagram showing the average molar ellipticity in the range of 195 to 200 nm.

 FIG. 23 shows a structure induced on the surface of DLPA / DMPC ribosome; FIG.

 FIG. 24 is a photograph showing an FE-TEM observation image of a complex of ribosome and poly-L-lysine. ,

 FIG. 25 is a photograph showing an FE-TEM observation image of a complex of ribosome, poly-L-lysine, and poly-aspartic acid.

 FIG. 26 is a diagram showing the ribopotential of ribosomes when each polypeptide is laminated. ρΗ7.4, 25 ° C and DLPA / DMPC). 5 / 0.5. FIG. 27 is a photograph showing FE-TEM observation images of a complex of ribosome and poly-L-lysine and a mixture of ribosome, poly-L-lysine and poly-aspartic acid. In the scale of the figure, (a) is 0.2 III and (b) is 100 nm.

 FIG. 28 is a diagram showing an FE-TEM observation image of ribosome and a mixture of ribosome and a complex of poly-L-lysine. The scale bar in the figure is 0. Ι ΠΙ.

 FIG. 29 is a photograph showing an FE-TEM observation image of a ribosome laminated with poly-L-lysine, poly-aspartic acid and poly-L-lysine in this order. In the scale bar in the figure, (a) is 100 nm and (b) is 20 nm.

 FIG. 30 is a photograph showing an FE-TEM observation image of a ribosome laminated in the order of poly-L-lysine, poly-aspartic acid, poly-L-lysine, and poly-aspartic acid. In the scale bar in the figure, (a) is 100 nm and (b) is 20 nm.

FIG. 31 is a diagram showing the ribopotential of ribosomes when the respective polypeptides are laminated. Figure 32 is a diagram showing a ribosome in which two or more layers of polymer are stacked on the surface containing semiconductor nanoparticles (nanoparticle phosphors).

 FIG. 33 is a diagram showing an adsorption isotherm of polyallylamine on a liposome. BEST MODE FOR CARRYING OUT THE INVENTION

 The liposome of the present invention in which the compound containing at least one polymer is laminated on the surface is a ribosome in which two or more layers of the compound containing at least one polymer are laminated on the surface of the ribosome.

 In the present invention, the term “ribosome” refers to a closed vesicle composed of a membrane-like structure composed of a lipid layer assembled in a membrane and an internal aqueous layer, and a vesicle.

(yes ic le). Further, in the present invention, a positively charged particle or a positively charged polymer refers to a particle or a particle having a larger positive charge than a negative charge in an aqueous medium near physiological pH, that is, pH 6.5 to 7.5. Polymer refers to negatively charged particles or negatively charged polymers.Particles or particles that have more negative charge than positive charge in aqueous medium near physiological pH, i.e., pH 6.5 to 7.5. Bolimer. A polymer is a compound in which the main chain in the molecule is bonded by a covalent bond and has a relatively large molecular weight, for example, a compound having a molecular weight of about 10,000 or more.One or several types of structural units are repeatedly bonded. That is, it includes a polymerized polymer. In a ribosome in which a compound containing at least one polymer of the present invention is laminated on two or more layers, the compound laminated on the liposome contains at least one polymer, and further includes a low-molecular compound, an organic or inorganic crystal, and titanium. Inorganic fine particles such as compounds, magnetic particles, and fluorescent nanoparticles may be laminated. Further, all of the compounds to be laminated may be polymers. For example, at least one polymer consisting of n layers (n is a natural number of 2 or more)

In the ribosome in which the compound containing-is laminated, the number of polymer layers out of the n layers is 1 to n, and the other layers may be layers of compounds other than the polymer. Further, the n polymer layers may be present in any number of layers counted from the inside. Hereinafter, the case where all of the compounds to be laminated are polymers will be described. However, it is not necessary that all of the compounds to be laminated are polymers, and low molecular compounds or the like may be included. That is, in the following description, the term "polymer" is used to refer to "including at least one polymer. Can be replaced with “compound other than polymer” or “compound other than polymer”. '

 In the ribosome having the polymer of the present invention laminated on the surface, the first layer of polymer interacts with and binds to the ribosome surface, and the second layer of polymer interacts with and binds to the first layer of polymer. . Further, the polymers are layered one after the other by interacting the polymer with the outermost layer of the polymer. The interaction between the liposome and the polymer may be an electrostatic interaction or an intermolecular interaction such as van der Waals attraction, or may be a bioaffinity or covalent bond. For example, there are complementary hydrogen bonds and the like found in the DNA double helix. Preferably, the polymer is laminated by electrostatic interaction. When laminating by electrostatic interaction, the ribosome surface must be negatively or positively charged. If the liposome surface is negatively charged, the first layer polymer should be positively charged and the second layer polymer should be negatively charged. Conversely, if the ribosome surface is positively charged, the polymer in the first layer should be negatively charged and the polymer in the second layer should be positively charged. In this way, the positively charged polymer and the negatively charged polymer may be alternately laminated. In the case of lamination by covalent bonding, the ribosome and the polymer and the polymer and the polymer may be covalently bonded using an appropriate functional group or the like. Such functional groups include, for example, SH group, N¾ group, phosphate group, carbonyl group, thio group, thiocarboxylic acid group, disulfide group, sulfo group, carbonyl group, acyl group, hydroxyl group, ether group, amide group, Examples include an amino group, a nitro group, an imino group, a cyano group, a vinyl group, a phenyl group, a halogen group, an amidino group, an imidazole group, and a guanidino group. In addition, the laminate composed of polymer chains may be chemically cross-linked by using an appropriate cross-linking substance. For example, they may be covalently bonded via a linker reagent or the like. Covalent bonding can be performed by a known method.

Examples of the lipid constituting the ribosome of the present invention include phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylserines, phosphatidylinositols, long-chain alkyl phosphates, and gangliosides. , Glycolipids, phosphatidylglycerols, cholesterols, etc., and phosphatidylcholines include dimyristoylphosph Apatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and as disamines, dioleylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidyl Phosphatidic acid or long-chain alkyl phosphates such as diethanolamine, distearoylphosphatidylethanolamine (DSPE)

Tilphosphoric acid, etc., phosphatidylserines, such as dipalmitoylphosphatidylserine, phosphatidylinosylsitols, dipalmitoylphosphatidylinositol, etc. GT 1b, etc .; glycolipids: galactosylceramide, darkosylceramide, lactosylceramide, phosphatide, gloposide, etc .; phosphatidylglycerols: dimyristo

And distearoylphosphatidylglycerol are preferred. Of these, phosphatidic acids or long-chain alkyl phosphates, gandariosides, glycolipids, and cholesterols have the effect of increasing the stability of ribosomes, and are therefore preferably added as constituent lipids. Also, single-chain fatty acids, alcohols and the like may be mixed. Further, it may contain not only the above-mentioned lipid but also an amphiphilic compound composed of a hydrophilic part and a hydrophobic part. In this case, any of cationic, anionic, nonionic and zwitterionic ones can be used as the hydrophilic part, and a double-chain alkyl or the like can be used as the hydrophobic part. These lipids and amphipathic compounds may contain more than one, but the length of the fatty acids of the lipids (the number of carbon atoms in the carbon skeleton of the fatty acids) or the length of the hydrophobic portion of the amphipathic compounds should be the same. Is desirable. The liposome surface must be negatively charged if the first layer polymer is attached to the ribosome by electrostatic interaction. It is sufficient that the ribosome surface is positively or negatively charged as a whole, and the above-mentioned lipids constituting the ribosome may be mixed at an appropriate ratio so that the surface is positively or negatively charged as a whole. In order to make the ribosome surface negatively charged, it is sufficient to use many negatively charged lipids as the constituent lipids of the ribosome '. Examples of the negatively charged lipid include phosphatidic acid such as dilauroyl phosphatidic acid, phosphatidyl serine such as dipalmitoyl phosphatidyl glycerin, and phosphatidylinositol such as dipalmitoyl phosphatidylinositol. Further, in order to positively charge the ribosome surface, a large amount of positively charged lipid may be used as the lipid constituting the liposome. As positively charged lipids, alkylamines such as stearylamine, amide derivatives of cholesterol such as 3- / 3_ [N- (Ν ', N'-dimethylaminoethane) -pothamyl] cholesterol, Ν — Trimethylammonioacetyldidodecyl-D—like glutamate chloride Ν— a Trimethylammonioacetyldi (10- to 20-carbon alkyl or alkenyl) —D—Dalmidomeryl chlorides , Ν— [1— (2,3-dioleoxy) propyl] Ν よ う な, such as Ν, Ν, Ν—trimethylammonium chloride

[1-(2,3-di (alkyl or alkenyl having 10 to 20 carbon atoms) oxy) propyl] 'mono-, di-, tri-trimethylammonium chloride and the like.

 Alternatively, cholesterol or a derivative thereof used as a constituent lipid of a ribosome and a group having a charge bound thereto may be used. For example, CHMES (Choles teryl hemisucc inate) into which a carboxyl group has been introduced can be used.

 In addition, since the ribosome is used for various purposes in the present invention, it needs to have a certain strength. Therefore, it is necessary to select lipids constituting the ribosome so that the ribosome has the required strength. For example, the strength can be changed by changing the amount of cholesterol or a derivative thereof.

 The charge state and the phase transition temperature of the ribosome can be appropriately selected by changing the composition of the lipid constituting the ribosome.

The charge of the ribosome or polymer-bound particles can be measured, for example, using a Compactze overnight potential measuring device ZEEC0M (Microtech Nithion) to measure ζpotential and Ζ or ΕΡΜ (electrophoretic mobility). Ζ potential of particles If the Z and EPM are negative or positive, then a positive or negatively charged polymer may bind to the surface by interaction. '

 The phase transition temperature can be measured, for example, using a differential scanning 查 ultrasensitive calorimeter.

 The ribosome can be produced according to a well-known method, and examples thereof include a thin film method, a reverse layer evaporation method, an ethanol injection method, and a dehydration-rehydration method. It is also possible to adjust the particle size of the ribosome by using an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like. The method for producing the liposome of the present invention itself is briefly described. For example, first, a mixed solution containing lipids constituting ribosomes is distilled to form a lipid film on the inner surface of the container and dissolve it in a suitable buffer. Next, after freeze-thawing is repeated several times, ribosomes having a desired particle size can be obtained by extraclusion.

 Although the particle size of the ribosome of the present invention is not limited, a liposome of several tens of dishes to several / im can be used. It is preferably from 30 to 500 nm, more preferably from 50 to 300 nm, particularly preferably from 70 to 150 nm.

 The polymer to be laminated is not limited, and proteins, polypeptides, polyamino acids, polysaccharides, nucleic acids and the like can be used. Also, natural polymers and synthetic polymers can be used. Specifically, the proteins include, but are not limited to, biological proteins such as antibodies and albumin. Polyamino acids include poly-L-lysine, polyarginic acid, polyarginine and the like. Examples of polysaccharides include chitosan, hyaluronic acid, alginic acid, heparan sulfate, dextrin, pectin, glycogen, amylose, chondroitin, and the like. Nucleic acids include DNA and RNA. Examples of the polymer include polysilane, polysilanol, polyphosphazene, polysulfazene, polysulfide, polyphosphate, polydalcolic acid, polylactic acid, polyamide, poly-2-hydroxybutyrate, polyproprolactone, polyallylamine, and the like. These copolymers can also be used.

Furthermore, as described above, in the ribosome of the present invention, not all of the compounds to be laminated need to be polymers, but low molecular compounds, organic or inorganic crystals, titanium compounds, magnetic particles, fluorescent nanoparticles, etc. May be included. Low The child compound is not limited, and examples thereof include mercaptoacetic acid.

 The liposome of the present invention can also be directly applied to a living body such as a drug delivery system. When used in applications that are applied to living organisms, the polymers used do not give adverse effects or strong irritation to living organisms over a long period of time, such as polymers derived from living organisms. ) Polymers are preferred.

 In the case where the polymer layers are bonded by electrostatic interaction, a positively or negatively charged polymer may be used. For example, poly-L-lysine can be used as a negatively charged polymer and polyarginic acid can be used as a positively charged polymer. In addition, a polyamino acid composed of arbitrary amino acids and containing a large amount of negatively charged amino acids such as lysine, etc., and being positively charged as a whole, and a polyamino acid containing a large amount of positively charged amino acids such as arginic acid. Charged polyamino acids can be used. Furthermore, chitosan or the like can be used as a positively charged polysaccharide, and hyaluronic acid or the like can be used as a negatively charged polysaccharide. Nucleic acids are negatively charged and can be bound to positively charged ribosomes or compounds that are stacked on ribosomes and are positively charged.

 The molecular weight of the polymer to be laminated is preferably higher to some extent. For example, a polymer having a molecular weight of 30,000 or more is preferably used.

 The number of polymer layers to be laminated is not limited, and is preferably at least two layers. Further, by adjusting the particle size of the ribosome used, the type of the polymer used, and the number of polymer layers, the final size of the ribosome on which the obtained polymer is laminated can be adjusted. The thickness of the layer depends on the type of polymer used, but is generally several Mi for one layer, and about 5 mn or less even when three layers are laminated. The particle size of the ribosome on which the polymer is laminated is 50nn! A liposome of uniform size can be obtained without difference between ribosome particles at ~ 500 nm.

 When a plurality of polymers are laminated, the type of the polymer is not limited, and the same type of polymer or different types of polymers may be laminated. Also,

A plurality of polymers may be mixed and laminated in one layer.

If the stacking is based on electrostatic interactions, the first layer will have a positively charged polymer First, polymers having different charge states may be alternately laminated, such as a negatively charged polymer in the second layer. '

 The lamination of the polymer can be performed as follows.

 The above-mentioned purified polymer is mixed with the ribosome prepared by the above-mentioned method and stirred. The temperature at this time is not particularly limited, and the temperature can be appropriately set at around the phase transition temperature, at a temperature equal to or higher than the phase transition temperature, or equal to or lower than the phase transition temperature according to the phase transition temperature of the ribosome. In addition, the reaction time is not limited, and the reaction may be performed with stirring for several minutes to several hours. Further, the aqueous medium used is not limited. Next, free polymer not bound to the ribosome is removed by centrifugation or the like. Next, the polymer forming the second layer is mixed with the ribosome to which the polymer forming the first layer is bonded, and the mixture is stirred. If the bond is based on electrostatic interaction, the polymer mixed at this time is a polymer having a charge opposite to that of the polymer of the first layer. In this case, the reaction temperature and the reaction time are not limited, and can be appropriately determined. After the reaction, unbound polymer is removed by centrifugation. Hereinafter, by repeating this operation, a ribosome in which a plurality of layers of polymers are laminated can be produced.

 The ribosome on which the polymer of the present invention is laminated has a certain strength because the polymer is laminated on the surface, and its shape is maintained without breaking even under an environment where physical force is applied. In addition, since the surface has a uniform charge, even in a suspended state, the dispersed state can be maintained without agglomeration. In addition, by controlling the type of lipid used as the constituent lipid of the ribosome, the type of polymer to be laminated, and the number of layers, the entire ribosome on which the polymer of the present invention is laminated (the strength as a book can be controlled). For example, a ribosome having a low strength may be used so that the drug can be released when used as a drug delivery carrier.

Further, when laminating a polymer on the ribosome, another compound can be included in the laminated polymer. Other compounds include, for example, drugs, nanosized particles such as nanophosphor particles (semiconductor nanoparticles), physiologically active substances, dyes, fluorescent substances, pigments, and the like. Here, the “nano-sized phosphor” refers to a sulfide, oxide or nitride doped with luminescent ions and having a size of 50 nm or less, and a composite particle containing a nano-sized phosphor. Including. Do as nano-sized phosphor There are a loop type and a core-shell type. In the present invention, any nano-sized phosphor can be used. Examples of the sulfide, oxide or nitride include semiconductor materials such as zinc sulfide (ZnS), cadmium sulfide (CdS), zinc selenide (ZnSe), and zinc oxide (ZnO). Mn), copper (Cu), aluminum (A1), silver (Ag) or chlorine (C1). A plurality of ions may be doped. Each doping ion can have its own light emission. In this specification, for example, ZnS doped with Mn is referred to as “ZnS: Mn”, and ZnS doped with Cu and A1 is referred to as “ZnS: Cu, Al”. Also, terbium

Even if (Tb), thulium (Tm), europium (Eu), or fluorine (F) is doped as a simple substance or as a compound, it is possible to have a specific light emitting property. The nano-sized phosphor is sometimes called a nanocrystal, a nanocluster, a quantum dot, or the like, and includes those so-called in the present invention. The nano-sized phosphor that can be used in the present invention is not limited thereto, and is disclosed in JP-A-10-310770,

A. P. Al ivisatos et al. MRS Bull., No. 2, 18 (1998), T. Trindade et al.,

Chem. Mater. 13 3843 (2001), Tetsuhiko Isobe, Future Materials 3, 26 (2003), Tetsuhiko Isobe, Journal of the Illuminating Engineering Institute of Japan 87, 256 (2003), T. Isobe, Recent Res. Dev. Mater. Sci. 3, 441 (2002), Tetsuhiko Isobe, Applied Physics 70, 1087 (2001), Tetsuhiko Isobe, Surface Science 22, 315 (2001, Tetsuhiko Isobe, Functional Materials 19, 17 (1999), Tamotsu Senna et al. Display and imaging 7,

3 (1998) and the like can be used. Also, commercially available nano-sized phosphors can be used. Commercially available nanosize phosphors include, for example, Qdot (trademark) (Sumisho Bioscience Co., Ltd.). These compounds can be laminated together with the polymer when the polymer is laminated, provided that they are mixed when the polymer is laminated. At this time, it is necessary that the compounds included in these layers also have a charge so as to interact with the polymer. Nanoparticles or the like that do not have a charge originally may be mixed by, for example, binding a cationic surfactant having a charge and a vanionic surfactant to the surface. The surfactant used in this case is not limited, but examples thereof include quaternary ammonium salts and the like as the cationic surfactant, and alkyl sulfonates and the like as the anionic surfactant. Also, when laminating polymers, other compounds such as nano-sized phosphors are synthesized. Good. In this case, the synthesized compound is contained in the ribosome on which the polymer is laminated. Other compounds may be included in the polymer stacked on the ribosome, or may bind on the outermost polymer of the stacked polymer. In any case, in the present invention, it is referred to as "the polymer contains another compound". As described above, by incorporating another compound into the laminate, it is possible to impart the properties of the compound contained in the liposome in which the polymer of the present invention is laminated. For example, when a drug is included, the ribosome of the present invention can be used as a carrier for drug delivery, and when a nanophosphor is included, the ribosome of the present invention can have fluorescent properties. It can be used as a marker for detection.

 The ribosome on which the polymer of the present invention is laminated can be used for various applications by selecting the polymer to be laminated on the surface.

 Further, various substances can be used by encapsulating various substances in the ribosome. Examples of substances contained in the ribosome include drugs, magnetic particles, dyes, fluorescent substances, facial masses, and bioactive substances. Examples of the physiologically active substance include proteins such as biological proteins, DNA, and RNA.

 Alternatively, the liposomes of the present invention can be aggregated by mixing ribosomes having different surface charges or applying pressure to the ribosomes by centrifugation or the like, thereby causing electrostatic interaction of the surface polymer ゃ bridging. Solids can be made. In this case, it can be called a crosslinked ribosome. At this time, the laminate composed of polymer chains may be chemically cross-linked using a cross-linking substance. As the crosslinkable substance used at this time, a known substance may be used. At this time, by preparing the solid in a mold, a solid aggregate of ribosomes in which the polymer is laminated in an arbitrary shape such as a sheet or a tube can be prepared. Further, once the crosslinked liposome, which has become solid, is powdered, it can be used as a powder.

 For example, the use of the ribosome laminated with the polymer of the present invention includes the following.

 (1) Drug delivery carrier

For example, a drug is encapsulated in the ribosome and used as a carrier for a drug delivery system. Can be used. At this time, by selecting a polymer on the surface, it is possible to obtain a ribosome targeted at an appropriate tissue or organ. In this case, examples of the polymer in the outermost layer include a substance that specifically binds to a protein specifically expressed in a specific tissue or organ, for example, an antibody. When a ribosome having the polymer of the present invention laminated thereon is used as a carrier for drug delivery, it is necessary to release the encapsulated drug in the body. This can be achieved by controlling the strength of the entire liposome on which the polymer is laminated. Further, as the polymer to be laminated, a polymer that dissolves in a body fluid may be used, or a biodegradable polymer may be used. Such a polymer-laminated ribosome can also be used as a sustained-release drug carrier.

 (2) Vector

 In addition, genes such as DNA can be enclosed in ribosomes and used as vectors. Also in this case, it is possible to introduce a gene encapsulated in a specific cell by using a substance that specifically binds to a protein specifically expressed in a specific cell as the outermost layer polymer. .

 (3) Marker for substance detection

 Further, the liposome obtained by laminating the polymer of the present invention can be used for detecting a substance. The ribosome containing the nanoparticles as described above can be used as a tool for detecting a substance. At this time, a protein such as an antibody that specifically binds to a substance to be detected may be used as a polymer to be laminated on the outermost layer of the ribosome. When the substance to be detected is a nucleic acid such as DNA, a nucleic acid complementary to the nucleic acid may be used.

 (4) Cosmetics

 Furthermore, ribosomes on which the polymer of the present invention is laminated can be used as cosmetics. The liposome can have a uniform charge on the surface, has good dispersibility, and can be suitably used as cosmetics. At this time, a cosmetic pigment, a cosmetic pigment, or an appropriate drug may be encapsulated in the ribosome.

 (5) Brightener

The ribosome on which the polymer of the present invention is laminated can also be used as a brightener. The Since the ribosome is hollow, it has a high refractive index and can be suitably used as a brightener. '

 (6) Paint pigment

 Normally, titanium oxide is used as a white pigment of a paint, but ribosomes on which the polymer of the present invention is laminated can be used as a substitute for titanium oxide. The ribosome of the present invention can be used without deteriorating whitening performance or masking performance. In addition, it can be used as a paint pigment that exhibits a specific color by enclosing a dye or pigment inside the ribosome. When used as a paint pigment, it can be used as a water-based paint in a dispersed state, and can be used as a powder paint or a solvent-based paint in a crosslinked ribosome state as described above.

 (7) Paper coating agent

 It is also possible to add a ribosome on which the polymer of the present invention is laminated to a coated paper coating, and in this case, it is possible to improve the gloss and printability of the coated paper and the opacity of the lightweight paper.

 (8) Lightening agent for resin

 Since the ribosome on which the polymer of the present invention is laminated is hollow and lightweight, if it is mixed with, for example, a resin, the weight can be reduced without reducing the resin performance.

 (9) One sheet of thermal printer

 When producing the thermal paper, a liposome layer in which the polymer of the present invention is laminated between the thermal agent layer and the paper layer can be provided as a heat insulating layer. As a result, the sensitivity of the thermal paper can be improved.

 (10) Hemostatic material

 When blood needs to coagulate during surgery or to prevent adhesions, it can bind to each other to form aggregates. An anti-adhesion effect can be achieved.

 (11) Alternative organs, etc.

As described above, the ribosome on which the polymer of the present invention is laminated can be used as a crosslinked ribosome, and can be formed into an appropriate shape such as a film or a tube. In this way, the ribosome of the present invention can be used as an artificial skin, an artificial blood vessel, an artificial nerve guide tube, or the like. Can be used during surgery. At this time, it is desirable to use a biocompatible polymer as the polymer to be laminated on the ribosome.

 (12) Cell culture substrate

 The ribosome laminated with the polymer of the present invention formed into a film can also be used as a substrate for cell culture. In this case, a protein or polysaccharide serving as a scaffold for cell growth may be bonded to the outermost layer of the polymer.

 Hereinafter, a specific description will be given based on examples of the present invention. However, the present invention is not limited to the following examples.

 [Example 1] Preparation of ribosome and its characterization

 1. Method of ribosome preparation and its characterization

 The liposome was prepared and characterized by the following method.

 (1) Ribosome preparation

 1,2-Dilauroyl-sn-Glycero-3-Phosphate Monosodium Salt (DLPA) and L-«-Phosphatidylcholine, Dimyristoyl (DMPC) were used as the constituent lipids of the ribosome.

 A total of 10 mol of phospholipid, which is a component of the lipid membrane, was weighed into a 50 ml eggplant-shaped flask equipped with a pickpocket, and dissolved in a small amount of methanol. The pressure was reduced using a rotary evaporator to distill off the solvent, and a lipid film was obtained on the wall of the flask. The mixture was transferred to a desiccator overnight and dried under reduced pressure for 6 hours using a vacuum pump to completely remove the solvent. next

1 ml of Buffer (mainly lOmM HEPES, pH 7.4 in this example) was added and hydrated to form multilamellar vesicles (MLV). At that time, peeling and hydration from the wall surface of the lipid film were promoted by applying mechanical vibration to the bathtub-type sonike overnight. To obtain a lipid bilayer of closed vesicles by hydrating a lipid film composed of saturated lipids, the lipids must be in a liquid crystalline state. Using. Next, ribosomes were fused by freeze-thawing to form large ribosomes. The operation of freezing the suspension using liquid nitrogen, thawing at room temperature, and heating to the phase transition temperature of phospholipid + 10 ° C was repeated three times. Finally, single unilamellar vesicles (SUVs) of uniform size were prepared by the extrusion method. Ribosome size in Extruder (Avestin) It can be controlled by changing the pore size of the polycarbonate filter to be set (Fig. 1). '

 The freeze-thaw method is as follows.

 The phospholipids that make up liposomes are amphipathic molecules whose hydrophilic groups hydrate when suspended in an aqueous solution. On the other hand, hydrophobic groups are pushed out of the water environment and aggregate with each other. Therefore, a lipid bilayer having a bilayer structure is formed. When the ribosome suspension thus formed is frozen, the hydrated water molecules are dehydrated to form ice. In the next melting process, the water molecules bind to the hydrophilic groups again, but it is thought that the bilayer rearranges in this process, and liposome fusion occurs when the bilayer rearranges.

 The extrusion method is as follows.

 This is a type of extrusion method used to prepare SUVs and LUVs of uniform size and to regulate the size of ribosomes by passing MLV through pores of a certain size. Compared to other methods, it is easier to operate and can be processed more quickly, has little degradation of lipids, and has better reproducibility. In addition, a relatively high-concentration lipid solution can be used, and there are advantages such as a high yield.

 (2) Particle size measurement by dynamic light scattering (DLS)

Dynamic Light Scattering (DLS) is a method to obtain particle size and particle size distribution by fluctuation of light scattering due to micro Brownian motion of particles. Has a measuring range of ~ 5 m. When laser light is applied to a system suspended with particles in a solvent, the scattering intensity changes over time due to Brownian motion of the particles. The light scattered by the non-uniformity in the solvent fluctuates in its non-uniformity itself, and because of the movement, the light spreads due to a change in the frequency. Here, the frequency change is mainly Rayleigh scattering in which the center frequency of the scattered light is equal to the frequency of the incident light, and reflects thermal fluctuations that have no inherent frequency. The shift of this frequency is extremely small compared to the frequency of the incident light, and the beat signal is transmitted to the photomultiplier using a method that mixes homodyne light with an optical system using a pinball and generates a beat (interference). To perform photoelectric exchange. The output signal of the photomultiplier tube becomes a pulse diffused from each other (this pulse is proportional to the amount of light). By calculating the number of pulses, the particle You can get information about exercise. The method of performing such photon calculations and taking the correlation of the photoelectron pulse train is called photon correlation spectroscopy (PCS).

When the secondary scattering intensity-time correlation function g ⁽²⁾ (r) is obtained from the photon pulse train obtained by the measurement, a correlation function with a long correlation time is obtained for large particles, and a correlation function with a short correlation time is obtained for small particles. Can be This correlation function contains information on the translational motion of suspended particles, and the particle size and particle size distribution can be obtained by using the calculation formula. Regulation e- 2 c

The classified quadratic correlation function g ( ² ) can be expressed as follows.

T: correlation function

 β: constant depending on experimental conditions

g ⁽¹⁾ (r): Normalized first-order correlation function (correlation function of scattered electric field) ''

 Here, the first-order correlation function is expressed as follows for spherical particles.

Number 2 g ^(l) = exp [-2q ² Dr) (Equation 2)

Number 3 m

 sin (Equation 3)

D: translational diffusion constant

 n: Refractive index of solvent

λ ₀ : wavelength of one laser beam

Θ: scattering angle The translational diffusion constant D can be easily obtained from the above equation, and the particle diameter can be obtained from the Einstein-Stokes equation. '

 Number 4

D = (Einstein-Stokes equation) (Equation 4) r: Stokes radius of particle

 D: viscosity coefficient of solvent

 k: Boltzmann coefficient

 T: Absolute temperature

To obtain the translational diffusion coefficient D from g ( ² ) (te) obtained by the measurement and the above equation, the quadratic correlation function is logarithmic, and in histogram analysis, the histogram is displayed by dividing the analysis particle range. . As a result, a scattering intensity distribution can be obtained, but it is possible to obtain a weight distribution relating to the particle diameter by correcting with a weight distribution conversion coefficient. A number average distribution can also be obtained from this weight distribution.

 In this study, the prepared ribosome suspension was diluted to an appropriate concentration with Buffer, and the measurement was performed using PAR-II (Otsuka Electronics) under the condition of 50 accumulations.

 (3) Measurement of electrophoretic mobility (EPM) and ζ potential

 If there are acid or base sites on the particle surface, the particle surface will be positively or negatively charged, and these can be sites for adsorption and exchange of ions. In an aqueous electrolyte solution, the electrolyte ions are attracted to the surface charge of the particles, forming an electric double layer. When particles in this state are placed in an electric field, they move in the direction of the oppositely charged electrode. This phenomenon is called electrophores is.

Electrophoretic mobility (EPM) measurement is a method of determining the charge state of the particle surface by placing the particle in an electric field and measuring the speed of its movement. When particles are dispersed in an electrolyte and an external electric field is applied, the particles undergo electrophoresis. At this time, the viscosity of the liquid becomes a resistive force, the viscous force and the force received from the electric field are balanced, and the particles move at a constant speed. By measuring the speed of this movement and dividing by the strength of the electric field, the EPM of the particles is determined. Number 5

EPM _(v ., _ = Vx (Equation v: Moving speed (Π · sec " ¹ )

 d: distance between electrodes (cm)

 V: Voltage (V)

 ζ Potential is expressed as follows.

ε: dielectric constant of aqueous solution

 7 ?: Viscosity of aqueous solution

 In this study, the リ ボ and ζ potentials of the prepared ribosomes were measured using a Compactze overnight potential measuring device ZEEC0M (Microtech NITION) at a solution temperature of 25 ° C and a migration voltage of 20 mV.

(4) Measurement of transition temperature _Tc by differential scanning ultra-sensitive calorimeter (Nano-DSC) In this study, the ribosome phase transition temperature ( _TG ) was measured by differential scanning ultra-sensitive calorimeter ( _TG ). The measurement was performed using Nano-DSCI I) (CSC).

 Scanning calorimetry is a method for measuring the flow of heat in and out of a material as the temperature changes. As one of the thermal analysis methods, the signal observed when equilibrium is present at each temperature corresponds to the heat capacity. Targets include temperature transition (thermal denaturation) of proteins and biopolymers. In addition to being able to easily measure the thermal denaturation temperature, it is possible to directly determine thermodynamic quantities such as changes in enthalpy and heat capacity. It is also possible to obtain information on the domain structure.

In the Nano-DSC used in this study, the thermal insulation of the cell was not controlled in order to suppress the noise and facilitate the temperature drop, and the temperature of the jacket was increased by the thermoelements installed in the jacket surrounding the cell. The temperature follows the system. There is a sample cell and a comparison cell. The comparison cell contains the solvent (Buf fer used in this study). It is. If heat flows in or out during the temperature rise, the heat is compensated by a compensation heater attached to the cell so as to cancel it, and both cells are controlled to keep the same temperature. The compensation heat flow at this time is recorded.

 The ribosome phase transition temperature is as follows.

 It is known that when a liposome is formed from a lipid consisting of a saturated hydrocarbon chain, a liposome takes a gel phase with reduced membrane motility at low temperatures. In the gel phase, the side chains of the lipid molecules are ordered. As the temperature of the lipid bilayer in the gel phase is increased, the liquid crystal phase (iduid crystal lin phase) is reached above a certain temperature. The temperature at which the gel phase changes to the liquid crystal phase is called the gel-liquid crystal transition temperature, or the phase transition temperature () (Figure 2).

When the temperature is above T _c , the lipid molecules on the ribosome surface are rich in mobility, and the fluidity (fluidity) of the membrane is high. As shown in Fig. 3, in the liquid crystal phase, lipid molecules move in two dimensions in the membrane, lateral diffusion (lateral diffusion), and anisotropic axial rotation in the membrane (anisotropic axial rotation). , The flip-flop (fl ip flop) moves from one side of the double layer to the other.

 From these facts, it is considered that the membrane fluidity of liposomes depends on temperature.

 (5) Measurement of phospholipid concentration

 In this example, the concentration of ribosomes produced by measuring the concentration of phospholipid was examined using phospholipid-Test Co. (Wako Pure Chemical Industries, Ltd.). 'This kit measures the phospholipid concentration by the permanganate incineration method.

 In general, phospholipids are quantified by scientific methods, but have disadvantages such as high temperature heating conditions, complicated operations, and use of dangerous chemicals. In contrast, this permanganate incineration method has an advantage in safety that phospholipids can be easily and completely decomposed in a boiling water bath.

When a ribosome suspension composed of phospholipids is used as a sample and trichloroacetic acid is added thereto, the phospholipids precipitate together with the protein. After centrifugation, the supernatant containing the dissolved inorganic phosphorus is removed to separate the phospholipids together with the protein. Sulfuric acid and permanganate are added to the separated precipitate and heated in a boiling water bath to remove most of the organic matter. Is oxidatively decomposed to produce phosphoric acid, which is also a component. When ammonium molybdate and a reducing agent are added to the inorganic phosphorus generated here, molybdenum blue is formed and exhibits a blue color. By measuring the absorbance of this blue color, the phospholipid concentration in the sample is calculated.

 In practice, weigh 0.1 ml of the ribosome suspension in a test tube, add 0.4 ml of sulfuric acid, and heat in a boiling water bath for 10 minutes. While mixing, add 2. Oml of potassium permanganate, an oxidizing agent, and heat for another 30 minutes. After leaving at room temperature for 10 minutes, add 2.0 ml of ammonium molybdate, a decolorizing agent, and mix using a mixer. Add sodium sulfite as a color former and heat in a 37 ° C water bath for 20 minutes. Finally, cool the solution from outside the test tube with running water for 5 minutes, measure the absorbance using a 660 nm filter, and calculate the phospholipid concentration.

 (6) Preparation of ribosomes of different sizes

 In this study, ribosomes are produced by extrusion method. With this method, it is possible to control the size of the ribosome produced by the pore size of the poly-one-ponate filter. Therefore, using a liposome with a composition of DLPA / DMPC = 0.5 / 0.5 using a polycarbonate filter with a pore size of 50 nm, 100 nm, and 400 nm, liposomes of different sizes were prepared, and the particle size was determined by dynamic light scattering. We examined the control of liposome size by measuring.

 (7) Observation by field emission transmission electron microscope (FE-TEM)

 A transmission electron microscope (TEM) is a device that irradiates a sample with an electron beam and mainly observes its internal structure. In addition to the shape and surface structure of the sample, the degree of aggregation of the sample, Observation of crystal patterns, existence of lattice defects, crystal orientation, etc. is possible.

 When a sample is irradiated with an electron beam, electrons that pass through the sample (transmitted electrons) and electrons that are scattered by interaction (scattered electrons) are generated. Normally, the scattered electrons are cut by the objective movable aperture, and a bright-field image that forms only transmitted electrons is observed. On the other hand, when scattered electrons are imaged, a dark-field image is obtained.

The principle of TEM is the same as that of an optical microscope, but observation is performed in a high vacuum. An electron beam is used as the light source, and an electromagnetic lens is used as the lens. Electron beam is longer wavelength than visible light Is short, so observation at a higher magnification than with an optical microscope is possible. Electromagnetic lenses generate a magnetic field that distributes in a convex shape when a current flows through a coil, and acts as a convex lens for electrons. In TEM, it is necessary to flake a sample to 0.1 xm or less, which allows electron beam transmission. Therefore, sample preparation such as ultra-thin sectioning and ion milling is used for thinning. There are several types of electron beam sources.The general type is a tungsten hairpin type, which generates electrons by passing a current through a tungsten filament, and the high-resolution type generates an electron by applying an electric field to a Si chip. A field emission type transmission electron microscope (FE-TEM) is used. FE-TEM is extremely effective in evaluating materials on a micro level, and is used in fields such as ceramics, metals, semiconductors, polymer materials, and biological samples.

 In this example, the ribosomes prepared were negatively stained to observe the shape by FE-TEM.

 Negative staining is as follows.

 In the negative staining method, unlike the general staining method, the sample and the staining agent do not react, and the density between the sample and the indicator film is reduced by surrounding the sample with a low-molecular compound, which is structured and has a high density. This is a method for observing the sample size and surface microstructure. That is, when the electron beam is irradiated with the dye that does not transmit the electron beam excluded from the lipid association site, the association site is exposed more strongly. Therefore, the bilayer structure appears as a white band.

 In this example, ribosomes are negatively stained and observed.

 Carbon deposition was performed on a copper grid coated with a collodion film, and a ribosome suspension diluted to about 1. OmM was dropped onto the dalid. After 1 to 2 minutes, the excess liquid was sucked up using a filter paper, and a lw% ammonium molybdate solution was added dropwise as a staining agent to remove the excess liquid in the same manner. Immediately after the preparation of this sample, observation using FE-TEM was performed.

 2. Production of ribosomes and results of their characterization

 The results of the produced characterization were as follows.

(1) Confirmation of ribosome size uniformity DLPA / secret O0 / 1.0, 0.1 / 0.9, 0.5 / 0.5, 1.0 / 0 Composition of liposomes (molar ratio, respectively) was prepared to control the size of ribosome. As a result of measurement by a dynamic light scattering method, ribosomes of any composition were monodispersed at about 約 ηπι (confirmed that D _ff / D _N ½1.10).

 (2) Measurement of phospholipid concentration

 • Creating a calibration curve

 In the present embodiment, the measurement of the phospholipid concentration is calculated by using a phospholipid-Test Corporation.

To prepare a calibration curve, dilute the phospholipid standard solution enclosed in the phospholipid-test container to a concentration of 150, 300, 450, and 600 mg / dl using 5% trichloroacetic acid in acetic acid, and dilute them. A calibration curve was created using the data (Fig. 4). Since linearity and the like R ² value to an approximate curve was obtained, used in and after the experiment Figure 4 with a calibration curve.

 • Measurement of phospholipid concentration in ribosome suspension

 Consider the phospholipid concentration of the ribosome suspension prepared with the compositions DLPA / DMPO0 / 1.0, 0.1 / 0.9, 0.5 / 0.5, 1.0 / 0. The prepared ribosome suspension was diluted two-fold and weighed 100, and the phospholipid concentration was measured using as a sample.

 In the case of ribosomes made only with DMPC, the absorbance is 0.170, the concentration is 340 mg / dl from the calibration curve, and the actual concentration is 680 mg / dl (6.8 g / l) due to double dilution. At that time. Therefore,

 Number 7

⁶ · ⁸ ( ^{695 '96} Γ ^{9 '77 χ 1} ° ^-3 (" ⁹ · ⁷⁷ (ι). Similarly, if the phospholipid concentration in the liposome suspension of another composition is measured, DLPA / DMPC = 0. 1 / 0.9 for 9.2 machines, 0.5 / 0.5 for 10.46 mM, DLPA ribosome

It was 10. 11 lmM (when mixed, it was calculated using the average molecular weight of DLPA and DMPC). All liposomes are prepared and weighed so that the phospholipid concentration is 10 mM, so the ribosome of any composition is calculated to be about 10 mM, although there are some errors. From this, it can be said that a liposomal suspension was obtained with a yield of almost 100%. The phospholipid concentration is determined in the subsequent experiments using such a method.

(3) Measurement of ribosome electrophoretic mobility

 DLPA / hidden C = 0 / 1.0.0, 0.1 / 0.9, 0.5 / 0.5, 1.0 / 0. Create ribosomes with the composition of 1.0 / 0, and use ZEECOM for electrophoretic mobility (EPM ) And ζ potential were measured. As a result, it can be seen that the ribosome containing ΡΑ shows a negative charge, and that the absolute values of ΕΡΜ and ζ potentials increase as the fraction increases (Table 1). This suggests that the negative charge on the ribosome surface increases with the amount of the negatively charged lipid ΡΑ. In addition, since う る can be an adsorption site having a negative charge on the ribosome surface, it is conceivable that the number of adsorption sites increases with the amount of ΡΑ charged.

 Table 1 Ribosome electrophoretic mobility (Ε Ρ Μ) and ζ potential

： Potential / EPM / m 's ^-1 ' cm-

DLPA / DMPC

 mV ν- '

 0: 1 0.000 0,000

 0.1: 0.9 -56.080 -4.376

 0.5: 0.5 -93.295 -7.281

 1: 0 -136.595 -10.657

(4) Measurement of ribosome phase transition temperature

 DLPA / DMPC = (a) 1.0 / 0, (b) 0.5 / 0.5, (c) A ribosome was prepared with the composition of 0 / 1.0, and the phase transition temperature using Nano-DSC was determined. Figure 5 shows the measurement results. The phase transition temperatures were (a) 30.6 ° C, (b) 29.2 ° C, and (c) 24.5 ° C. The ribosome peaks (a) and (c), which consist of a single lipid of PA and PC, show sharp peaks, whereas the ribosome peak (b), which consists of two types of lipids, PA and PC, shows a sharp peak. It shows a gentle peak. In addition, since only one peak appears, it is considered that the ribosome consisting of the two components, PA and PC, does not form a domain and is undergoing slow metastasis.

 (5) Preparation of ribosomes of different sizes

In this embodiment, ribosomes are produced by the extrusion method. We attempted to produce liposomes of different sizes by using a pore size filter with a pore size of 50 nm, 100 nm, and 400 nm. DLPA / DMPC = 0.5 / 0.5 composition.

 Table 2 summarizes the size of the ribosomes produced.

 Table 2 Diameter of liposomes

 Filter pore size

/ nm Liposo ^ — diameter of the membrane / nm D _W / D _N

50 68.1 1.08

100 103.2 1.09

400 161,7 1.18

In the case of lOOnm size, it can be said that ribosomes of about the same size as the pore size of the filter are monodispersed. In the case of 50 nm, the particle size is slightly larger, but it can be said that the liposome is produced in a monodispersed state. It is thought that ribosomes with a size close to the pore size of the filter can be produced by devising, for example, increasing the number of extrusion operations performed by the extension zone. At 40 Onm, the polydispersity is higher than the former, and ribosomes are produced in a size smaller than the pore size. This is considered to be one of the causes of the lack of a sufficiently large MLV during the freeze-thaw operation in ribosome production. For this reason, the same production was performed by increasing the number of freeze-thaw operations, but the result was not as large as the difference between the results and the slightly larger size.

 For this reason, in systems using DLPA and DMPC as constituent lipids, 50ηπ! It can be said that liposome fabrication in the range of about 150 nm is possible. In addition, it can be said that lOOnm-sized liposomes can be easily manufactured. In addition, it is considered that the width of size control can be changed by changing the constituent lipids and the operating conditions during preparation.

 (6) Observation of ribosome shape

 Negative staining was applied to the prepared ribosome with the composition of DLPA / DMPC = 0.5 / 0.5 and FE-

Fig. 6 shows the results of TEM observation. By performing negative staining, the lipid membrane itself You can see that it is not dyed and is white. The collapse of the shape is considered to be the effect of the drying process. Overall, it can be said that the ribosome remains spherical.

 As described above, by producing ribosomes using the extrusion method, ribosomes having a uniform size of about 100 nm and a concentration of about 10 mM could be produced. The difference in surface charge was confirmed by changing the composition of DLPA and DMPC. We found that the higher the proportion of DLPA, the more negatively the surface was. From this, it can be said that ribosomes containing DLPA as a constituent lipid have adsorption sites utilizing electrostatic interaction.

 [Example 2] Adsorption of polypeptide on liposome surface

In this example, the adsorption of a polypeptide to the surface of a ribosome of type I will be examined. As described in Example 1, it is known that ribosomes containing DLPA in the constituent lipids are negatively charged. Therefore, particles with different surface charges are obtained by applying poly-L-lysine (P _Lys ), a polycation, and poly-L-aspartic acid (P _Asp ), a holion, to the ribosome surface in a layer-by-layer manner. Was attempted. Figure 7 shows a conceptual diagram of Layer-by-Layer.

 We also report on the fabrication of structures using liposomal-polypeptide complexes with different surface charges and utilizing electrostatic interaction.

 1. Adsorption of polypeptide to liposome surface

 Adsorption of the polypeptide on the liposome surface and examination of the obtained liposome were performed by the following methods.

(1) Adsorption of poly-L-lysine (P _Lys ) to liposome surface

By containing the acidic lipid DLPA as a constituent lipid, the ribosome is negatively charged and has an adsorption site (see Example 1). The polycation poly-L-lysine.Plys (P _Lys ) was allowed to act on the ribosome surface, and its behavior was investigated by adsorption by electrostatic interaction. It is generally said that ribosomes consisting only of PA are less stable than ribosomes consisting only of PC. Therefore, in this study, we examined the adsorption of P _Lys mainly using DLPA / DMPO 0.5 / 0.5 ribosomes. (i) Adsorption measurement

A ribosome solution (final phospholipid concentration: 0.5 mM) and a _Lys solution of a predetermined concentration were reacted under stirring conditions of 25 ° C, 700 rpm, 30 min, and pH 7.4. After the reaction, ultracentrifugation was performed at 4 ° C, 70, 000 rpm, and 45 min using a small-sized ultracentrifuge CS 100GX (Hitachi Machine) to precipitate the liposome-P _Lys complex from the mixture. . The concentration of the supernatant was calculated PL _ys concentration adsorbed by measuring using a CBQCA.

 Here, the CBQCA method is one of the protein quantification methods. In this method, 3- (4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA), which has been used as a reagent for derivatization of amines in a mouth, is used. Although CBQCA is non-fluorescent in aqueous solution, in the presence of cyanide, CBQCA reacts with primary amines in proteins to form very fluorescent derivatives (Figure 8). Dilute the protein-containing sample with an appropriate buffer, add KCN, and start the reaction by adding CBQCA. After incubating for 1 hour (up to 5 hours if necessary), measure the fluorescence intensity of the sample at the absorption / emission wavelength of 465/550 nm.

 (i i) Examination of reaction time

The reaction time was examined using ribosomes having a composition of DLPA / DMPC = 1.0 / 0, 0.5 / 0.5, and 0 / 1.0 and P _Lys . 25 ° as ribosome suspension 500 / z L (final phospholipid concentration 0. 5 mM) and P _s solution 500 L (final concentration 50 ppm) (:., Was reacted under stirring conditions of 700rpm this time, the reaction time The comparison was made at 20 min and 80 min.

 (i i i) Creation of adsorption isotherm

10 mM HEPES pH 7. 4 with DLPA / DMPO0. 5/0. To produce ribosomes 5 composition was prepared an adsorption isotherm by changing the concentration of PL _ys added.

500 L of ribosome solution (final phospholipid concentration: 0.5 mM) and 500 L of P solution were reacted under stirring conditions of 25 ° C., 700 rpm, 30 min, and pH 7.4. After the reaction, 4 ° C, 70, OOOrpm , performs ultracentrifugation under the conditions of 45Pye, the P _s concentration of the supernatant was adsorbed by measuring using a CBQCA! _{The ys} concentration was calculated. This operation was performed at each concentration to obtain an adsorption isotherm.

 (iv) Purification method

Purification by ultrafiltration to obtain the prepared l iposome-P ^ complex in a monodispersed state saw.

Using an ultrafiltration filter (Amicon Ultra-4, molecular weight cut off 30, 000), perform centrifugation at 25 C, 3000 rpm, 2 min. L iposome-? ^ The complex and the non-adsorbed _PLys were separated (Fig. 9). Dilute the concentrated liposome-P _LYS using HEPES and perform the same concentration. By repeating this operation seven times, liposome-P _Lys and P _Lys were completely separated.

 The particle size of the purified iposome-P ^ was measured using dynamic light scattering.

(V) Observation of secondary structure of P _Lys using circular dichroism spectrometry (CD)

 Circular dichroism spectrometry is one of the measurement methods used in the study of the three-dimensional structure of proteins and nucleic acids. It is also used as a means to determine the chirality of small molecule biological materials.

 Planar polarized light (linearly polarized light) is a combination of left and right polarized light having the same amplitude and frequency rotating left and right toward incident light, and is expressed as follows using complex numbers.

 Number 8

e ^lwt = cos cot + is t β ~ ^1ϋ ~ = cos c-isin ca (Equation 7) e ^l0X + e ~ ^{ia (} -2cos

When this plane-polarized light passes through an optically active substance, the extinction coefficients of left and right circularly polarized lights are different, so that the speed and the absorbance of the light passing through each substance are different. This phenomenon of different passing speeds is called circular dichroism (CD). FIG. 10 shows how incident light of plane polarization passes through the optically active substance and becomes elliptically polarized light. When viewed from a direction perpendicular to the direction of travel, plane polarized light oscillates on a straight line as a result of superimposing left and right circularly polarized light (A or A _R ). Therefore, plane polarized light is also called linearly polarized light. When passing through an optically rotatory substance, the plane of polarization rotates due to the different extinction coefficients of left and right circularly polarized light. The angle is called Q: (In the case of Fig. 10, the extinction coefficient of left-handed circularly polarized light is larger than that of right-handed circularly polarized light, and the right-handed circularly polarized light is transmitted faster. As a result, the plane of polarization rotates rightward ( Permeation of right-handed substance)). Also, when transmitting a circular dichroic substance, the absorbance of the left and right circularly polarized light is different, so that the amplitude of the left and right polarized light after transmission is different, and the locus of the polarization plane is elliptical. (In the case of Fig. 10, since the absorbance of left-handed circularly polarized light is larger, the amplitude of left-handed circularly polarized light is smaller than that of right-handed circularly polarized light after transmission through the sample. As a result, their superposition becomes elliptically polarized light. ) CD is expressed as Δ £ = s _L- £ _R using the molecular extinction coefficient for left and right circularly polarized light (8 _R ). In addition, since the absorbances for the left and right circularly polarized lights are different, the amplitude of the circularly polarized light also differs, and the transmitted light becomes elliptically polarized light. The size of the CD is also represented by the angle 0 (ellipticity) defined by the ratio of the minor axis to the major axis of the ellipse. The molar ellipticity [0] (molar, ellipticity) is represented by the following equation.

 Number 9 '

[θ]-cm ¹ · ώηοί—ή (Equation 8)

c: molar concentration of sample (mol · cm- ³ )

 1: Optical path length (cm)

 θ ~] and △ ε are denoted with a positive or negative sign, and are also referred to as a positive cotton effect and a negative cotton effect, respectively.

In this study, we investigated the secondary structure of the P _TYS by performing circular dichroism measurements in the far ultraviolet region using circular dichroism measuring apparatus (J- 720W1 JASC0). A 300-400 L sample was injected into a cell with an optical path length of 1, and the molecular ellipticity [0] was measured in the far ultraviolet region (190 M! -250ηπι). The measurement was performed at a measurement speed of 20 nm / miii and a scan count of 10 to 30 times. Observation of secondary structure of P

It is known that P _Lys has three secondary structures, ie, a helix, a three-sheet, and a random coil, depending on the temperature and pH, and that it has a random structure at physiological pH. Therefore, each structure was prepared by the following method, and CD spectrum measurement at 190 to 250 nm was performed. Random coil: dissolved in pH 7.4 buffer

 α helix: soluble in pH 1.4 buffer

 Sheet: Dissolve in pH11 buffer, heat at 65 ° C for 10 minutes, then cool to 20 ° C. Using each solution, measurement was performed under the conditions of a measurement speed of 20 nm / min and a scan count of 10 times.

• P _Ty associated with ribosome adsorption. Secondary structure change

CD spectra were measured to study the change in secondary structure due to the adsorption of P _Lys to ribosomes.

 A liposome with the composition of DLPA / DMPC = 1.0 / 0, 0.5 / 0.5, 0 / 1.0 was prepared. (Note that the liposome used for CD measurement was made with HEPES, and the background noise of the CD spectrum became large. Therefore, 200 L of this ribosome suspension (final phospholipid concentration: 0.5 mM) and 200 L of a solution of a predetermined concentration were added at 25 ° C, 700 rpm, and 30 min. The reaction was carried out under stirring conditions of pH 7.4. Using this solution, the CD spectrum was measured at a cell temperature of 25 ° C, a measurement speed of 20 nm / iniii, and 30 scans.

(2) Adsorption of poly-aspartic acid (P _Asp ) on liposome-P _Lys surface

The adsorption of poly-aspartic acid (P _Asp ) on the surface of the prepared liposome-P _Lys complex is examined.

The liposome-? The complex was studied. By adsorbing the polycation, P _Lys , on the ribosome surface containing PA as a constituent lipid, the surface potential was reversed, and the complex was found to be positively charged. Therefore, we attempted to adsorb the polyanion P _Asp to the liposome-P _Lys surface as the second layer adsorption. 1 Preparation of iposome-P _Lys -P _Asp

 DLPA: DMPC = l: l (by mol) liposome 500 1 (final phospholipid concentration 0.5mM)

The P _Lys solution 500 ^ 1 (final _Pis concentration 400 ppm) was _reacted for 30 min at 25 ° C, 700 rpm, and pH 7.4. The reaction solution was filtered using an ultrafiltration filter (fraction molecular weight: 100, 000) for 2 min.

Purification was performed by concentrating and redispersing 7 times at 25 ° C and 3000 rpm. Made

11 0301116-? ^ Solution 500 ^? ^ Solution 500 // 1 (final concentration 40 (^ 111) was reacted under the same reaction conditions. To remove polyion complex, 15miii, 25T :, 15, After centrifugation under the condition of OOOrpm, the same purification was performed using an ultrafiltration filter. '

 (3) Fabrication of ribosome-polypeptide complex structures with different charges

Until now, a negatively charged ribosome as a template is coated with P _Lys or P _Asp to have a strong ion potential 1 iposome-? We have produced a complex, 1 iposome-P _Lys -P _Asp with anionic properties. Therefore, an attempt was made to fabricate a structure using these charges.

The l iposome_P _Lys solution 200 1 and the l iposome-P _Lys -P _Asp solution 200 1 (both having a final phospholipid concentration of 0.5 mM) were reacted under the conditions of 30 min, 25 ° C, and 700 rpm. Also, a ribosome having a negative charge]) LPA / DMPC). 5 / 0.5 and 1 iposome-P _Lys were reacted under the same conditions. The sample thus obtained was negatively stained and observed by FE-TEM.

 2. Ribosome adsorbed polypeptide

 The results of the preparation and examination of the ribosome to which the polypeptide was adsorbed were as follows.

(1) Adsorption of poly-Llysine (P _Lys ) on the ribosome surface

 (i) Adsorption amount

 • Creating a calibration curve

In this study,

Was calculated. Final P _Lys concentration of 5 using a pH 7. 4 of HEPES for preparation of the calibration curve, 10, 20, 50, 100, 200, 500, to prepare a P _s solution LOOOppm. A calibration curve was created using these data (Fig. 11). From the calibration curve, it can be said that PL _ys can be quantified using CBQCA. Regarding the measurement range, 500 ppm and 100 ppm deviate slightly from the calibration curve, but are considered to be within the error range from the R ² value. According to the literature, the measurement range of protein quantification using CBQCA is 0. Olppn! Since it has been reported that detection is possible up to about 1500 ppm, it is possible to quantify P! ^ In this range. • Calculation of adsorption amount

Cross-sectional area at the interface with DLPA a _D = 0.6 nm ² · Length when all C-C bonds are in the transformer l _c = l. 43 nm, and for DMPC a. = 0.65 nm ² · l _c = 1 80 nm (Fig. 12). At this time the diameter The number of lipid molecules contained in one lOOnm ribosome is calculated by dividing the sum of the ribosome outer surface area and inner surface area by the area occupied by one lipid molecule.

 Number 10

^Marauder : ^{4 02 +47} ' ¹⁴² ) ₌ ^9.89 _{X 10} 4 (pieces)

 0.6

Marauder _PC: 50 ² ÷ 46.4 ² ) ₄

 0.65, represented as no.

 Here, consider the case of a binary system consisting of DLPA and DMPC. DLPA / DMPO0.5: For ribosomes with a composition of 0.5 (by mol), the total number of DLPA and DMPC contained in one liposome is

^^ / ^ iv r ^ -u ^ .u ^ ;. 50 +46 X ₄ ^

 '(0.6 + 0.65),

(4.68 XH) ⁴ to 2 = 9.36 χ 10 ⁴ (pieces). From this, the number of liposomes per 1 mol of lipid is expressed as follows.

 Number 1 2

^ —- = 6.43

9.36 ^ IO ⁴

N _A : Apogadro number

When making ribosomes of DLPA / DMPC = 0.5: 0.5 (by mol), each lipid is weighed and mixed 5.0 x 10-—ol, so the number of ribosomes formed in this system is

Number 1 3 ^{(6.43 x 10 18) χ (} 1.0 x 10 one ^6} = 6.43 ^χ 10 ¹³ (pieces)

Actually, the final phospholipid concentration is 6.43 × 10 ¹² (pieces) because it is used at 1/20 the concentration at the time of preparation. Therefore, the total surface area of the ribosome in the system is

 Number 1 4

4π X 50 ² X (3.22 χ 10 ¹² ) = 1.01 χ 10 ¹⁷ rnm ² )

It is expressed.

Assuming that 50 ppm of P _Lys was adsorbed on the liposome surface with a composition of DLPA / DMPC = 0.5 / 0.5, the amount of adsorption was

 Number 1 5

It is expressed.

Based on the I \ _ys concentration quantified by CBQCA method in the present embodiment, it calculates the by Ri adsorbed amount calculation as described above.

 (ii) Examination of reaction time

The reaction was carried out using ribosomes having a composition of DLPA / DMPOL 0/0, 0.5 / 0.5, 0 / 1.1.0 and P _Lys for a reaction time of 20 min and 80 niin. Table 3 summarizes the adsorption amount.

Table 3 Time dependence of sorption

 PA morph fraction time / adsorption

 Min 1 ng * cm ""

 0 20 0.00

 0.5 20 48.16

 1.0 20 48.09

 0 80 0.00

 0.5 80 45.54

 1.0 80 49,50

From these results, it is considered that when the mole fraction of PA was 1.0 or 0.5, almost all of the charged _Lys had absorbed. Since there is no significant difference between 20 mm and 80 min, the reaction time in these systems is considered to be sufficient for 20 min.

For this reason, the subsequent stirring conditions for the reaction of ribosomes with P _Lys were 25 ° C, 700 rpm, 30 min, and pH 7.4.

 (i i i) Creation of adsorption isotherm

ΙΟπιΜ HEPES pH7. 4 DLPA / DMPC = 0 using. 5/0. Ribosomes 5 composition was prepared, when varying the concentration of P _tys added (final concentration 5, 10, 20, 50, 100 , 200, 400, 600, 800, 1000, and 2000 ppm) are shown in Table 4 and the adsorption isotherm created based on the _Ps concentration is shown in Fig. 13 (adsorption amount). The numerical value of is the average value of the same experiment performed four times.)

Table 4

Change in adsorption amount depending on polymer concentration

One ribosome One ribosome Charge P _Lys concentration PLy _S adsorption amount Per PLy _S adsorption amount

/ ppm / ppm / ng · cm— ²

Number of PLy _S molecules PLYS occupied area / nm ²

5 5.00 4.95 18.70 1275.98

10 10.00 9.90 37.39 2551.96

20 20.00 19.80 74.78 5103.91

50 50.00 49.50 186.96 12759.78

100 94.85 93.91 354.65 24204.67

200 127.09 125.83 475.22 32433.45

400 130.32 129.02 487.26 33255.82

600 137.21 135.85 513.05 35015.40

800 140.04 138.65 523.63 35737.60

1000 132.20 130.89 494.29 33735.59 '

1500 130.00 128.71 486.09 33175.43

2000 143.24 141.82 535.58 36553.59

From FIG. 13, it can be seen that the adsorption amount is almost constant. This adsorption isotherm is of the Langmuir type, and it is considered that almost all adsorption sites on the liposome surface are buried at 130 to 140 ng m ² . There is a part where the amount of adsorption slightly fluctuates, but this is due to the liposome used, where the PA molecules that become the adsorption site are oriented toward Balta. Therefore, it is considered that there is some difference in the number of adsorption fields of P _Lys .

 Table 5 below summarizes the changes in the state of the liposome-P complex before and after separation by ultracentrifugation.

Table 5 Changes in the state of the ribosome-P _LYS complex

State of π liposome-P _LYS complex

PLVS; cinnabar / ppm —

 20

 50

 100

 200

 500 Membranous sediment

 1000 Membranous sediment

 1500 Membranous sediment

 2000

 ノ, ノ ノ ノ 777, s Membranous sediment

 ο. 3τ- κ κ ,, ゝ \

From these results, when the charged amount of P was 5 to 10 ρππ, the particles were redispersed even after separation by ultracentrifugation and were monodispersed at about 100 nm, whereas they were agglomerated at 20 to 100 ppm. Further, when the content was 200 ppm or more, a film-like sediment was generated (Fig. 14).

 Suspicion

Such a difference is considered to be caused by fi that the amount of _Lys adsorbed on the ribosome is different. As shown in FIG. 1 5, for the case of charge of. 5 to 10 ppm small area occupied by the PL _ys to the surface of the liposome, reduce the influence of positive charge, negative charge as a whole liposome Is considered to have a state. Therefore, it is considered that the monodispersion exists even after the separation operation by ultracentrifugation due to the repulsion between the negative charges. On the other hand, in the case of 20 to 100 ppm, there are a portion where P _Lys is adsorbed and a portion where it is not, so it is considered that there are a portion showing a positive charge and a portion showing a negative charge on the ribosome surface. Therefore, it is considered that aggregates are easily formed after the ultracentrifugation operation due to the electrostatic interaction. If the concentration exceeds 200 ppm, excess will be present in the system. After ultracentrifugation in such a system, a film-like sediment is formed. As will be discussed later, it is probable that such membrane-like precipitates have been formed due to bridging between ribosomes with excess P _Lys .

 (iv) Examination of film-like sediment

As described above, liposome-P _Lys complex is formed in the presence of excess P, It was found that a membrane-like sediment as shown in Fig. 14 was formed when this purification was attempted. In order to examine the state of ribosomes in the sediment, we prepared ribosomes containing 1-hydroxypyrene-3,6,8-trisul fonic acid (HPTS), a fluorescent substance, and performed similar experiments. By doing so, we examined the state of ribosomes in the membrane.

When liposomes were hydrated, they were hydrated using a 10 mM HPTS solution (pH 7.4), and purified using gel filtration and ultracentrifugation to obtain HPTS-enclosed ribosomes. HPTS-encapsulated liposome 5001 (final phospholipid concentration 0.5 mM) and P _Lys solution 500 x 1 (final concentration 100 ppm) were reacted for 30 min at 25 ° C, 700 rpm, and pH 7.4. The reaction solution was ultracentrifuged at 45 and 4 min at 70 and OOO rpm. Figure 16 shows the film-like sediment generated at this time. It is a film-like sediment formed in the case of).

 According to FIG. 16 (a), the membrane was green, but the supernatant after ultracentrifugation was also green. This suggests that the ribosome may have collapsed during membrane formation. The green membrane formed in (a) may be green due to the binding of HPTS and Ι ^ 'leaked out by ribosome breakdown due to electrostatic interaction.

For this reason, ultracentrifugation of the system in which ribosomes and excess P _Lys were present causes the ribosome to collapse and crosslink the surrounding P _Lys to form a membrane-like sediment as shown in Figure 16. It is thought that it is. In addition, it was found that these film-like sediments were relatively stable and could maintain their shape in HEPES at pH 7.4. Furthermore, it was confirmed that the shape of this film-like sediment collapsed in pH 3.6 MES.

 (v) Examination of purification method

 Purification using ultracentrifugation covered almost the entire ribosome surface as described above.

One iposome-complex could not be obtained in a monodisperse manner. Therefore, purification using ultrafiltration was attempted. The l iposome-P solution was purified and concentrated seven times using an ultrafiltration filter (fraction molecular weight: 300, 000) at 25, 3,000 rpm and 2 min, and the l iposome-P was purified. .

L iposome- using dynamic light scattering? The particle size was measured and found to be monodisperse at about 100 nm. was gotten. When the phospholipid concentration was measured before and after purification, it was slightly reduced but the loss was small.

It can be said that P _Lys and the liposome-P complex can be separated in a monodispersed state with a small loss.

 In subsequent experiments, purification of the liposome-complex was performed using an ultrafiltration filter.

(vi) Examination of secondary structure of P _Lys using CD

• Observation of secondary structure of P, _y ,

It is known that P _Lys has three secondary structures: α helix, j3 sheet, and random coil (Fig. 17). Fig. 18 shows the results of CD spectrum measurement at 190 to 250 nm for each structure.

As for the ellipticity of each secondary structure, the random coil has a maximum at 195 nm, the α-helix has a minimum at 222 nm and 208 nm, a crossover at 200 mn, a maximum at 191 mn, and a j6 sheet has a minimum at a crossover of 206 to 207 nm. · We know we have a maximum. When this value is compared with Figure 18, almost the same CD spectrum is obtained. Thus, it can be confirmed that each secondary structure of _PLys is formed. Thereafter, Fig. 18 was examined as the reference spectrum of the secondary structure.

 • Secondary structure change of the ribosome upon adsorption to ribosome

DLPA / DMPC = 1.0 / 0, 0.5 / 0.5, 0 / 1.0 Reaction of ribosome solution (final phospholipid concentration 0.5mM) and solution (final _Lys concentration 50m) Fig. 19 shows the results of CD spectrum measurement. Figure 20 shows the average of the molar ellipticity in the region of 195-200 nm, which is characteristic of random structure and] 3 structure. It is known that in this region, the random structure has a negative molar ellipticity and the β structure has a positive molar ellipticity.

This I \ _ys so far adsorbed on the ribosomal surface with the configuration lipid ΡΑ, the ribosomes were made only at the PC has been confirmed that no adsorption. From FIGS. 19 and 20, it can be seen that those with a PA fraction of 1.0 and 0.5 in which P is adsorbed have a / 3 structure, and those with only PC have a random structure.

Next, a ribosome solution (final phospholipid concentration: 0.5 mM) with the composition of DLPA / DMPO 0.5 / 0.5! ^ Solution (final P ^ concentration 400 ρριιι), before and after purification by ultrafiltration The spectrum measurement results are shown in Fig. 21 and the average of the molar ellipticity is summarized in Fig. 22. From this result, it can be seen that before purification, the protein had a random structure, whereas after purification, it had a) 3 structure. This is because P _{Lys, which} had not been adsorbed before purification, had a large number of random structures, whereas purification using ultrafiltration removed them, leaving a / 3 structure on the ribosome surface. This indicates that the adsorbed P ^ remains. From these results, it was confirmed that the secondary structure of _Iys changed from a random structure to an 8) structure by adsorption to ribosomes.

• Ribosomes and P _{T y} . The interaction of

From the results in Fig. 19 to Fig. 22, it was found that the secondary structure of P was changed from a random structure to a (3) structure with the adsorption to the ribosome surface. Is known to show the j6 structure in the high pH region as shown in Fig. 17, but when adsorbed on the ribosome surface, it adopts the jS structure even though the pH is near neutral. Adsorption of P _s is caused by electrostatic attraction between the phosphate moieties of DLPA a constituent lipid of Amino groups and ribosomal lys ine residues of the side chains of P _s. If only to produce a ribosome DLPA, P _Lys changes to the / 3 structure interactions at multiple points for many adsorption sites Bok respect lys ine residues of P _s. On the other hand, in the case of DMPC, because of the presence of the choline group, the electric charge of the phosphate moiety is canceled out, indicating neutrality in charge. This does not mean that electrostatic attraction works. Furthermore, the fact that the choline group is structurally bulky may also prevent P _Lys from approaching.

Considering the case of liposome and P _Lys in a two-component system of DLPA and DMPC, it is considered that the change to the i3 structure does not appear unless DLPA is present to some extent. Also, PL _ys if any portion DLPA in Liposomes one beam surface are continuous induces interact i3 structure from electrostatic attraction, but if the part if any DMPC would be present remains random It is possible (Fig. 23). From this, as shown in Fig. 19, when the content of DLPA increases, the j3 structure becomes richer because the ratio of the continuous sequence of DLPA is large on the ribosome surface, so that it becomes easier to adopt the / 3 structure. It can be said that it is.

In this study, the adsorption experiment was performed at room temperature (25 ° C). This temperature is slightly lower than the phase transition temperature of the ribosome, and is the temperature at which the gel phase begins to partially change from the gel phase to the liquid crystal phase. This temperature must be clearly different from the phase transition temperature. It can be said that the difference in the adsorption behavior is caused by the removal. If the T> T _e, continuous to become lipid molecules easy to move in the lipid membranes, in order to become easily take contiguous sequence of DLP'A, that T <immobile lipid molecules if T _e It becomes difficult to take the array.

 From these points, it can be said that the temperature conditions and lipid composition in the adsorption of are factors that greatly affect the adsorption behavior.

 (vi i) Shape observation and 形状 potential measurement using FE-TEM

The observation results using the prepared l iposome- I _ys complex FE- subjected to negative staining in the TEM are shown in Fig 4. Figure 24 shows that the liposome-P ^ complex exists with a relatively spherical shape.

The ζ potential of the liposome-P _Lys complex showed a positive charge at 70.865 mV. DLPA / DMPC used as template = 0.5 / 0.5 The ribopotential of the ribosome had a negative charge of -73.585 mV. You can see that is happening.

(3) Adsorption of poly-aspartic acid (P _Asp ) on l iposome- P _Lys surface

(i) Preparation of l iposome- P _Lys _P _Asp

Adsorption experiments were performed using _polyanion Poly-L-aspartic acid (P _AsD ) as an adsorbate on the surface of the prepared liposome-P _Lys .

 DLPA: DMPC = 1: 1 (by mol) composition of ribosome 500 1 (final phospholipid concentration 0.5 mM) and? Solution 500 1 (final? 1 ^ concentration 400400111) at 301 ^ 11, 25 ° C, The reaction was carried out under the conditions of 700 rpm and pH 7.4, and the mixture was concentrated and re-dispersed seven times under the conditions of 3000 rpm at 25 ° C for 2 min using an ultrafiltration filter (fraction molecular weight: 100, 000) to purify. Done.

The liposome-P _Lys solution 5001 thus prepared and the P _Asp solution 500_ί1 (final P _Asp concentration: 400 ppm) were reacted under the same reaction conditions. At this time, it is possible that P _Asp could form a polyion complex if it was not completely removed in addition to the surface of the 11003011½-? ^ Complex. Therefore, centrifugation was performed under the conditions of 15 min, 25 ° C, 15 and OOOrpm to remove the polyion complex. Further, the supernatant after centrifugation was subjected to the same purification using an ultrafiltration filter.

The particle size of the 1 ipsome-P _Lys -P _Asp complex thus obtained was measured by a dynamic light scattering method and found to be monodisperse at about 100 nm. (ii) FE-TEM observation of shape and measurement of ζ potential

Fig. 25 shows the results of negative staining of the prepared liposome-P ^ -P _Asp complex and observation using FE-TEM. From Fig. 25, it can be seen that the liposome-P _Lys -P _Asp complex exists while maintaining a spherical shape.

The ζ potential of the liposome_P _Lys -P _Asp complex was negative at -61.525 mV. Comparing with the ipotential of the l iposome-P ^ complex, it can be confirmed that the surface charge has been inverted due to the adsorption of P _Lys .

 (4) Evaluation of each ribosome by ζ potential

Changes in テ ン potential and の of l iposome, l iposome-P _Lys and l iposome-P _Lys -P _Asp prepared so far are summarized in Table 6 and FIG. 26.

変 化 From the change in potential, it can be seen that the adsorption of P _Lys and P _Asp on the template ribosome reverses the surface charge.

In this way, it is possible to obtain a monodispersed ribosome-polypeptide complex by reacting the ribosome surface with P _Lys or P _Asp and purifying it using ultrafiltration. In the present study, adsorption was performed up to the second layer at present, but it is possible to add more layers using the same method. It seems that more layers can be obtained by stacking layers.

 Table 6 Changes in ζpotential and ΕΡΜ

^ Potential 1 EPM 1 μπι- s " ¹ -cm *

Sample mV v- ^!

 Liposomes -73.585 -5.742

Ribosome-P _Lys 70.865 5.531 +

 Ribosome

 -61.525 -4.801

 -PLys Asp

(5) Preparation of ribosome-polypeptide complex with different charges

• About the structure consisting of l iposome- Ρ and l iposome- P, _y -P ^

The 1 ipos ome-P _Lys -P _Asp with 1 i po s ome-P _Lys and Anion of having a cationic were prepared. Therefore, these are mixed to form a structure, which is observed by FE-TEM. became.

 Fine granular agglomerates could be visually observed in the solution after the reaction. The sample obtained in this way was negatively stained and observed by FE-TEM. The results are shown in FIG.

 From Fig. 27, it can be confirmed that ribosomes of about 100 nm are lined up. Some of the shapes are slightly collapsed, but this is probably due to the drying process. On the whole, it can be said that the ribosome exists while maintaining almost spherical shape. In addition, when this sample was allowed to stand at room temperature, fine particles formed larger aggregates and settled as time passed.

• About the structure consisting of 1 iposome and 1 iposome-P _r „

The structure was attempted by mixing liposomes with the composition of DLPA / DMPO 0.5 / 0.5 with anionic properties and lipsome-P _Lys with cationic properties, and observed by FE-TEM. Also in this case, fine granular aggregates were visually confirmed in the solution after the reaction. Figure 28 shows the FE-TEM observation results.

From FIG. 28, it was confirmed that ribosomes of about 100 nm were linked. In addition, when this sample was allowed to stand still at room temperature, it was observed that fine particles formed larger aggregates and settled as time passed. These aggregates are composed of ribosomes with DLPA as a constituent lipid, l iposome_P _Lys , and l iposome-

It is thought that due to the difference in the surface charge of PLy _S -P _AsP , aggregates were formed due to the electrostatic interaction.

By creating particles with different surface potentials, such as liposomes, liposome-P _Lys , and 1 iposome-P _Lys- P _Asp , which have DLPA as a constituent lipid, we can use electrostatic interactions to construct structures using electrostatic interactions. It was suggested that it is possible to produce.

 In this example, the adsorption of polypeptide on the ribosome surface using Layer-by-Layer was examined.

Using a ribosome having a negative charge as a template, poly-L-lysine (P _Lys ), which is a polycation, was adsorbed as a first layer adsorbate. From the adsorption isotherm of Langmuir type created from the adsorption amount measurement using CBQCA, it is considered that almost all adsorption sites on the ribosome surface are buried at 130 to 140 ng / cni ² . In addition, P has a secondary structure during adsorption. Was changed from random to three sheets. Furthermore, the liposome-P ^ complex was obtained in a monodispersed state by purification using ultrafiltration.

Using a polyanion, Poly-L-aspartic acid (P _Asp ), as a second layer adsorbate, it was adsorbed on the surface of 1 iposome_P _Lys . The potential of these liposome-polypeptide composite particles was confirmed to change with each adsorption from the measurement of the potential. This also indicates that the polypeptide is adsorbed on the surface. The ribosome-polypeptide complex thus prepared was negatively stained, and the shape was observed by FE-ΤΕΜ. As a result, the ribosome-polypeptide complex was observed to be spherical.

 In addition, using a ribosome-polypeptide complex with different charges, we were able to construct a structure using electrostatic interaction. It was observed that ribosomes were linked in the structure while maintaining their morphology. By introducing factors that control shape and arrangement into this experimental system, it can be said that a more ordered structure can be produced.

 [Example 3] Preparation of ribosome-polypeptide complex

 Liposomes having a multilayer structure were prepared using a ribosome having a composition of DLPA / DMPO 0.5 / 0.5 as a template.

(1) First layer (production of l iposome-P _Lys )

l iposome 500 1 (final phospholipid concentration 0.5 mM) and Poly-L-lysine (P _Lys ) 500 1 (final P _Lys concentration 400 ppm) at 25 ° C, 700 rpm, 30 min, pH 7.4 The reaction was followed by purification by ultrafiltration at 25 ° C., 3000 rpm, 2 min. At this time, concentration and redispersion were performed seven times.

(2) 2nd layer (production of l iposome-P _Lys -P _Asp )

(1) l obtained in iposome _P _Lys 500 1 (final phospholipid concentration 0. 5 mM) and Poly-L - aspart ic acid ( ? Lys) 500 x 1 in (final P _Lys concentration 400 ppm) to 25, 700 rpm, The reaction was carried out under the conditions of ρΗ7.4 for 30 min, followed by centrifugation at 25 ° C, 15000 rpm for 15 min to remove the polyion complex. Furthermore, purification was performed by ultrafiltration under the conditions of 25 ° C, 3000 rpm, and 2 min. At this time, concentration and redispersion were performed seven times. As a result, a ribosome having two layers of polymer laminated on the surface was obtained. (3) Third layer (preparation of l iposome-P _Lys -P _Asp -P _Lys )

The l iposome-P _Lys -P _Asp 500 1 (final phospholipid concentration 0.5 mM) and Poly-L-lysine (P _Lys ) 500 1 (final P _Lys concentration 400 ppm) obtained in (2) at 25 ° C The reaction was performed under the conditions of, 700 rpm, 30 min, pH 7.4, and then centrifuged at 25, 15,000 rpm, 15 min to remove the polyion complex. Furthermore, purification was carried out by ultrafiltration at 25 ° C, 3000 rpm and 2 min. At this time, concentration and redispersion were performed seven times. As a result, ribosomes with three layers of polymer laminated on the surface were obtained. Figure 29 shows the FE-TEM observation image.

(4) Fourth layer (preparation of l iposome _P _Lys -P _Asp -P _Lys -P _Asp )

L iposome- P _Lys -P _Asp -P _Lys 500 1 (final phospholipid concentration 0.5 mM) and Poly-L-aspartic acid (P _Lys ) 500 1 (final P _Lys concentration 400 ppm) obtained in (3) ) Was reacted at 25 ° C, 700 rpm, 30 min, pH 7.4, and centrifuged at 25 ° C, 15000 rpm, 15 min to remove the polyion complex. Furthermore, purification by ultrafiltration was performed under the conditions of 25 ° C, 3000 rpm, and 2 min. At this time, concentration and redispersion were performed seven times. As a result, a ribosome having four layers of polymer laminated on the surface was obtained. Figure 30 shows an FE-TEM observation image.

 The results of the measurement of the zeta potential of the ribosome having a multilayer structure thus obtained were as follows. Figure 31 shows the graph.

Table 7 Sample ^ potential (mV) —mobility charge liposome -127.03 9.902 liposome-P _Lys 83.01 6.478

liposome-P _Lvs -P _Asp -81.66 6.373

liposome-P _L y _S -P _AS p-PLys 52.12 4.068

liposome-P _LYS -P _Asn -PLYs-PA _SD -61.99 4.837

[Example 4] Adsorption of polyallylamine and mercaptoacetic acid on liposome surface and lamination of fluorescent semiconductor nanoparticles

In the present example, polyallylamine and mercaptoacetic acid are adsorbed on the surface of a ribosome of type I, and the lamination of semiconductor nanoparticles (nanophosphor particles) is examined. As described in Example 1, ribosomes containing DLPA as a constituent lipid are negatively charged. I know you are. Therefore, we attempted to make particles with different surface charges by layer-by-layering polyacrylamine (PAA) and mercaptoacetic acid on the ribosome surface. Figure 32 shows a conceptual diagram of Layer-by-Layer in this case.

 (1) Adsorption of polyarylamine on liposome surface

 By containing the acidic lipid DLPA as a constituent lipid, the liposome (liposome) is negatively charged and has an adsorption site (see Example 1). We investigated the behavior of ribosomes by allowing polyallylamine, a polycation, to act on the ribosome surface and adsorbing them by electrostatic interaction.

 (i) Adsorption measurement

 A ribosome solution (final phospholipid concentration: 0.5 mM) and a predetermined concentration of a polyallylamine solution (500 × L) were allowed to react at 25 ° C., 700 rpm, 30 min., A predetermined concentration and a predetermined pH under stirring conditions. After the reaction, the mixture was centrifuged using an ultrafiltration filter (Amicon Ultra-4, molecular weight cut-off 30,000) at 25 ° C, 3000 rpm, and 2 min, to obtain 1 iposome-polyarylamine from the mixed solution. The body sank. The concentration of adsorbed polyarylamine was calculated by measuring the concentration of separated polyallylamine using CBQCA.

 (i i) Preparation of adsorption isotherm

 Using 10 mM HEPES pH 7.4, ribosomes with the composition of DLPA / DMPO 0.5 / 0.5 and DLPA / MPC = 0.75 / 0.25 were prepared, and the concentration of polyallylamine added was changed to pH6. Adsorption isotherms were created at 48 (PAH because of the positive charge associated with the proton) and pH 8.50 (PAA in this case).

 Ribosome solution 500 L (final phospholipid concentration 0.5 mM) and PAA or PAH solution 500 ^

L was reacted at 25 ° C. under stirring conditions of 700 rpm for 30 min. After the reaction, centrifugation was performed using an ultrafiltration filter (Amicon Ultra-4, molecular weight cut off 30,000) at 25 ° C, 3000 rpm, and 2 min. The complex was allowed to settle. The concentration of adsorbed PAA or PAH was calculated by measuring the concentration of the separated PAA or PAH using CBQCA. This operation was performed for each god and an adsorption isotherm was obtained. The results are shown in FIG. As the negative charge of the ribosome increased, the amount of polyallylamine adsorbed increased, indicating that PAH with higher positive charge adsorbed PAA and PAH more easily. (lii) Mercaptoacetic acid adsorption on ribosome-polyallylamine complex

 Using 10 mM HEPES pH 7.4, a ribosome (final concentration: 1.5 mM) having a composition of DLP'A / DMPC = 0.5 / 0.5 was prepared, and polyallylamine was added to a final concentration of 500 ppm. The adsorption was performed at 25 ° C for 30 minutes under stirring at 700 rpm. A ribosome-polyallylamine complex was obtained by ultrafiltration at 25 ° C. To 0.8 mL of the 1.5 mM complex, 200 L of 4000 ppm mercaptoacetic acid was added, and the mixture was adsorbed at 4 ° C under stirring at 500 rpm for 20 minutes.

 (2) Sample preparation for semiconductor nanoparticle fabrication

Cation solution _{(0. 09M Zn (CH3COO) 2} 0. 01M Mn (CH 3 C00) 2)

396.68 mg of zinc acetate dihydrate was added to 18 ml of ultrapure water, and dissolved using a magnetic mixer (0.1 M Zn (C¾C00) ₂ ). Meanwhile, 49.20 mg of manganese acetate tetrahydrate was added to 20 ml of ultrapure water and dissolved using a magnetic stirrer (0.1 M Mn (CH3COO) ₂ ).

After sufficiently dissolving each, the two aqueous solutions were mixed to prepare a cation solution. Anion solution (0.1 M Na ₂ S)

 488.28 mg of sodium sulfide nonahydrate was dissolved in 20 ml of ultrapure water.

Dispersion stabilizer (0.1 M C ₆ H ₅ Na ₃ 0 ₇ )

 Sodium sodium citrate hydrate 295. (kg was dissolved in 10 ml of ultrapure water.

(3) Conjugation of ZnS: Mn2 ⁺ nanoparticles to ribosome-polyallylamine-mercaptoacetic acid complex

 4 ml of ultrapure water (30 ° C) was added to the flask, and the atmosphere in the flask was replaced with nitrogen for 90 minutes.Then, 0.1 M sodium citrate dihydrate 0.2 ml, 0.1 M sodium sulfide nonahydrate 0.32 ml, acetic acid A mixed solution of zinc dihydrate and manganese acetate (Π) tetrahydrate (9: 10.1 M, 0.4 ml) was added in that order, and the mixture was reacted in a nitrogen atmosphere. The reaction time was 45 minutes, and then ribosomes and various ribosome complexes (lipid amount: 3.0 mol) were added and reacted for 2 hours.

 (4) Characteristics evaluation method

The obtained composite sample was excited using an Xe lamp, and the fluorescence intensity emitted from the sample was measured using a spectrofluorometer. As the excitation wavelength at that time, the excitation spectrum was measured at a fluorescence wavelength of 580 m using a spectrofluorometer for each sample, and the peak wavelength of the excitation spectrum was used. It was found that the ribosome, ribosome-polyallylamine complex, and ribosome-polyallylamine-mercaptoacetic acid complex 'all adsorb semiconductor nanoparticles. The sample obtained by conjugation to the liposome-PAH complex showed the highest fluorescence intensity, but also resulted in high cohesion. It was found that the ribosome-PAH-mercaptoacetic acid complex hardly aggregated and gave a sample with high fluorescence intensity (Table 8). Table 8

 Sample fluorescence intensity

① Liposome-ZnS: Mn ^2T com lex 805.7

(DLiposome-PAA-ZnS: Mn ²⁺ complex 119. 7

③ Liposome-PAH_ZnS: Mn ²⁺ complex 930.7

④ Liposome- PAA_mercaptoace tic ac id-ZnS: Mn ²⁺ com lex 581.2

⑤ Liposome-PAH- mercaptoacet ic ac id-ZnS: Mn ²⁺ complex 625.1 Industrial applicability

 The ribosome on which the polymer of the present invention is laminated can be manufactured at any size and any strength.When a charged polymer is used as the polymer, ribosome particles that can be uniformly dispersed can be obtained. Can be. Furthermore, it is possible to encapsulate a specific substance inside the ribosome, or to include a specific substance in the polymer to be laminated. Such a hollow particle in which a polymer is laminated on a ribosome is a particle that has not existed conventionally, and a ribosome in which the polymer of the present invention is laminated cannot be achieved by the conventional technology. Having.

 All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

 The scope of the claims

I. A liposome in which two or more layers of a compound containing at least one polymer are laminated on the surface.

 2. The liposome according to claim 1, wherein the compound is a biocompatible polymer.

 3. The liposome according to claim 1, wherein the compound is a biodegradable polymer.

 4. The liposome according to any one of claims 1 to 3, wherein the compound is at least one selected from the group consisting of proteins, polyamino acids, polysaccharides, and nucleic acids.

 5. The ribosome according to any one of claims 1 to 4, wherein a negatively or positively charged compound is alternately stacked on the positively or negatively charged ribosome by electrostatic interaction.

 6. The ribosome is a negatively charged ribosome, and the ribosome comprises at least one lipid selected from the group consisting of phosphatidic acid, phosphatidylglycerols, phosphatidylserines, and phosphatidylinositols as constituent lipids of the ribosome. The ribosome according to claim 5.

 7. The liposome according to claim 6, wherein the constituent lipid of the ribosome is dilauroylphosphatidic acid (DLPE).

 8. The negatively charged compound is selected from the group consisting of poly-L-lysine, hyaluronic acid and nucleic acid, and the positively charged compound is selected from the group consisting of poly-L-arginic acid and chitosan. 8. The liposome according to any one of 5 to 7.

 9. The ribosome according to any one of claims 1 to 4, wherein a compound containing at least one polymer is alternately stacked on the ribosome having a reactive functional group via a chemical bond.

 10. The ribosome according to any one of claims 1 to 4, wherein the compounds are alternately stacked by utilizing the intermolecular force acting between the ribosome and the compound or between the compound and the compound.

II. The ribosome according to claim 10, wherein the intermolecular force is due to complementary hydrogen bonding of the nucleic acid.

12. The ribosome according to any one of claims 1 to 11, wherein a substance selected from the group consisting of a drug, a fluorescent substance, a bioactive substance, and a dye is encapsulated in the ribosome.

 13. The liposome according to claim 12, wherein the physiologically active substance is a protein or DNA.

 14. The liposome according to any one of claims 1 to 13, wherein the semiconductor nanoparticles are contained in a compound containing at least one polymer laminated on the ribosome surface.

 15. Include at least one polymer on the surface, including preparing a positively or negatively charged ribosome and alternately binding a negatively or positively charged compound to the ribosome surface by electrostatic interaction A method for producing ribosomes in which two or more layers of compounds are stacked.

 16. The method according to claim 15, wherein the compound is a biocompatible polymer, wherein two or more layers of the compound are laminated on the surface.

 17. The compound according to claim 15 or 16, wherein the compound is at least one selected from the group consisting of a protein, a polyamino acid, a polysaccharide, and a nucleic acid. A method for producing the laminated ribosome.

 18. The ribosome is a negatively charged ribosome, and the liposome comprises at least one lipid selected from the group consisting of phosphatidic acid, phosphatidylglycerols, phosphatidylserine, and phosphatidylinositol as lipids constituting the ribosome. A method for producing a ribosome in which two or more layers of a compound containing at least one polymer are laminated on the surface according to any one of claims 15 to 17.

 19. The liposome constituent lipid is dilauroylphosphatidic acid (DLPE), wherein the compound containing at least one polymer on the surface according to any one of claims 15 to 18 has two or more layers. A method for producing stacked ribosomes.

20. The negatively charged compound is selected from the group consisting of poly-L-lysine, hyaluronic acid, and nucleic acid, and the positively charged compound is poly-L-arginic acid, chitosan. 10. A method for producing a liposome in which two or more layers of a compound containing at least one polymer are laminated on the surface according to any one of claims 15 to 19 selected from the group consisting of:

 2 1. A liposome obtained by chemically cross-linking a stack of compounds using a cross-linkable substance.

 22. A solid crosslinked ribosome obtained by assembling the ribosome according to any one of claims 1 to 14 by crosslinking a surface compound.

 23. The solid bridge-type ribosome according to claim 22, which has a sheet shape or a tube shape.